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Progestin inhibition of cell death in human breast cancer cell lines
Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 218–227
Progestin inhibition of cell death in human breast cancer cell lines Michael R. Moore a,∗ , James B. Spence a , Kelley K. Kiningham b , Joshua L. Dillon a a
Department of Biochemistry and Molecular Biology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, United States b Department of Pharmacology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, United States Received 29 March 2005; accepted 26 September 2005
Abstract Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17 surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 M. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment. © 2006 Elsevier Ltd. All rights reserved. Keywords: Progesterone; Apoptosis; Breast cancer; Chemotherapy; Estrogen
1. Introduction While estrogens have convincingly been shown to stimulate breast cancer [1,2], and antiestrogens are one of the main forms of adjuvant therapy [3], the role of progestins has been more controversial. Various laboratories have reported that progestins inhibit [4,5], stimulate for one turn of the cell cycle and then inhibit [6,7], have no effect [8], and stimulate breast cancer [9–13]. We were the first to report, in 1987, progestin stimulation of proliferation in the human breast cancer cell line T47D and have since confirmed and extended this observation in several other reports [9,14–19]. We have found that progestins not only stimulate proliferation, but also inhibit cell death [19]. Ory et al. later confirmed our observation of progestin inhi∗ Corresponding author at: Department of Biochemistry and Molecular Biology, Joan C. Edwards School of Medicine, Marshall University, 1542 Spring Valley Drive, Huntington, WV 25755-9320, United States. Tel.: +1 304 696 7324; fax: +1 304 696 7253. E-mail address: [email protected] (M.R. Moore).
0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.09.008
bition of cell death in human breast cancer cells, reporting its mediation by medroxyprogesterone acetate in T47D, MCF-7 and H466-B cells [20]. Many recent large-scale clinical studies have strongly strengthened the idea that progestins in general stimulate breast cancer. Schairer et al., in a study involving 46,355 postmenopausal women on hormone replacement therapy [HRT], comparing HRT involving combined estrogen–progestin to that with estrogen alone, found that the risk of breast cancer increased 8% per year of combined HRT as compared to 1% per year with estrogen alone [21]. Persson et al. drew a similar conclusion, that combined estrogen–progestin HRT gave a greater risk of breast cancer than estrogen alone, in their study of 11,231 Swedish women [22]. In still another study of 3534 women, Ross et al. concluded that combined progestin–estrogen HRT increased breast cancer risk significantly more than estrogen alone [23]. Yet another report by Li et al. describes their study of 1029 women, finding that combined progestin–estrogen HRT increases breast cancer risk substantially more than estrogen alone [24]. More recently, the Women’s Health Initiative study [25] stopped the arm
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of the study involving 16,608 postmenopausal women taking either both estrogen and progestin or placebo because “the test statistic for invasive breast cancer exceeded the stopping boundary for this adverse effect”. Further, Lydon et al. reported, using PR knockout mice, that mammary gland carcinogenesis is critically dependent on PR function [26]. Finally, the “Million Women Study”, involving 1,084,110 women aged 50–64 in the United Kingdom, found an increased risk of incident and fatal breast cancer for those women on HRT using combined estrogen–progestin than for those on estrogen only [27]. Progestins have been shown to inhibit cell death also in normal mammary epithelial cells in vivo. Berg et al. showed that both progestins and glucocorticoids (separately) prevent apoptosis in the lactating rat mammary gland [28]. Feng et al. showed this also in postweaning (mother’s) mouse mammary gland [29]. Moran et al. found that glucocorticoid inhibits apoptosis in the immortalized human mammary epithelial cell line MCF10A [30]. Hence, it appears that progestin inhibition of breast cancer cell death is an unfortunate holdover from its function in normal breast cells. Based on our previous report [19], we have begun to further characterize progestin inhibition of breast cancer cell death, both from serum withdrawal and from chemotherapeutic agents.
2. Materials and methods 2.1. Reagents R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20dione) was purchased from NEN Life Science (Boston, MA). RU486 (17-hydroxy-11-[4-dimethyl-aminophenyl1]-17␣-[prop-1-ynil]-estra-4,9-dien-3-one) was a gift from Dr. R. Deraedt of Roussel-Uclaf (Romainville, France). Progesterone, testosterone, dexamethasone, aldosterone, estradiol-17, doxorubicin and 5-fluorouracil were from Sigma–Aldrich. Antibody to activated caspase 3 was from Cell Signaling Technology, Beverly, MA. 2.2. Cell culture All human breast cancer cell lines (T47D, MCF-7, and MDA-MB-231), obtained from the American Type Culture Collection, were grown in Corning plastic flasks (Corning, NY) in 5% CO2 in air at 37 ◦ C. Routine growth medium was minimum essential medium, powdered (autoclavable) plus non-essential amino acids, 2 mM l-glutamine, 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin (Gibco) and 6 ng/ml insulin (Sigma). This medium contains phenol red. Cells were harvested by replacing the growth medium with Hank’s Balanced Salt Solution without calcium and magnesium but with 1 mM EDTA, incubating 10 min at 37 ◦ C, aspirating the cells and centrifuging. Cells were then frozen at −80 ◦ C for storage prior to making extracts. The
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human neuroblastoma cell line SK-N-SH was obtained from the American Type Culture Collection and grown at 37 ◦ C in a humidified atmosphere with 5% CO2 . Growth medium was minimum essential medium plus 10% fetal calf serum, 1 mM sodium pyruvate, 1% nonessential amino acids and 1% penicillin–streptomycin–neomycin (Gibco). 2.3. Cell death assays Routine cell death assays were performed by trypan blue exclusion manually, using a hemocytometer to determine the total number of live and dead cells in each flask, extrapolated from an actual count of around 1500 cells per flask. In most experiments, cell death was assessed after 6 days of serum withdrawal, although in some experiments after 5–8 days. This was simply a matter of convenience, as the phenomenon is visible to the naked eye by the greater turbidity in control cell medium due to floating dead cells from day 5 on. Apoptosis was confirmed by three different methods: fluorescence microscopy, immunoblotting for activated caspase 3, and fluorescence activated cell sorting. 2.4. Fluorescence microscopy Cells were grown to confluency in 25 cm2 flasks in triplicate for each treatment in routine growth medium. They were then treated with 10 nM R5020 or vehicle in serum-free, phenol red-free medium for 5 days with no medium change or additional hormone added. Cells floating in the medium and still attached to the flask were then combined as follows. Cells still attached to the flask were harvested by incubating 10 min in Hank’s Balanced Salt Solution without calcium or magnesium, but with 1 mM EDTA, and aspirated well to separate them into individual cells. These cells were then combined with those that had been floating, washed twice with 0.9% NaCl, and then suspended in the saline at >3 × 106 cells/ml. Next 2 l of 0.1 mg/ml acridine orange, 0.1 mg/ml ethidium bromide, in 0.9% NaCl were added. Approximately 10 l of dyed cells from each flask were then examined by placing on a microscope slide, covering with a 10 mm × 10 mm no. 1 cover slip, and observing with a Nikon Microphot SA microscope equipped for fluorescence imaging. At least 100 cells from each flask were then scored for apoptosis [31] by a scorer “blinded” as to the samples’ identity. 2.5. Immunoblotting for activated caspase 3 Cells were grown to confluency in fourteen 150 cm2 flasks in routine growth medium. After washing with serumfree, phenol red-free medium, 7 flasks each were treated with 10 nM R5020 or vehicle in serum-free, phenol red-free medium for 6 days with neither medium change nor additional hormone added. Following this, floating cells were harvested separately from cells still attached to the flask and pelleted by centrifugation. After removal of the supernatant, cell pellets were washed twice in 25 ml of ice cold phos-
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phate buffered saline. Cytoplasmic extracts were isolated as described by Dignam et al. [32] with the inclusion of protease inhibitors [pepstatin, aprotinin and leupeptin] at 1 g/ml in the extraction buffer. The phosphatase inhibitors NaF (5 mM) and Na3 VO4 (1 mM) were included. Protein concentration was determined by colorimetric assay (Bio-Rad Laboratories, Hercules, CA, USA); 125 g of control and R5020-treated and 200 g of positive control (+) protein were separated on a 12.5% SDS-PAGE gel and transferred to nitrocellulose. Transfer efficiency was assessed by incubation with 0.1% Ponceau. Western analysis was performed using a polyclonal antibody raised against a synthetic peptide corresponding to residues surrounding the cleavage site of human caspase-3 (1:1000, Cell Signaling Technology; Beverly, MA). 2.6. Fluorescence activated cell sorting Cells were grown to confluency in 25 cm2 flasks in triplicate for each treatment in routine growth medium. They were then treated with 10 nM R5020 or vehicle in serum-free, phenol red-free medium for 5 days with no medium change or additional hormone added. The cells floating in the supernatant were then pelleted at 300 × g for 5 min, washed once with phosphate buffered saline without calcium and magnesium and resuspended at about 107 cells/ml. To 100 l of this suspension in a 12 mm × 75 mm round bottom tube, 1 ml of ice-cold ethanol was then added dropwise, while vortexing slowly. The tubes were then capped and allowed to fix at 4 ◦ C for 18–24 h. They were then stored like this or stained with propidium iodide staining solution (50 g/ml propidium iodide, 1000 units/ml RNase A, 1 mg/ml glucose in phosphate buffered saline [without calcium and magnesium]). To stain the cells, they were first vortexed, and centrifuged at 1200 × g for 5 min. After discarding the supernatant, the cells were gently vortexed, 1 ml of the above staining solution was added, and the cells were again gently vortexed. The tubes were then capped and incubated with rocking at room temperature for at least 30 min. The cells were then analyzed in a Becton–Dickinson flow cytometer.
3. Results 3.1. Progestin inhibition of cell death in the progesterone receptor-rich human breast cancer cell line T47D We showed in earlier work [19] that under our conditions progestins increase the numbers of T47D human breast cancer cells in culture both by stimulating the rate of proliferation for many, probably unlimited numbers of doublings, and by simultaneously inhibiting cell death, acting as survival factors. This earlier work was done in charcoal-stripped serum-containing medium. In order to further characterize the phenomenon of progestin inhibition of cell death, we decided to use serum starvation as a reproducible way of
killing the cells and determine whether progestins and estrogens would serve as survival factors. Serum starvation has been used by many laboratories to examine the effects of various substances on cell death, including, among numerous others, studies on hepatoma cells [33], MCF-7 human breast cancer cells [34] and vascular endothelial cells [35]. The data of Fig. 1A show that the progestin R5020, at a physiological concentration, 10 nM, does indeed protect from cell death. However, much to our surprise, estradiol-17, at the same concentration, does not. This is in spite of the fact that we have previously shown that estradiol dramatically stimulates proliferation at this concentration [19]. In addition, the combination of estradiol and R5020 at these same concentrations inhibits cell death to the same extent as progestin alone (data not shown). The time point of 6 days mentioned in the legend was selected because after 6 days of serum starvation, the protective effect of progestin from cell death is readily apparent. That is, the medium of the control cells is turbid due to the floating dead cells, whereas that of the progestintreated cells is much clearer. Fig. 1B shows that the naturally occurring hormone progesterone, at the physiological concentration 100 nM, also protects from cell death. 3.2. Effect of the antiprogestin RU486 We have previously reported that RU486 has antiprogestin, progestin, and antiestrogenic effects on proliferation in T47D cells [15,16]. Fig. 1C shows that RU486, as expected, both attenuates progestin inhibition of cell death and slightly inhibits cell death on its own. This recapitulates its antagonistic/agonistic properties for proliferation we reported on earlier. We used 1 nM R5020 and 10 nM RU486 in these experiments so that RU486 could inhibit R5020s effect without its (RU486’s) concentration being so high that its progestin-like properties became too apparent. 3.3. Progestin concentration dependency In a previous report, we showed that progestin stimulation of T47D human breast cancer cell proliferation occurs in a dose–responsive manner in the physiological range of progestin concentration [15]. Fig. 1D shows that the same is true for progestin inhibition of cell death. The shape of the curves are very similar for stimulation of proliferation and cell death inhibition, both beginning at around 0.01 nM, becoming maximal somewhere around the nM range and then diminishing as the concentration goes through the M range. 3.4. Estradiol-17β does not inhibit serum-starvation-induced cell death at any physiological concentration Because of the surprising data above indicating that estradiol did not inhibit cell death at 10 nM, we tested the possibility that higher physiological concentrations would be effective. However, the data of Fig. 2 show that while the
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Fig. 1. Progestin inhibition of cell death. (A) Progestin inhibits cell death; estrogen does not. T47D human breast cancer cells were grown to confluency in triplicate flasks for each treatment. Growth medium was minimum essential medium [autoclavable] [powdered] with Earle’s salts, plus non-essential amino acids, 2 mM l-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin [GIBCO], 6 ng/ml insulin, and 10% fetal bovine serum. All other media described in this report are variations of this maintenance medium. The cells were then washed three times with serum-free, phenol red-free medium, and incubated in this for 6 days with 10 nM R5020, 10 nM estradiol-17, or vehicle [ethanol]. Fresh hormone or ethanol was added every 2 days without changing the medium; final ethanol concentration at day 6 was 0.03%. Cells in the supernatant and those still attached to the flask were then counted and assayed for viability with trypan blue. In this and all other figures error bars indicate S.E.M. unless otherwise indicated. By the Student–Newman–Keuls multiple comparison procedure, R5020 is different from control and estradiol at p < 0.003; control and estradiol are statistically the same. Representative of at least three independent experiments. (B) Progesterone inhibits T47D cell death. Cells were grown and treated in triplicate as in (A), except that progesterone [100 nM final concentration] was added each day, due to the short half-life of progesterone. The difference is significant at p < 0.01 by Student’s t-test. (C) RU486 antagonism of progestin inhibition of T47D cell death. Cells were grown and treated in triplicate as in (A). Ethanol [vehicle] concentration was the same in all flasks and was 0.06% at the end of the experiment. All differences are statistically significant at p < 0.02 by the Student-Newman-Keuls multiple comparison procedure except between RU486 and [R5020 + RU486], which are the same. Representative of several experiments. (D) Concentration dependence of progestin inhibition of cell death in T47D human breast cancer cells. T47D cells were grown to confluency in the same whole medium described in (A). Next, they were switched to serum-free, phenol red-free medium with either vehicle [0.1% ethanol, control], or the indicated concentration of the progestin R5020 for 6 days without medium change or further hormone addition. Cells were then analyzed for cell death as in (A). Average ± S.E.M. of three independent experiments. Single asterisk denotes statistically different from control by Student’s t-test, p < 0.05; double asterisks denote statistically different from each other by Student’s t-test, p < 0.01.
progestin R5020 at 10 nM dramatically inhibits, estradiol17 does not inhibit T47D human breast cancer cell death at any physiological concentration, from 0.001 nM to 1 M. This is inspite of the fact that we and others have reported that it significantly stimulates proliferation of these cells. In addition, as we stated above, combining estradiol with R5020 has no effect on the ability of the progestin to inhibit cell death. 3.5. Inhibition of cell death shows specificity for progestins in T47D cells
Fig. 2. Estradiol-17 does not inhibit T47D cell death at any physiological concentration. T47D cells were grown to confluency in whole medium as described in Fig. 1A. They were then serum starved by switching to serumfree, phenol red-free medium and incubated for 6 days, with fresh hormone at the indicated concentration or vehicle added every other day, as described in Fig. 1A. In the sample treated with R5020, the R5020 concentration was 10 nM. Representative of three independent experiments.
In order to further test the steroid hormonal specificity of this phenomenon, we treated the cells with 10 nM testosterone, dexamethasone, R5020 and aldosterone, and observed their effects on cell death. As shown in Fig. 3, only the progestin was effective. Thus, in T47D cells, only progestins, among the five major classes of steroid hormones, are protective from cell death at this concentration.
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Fig. 3. Hormonal specificity in T47D cells. Cells were grown and treated as in Fig. 1A, with or without 10 nM hormone, duplicate flasks for each treatment. Error bars indicate range. Representative of two independent experiments.
3.6. Progestin inhibition of cell death in human breast cancer cell lines with widely differing progesterone receptor contents We next wanted to determine if progestin inhibition of human breast cancer cell death was restricted to the progesterone receptor-rich cell line T47D or occurred in other lines as well. Fig. 4A shows that the phenomenon occurs in MCF-7 cells, which have an intermediate level of progesterone receptor. Once again, however, estradiol does not protect from cell death, at a concentration which has been shown to stimulate proliferation of this cell line [2]. The human breast cancer cell line MDA-MB-231 is progesterone receptor and estrogen receptor negative [36] and so we expected to find no progestin inhibition of cell death in this cell line. However, Fig. 4B shows progestin protection from cell death even in these cells, although once again estradiol had no effect. Because of this surprising result, we attempted to detect PR in these cells using the highly sensitive monoclonal antibody 1294 [37]. Although we could easily detect PR by immunoblot in T47D and MCF-7 cells, it was undetectable in the MDA-MB-231 cells (data not shown), as previously reported. Since progestins are known to bind to and to be capable of activating the glucocorticoid receptor, although with lower affinity than to the progesterone receptor [38], we reasoned that perhaps progestins were acting through the GR in the MDA-MB-231 cells, which are rich in glucocorticoid receptor [36]. We have since found glucocorticoid inhibition of cell death in these GR-rich cells (J.L. Dillon, C.M. Guetzloff, B.M. McCloud, C.M. Hoshibata, M.R. Moore, unpublished observations), although as shown in Fig. 3, it does not occur in T47D cells, which have a very low level of GR. Although this raises the possibility that progestin action in PR-negative MDA-MB-231 cells may be occurring through the GR, further experiments will be necessary to determine how progestins are acting in this PR-negative cell line.
Fig. 4. (A) Progestin inhibits cell death in MCF-7 human breast cancer cells; estrogen does not. MCF-7 cells, rich in ER and PR-positive, were grown to confluency in maintenance medium and then treated 7 days in serumfree, phenol red-free medium in triplicate with 10 nM estrogen, progestin or vehicle as described in Fig. 1A. R5020 is different from both control and estradiol at p < 0.006 by Student–Newman–Keuls multiple comparison procedure. Control and estradiol are the same. Representative of two experiments. (B) Progestin inhibits cell death in PR-negative, ER-negative MDA-MB-231 human breast cancer cells; estrogen does not. MDA-MB-231 cells were grown to confluency in maintenance medium and then treated 8 days in serum-free, phenol red-free medium as described in Fig. 1A. R5020 is different from control and estradiol at p < 0.007 by Student–Newman–Keuls multiple comparison procedure. Control and estradiol are the same. Representative of three experiments.
3.7. Progestin inhibition of apoptosis In a previous report, we showed that progestins protect from cell death, but did not determine if they were protecting from apoptosis [19]. In order to determine this, we used several methods. Fig. 5 A shows T47D cells stained with ethidium bromide and acridine orange to differentiate among live cells, dead apoptotic cells, and dead non-apoptotic cells by fluorescence microscopy. Fig. 5B shows quantitatively that this method reveals progestin protection from apoptosis. As a further test for progestin protection from apoptosis, we determined levels of activated caspase 3. Fig. 5C shows that there is much more activated caspase 3 in control cells than in progestin-treated cells. Once again, these data indicate that progestins are inhibiting apoptosis [39]. Finally, we used fluorescence activated cell sorting of propidium iodide-stained cells to test for cells with a subG1 content of DNA, another indicator of apoptosis. This assay also showed more cells with sub-G1 DNA content in
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the control cells than in the progestin-treated cells (data not shown). From all of the above data, we conclude that progestins protect T47D human breast cancer cells in culture from apoptosis. 3.8. Progestin protection from chemotherapy-induced cell death
Fig. 5. (A) Representative T47D cells scored for apoptosis by the method described in (B) [31]. Acridine orange stains live cell nuclei green. Ethidium bromide penetrates the membranes of only dead cells, staining the nuclei orangish red. Apoptotic cells are identified mainly by their condensed nuclei. L: live cell; D/NA: dead non-apoptotic cell; D/A: dead apoptotic. Pictured are one live cell and one dead non-apoptotic cell. All four of the other cells are dead apoptotic. (B) Progestin inhibition of apoptosis in T47D human breast cancer cells. Cells were grown to confluency in 25 cm2 flasks in triplicate for each treatment in whole medium as in Fig. 1A. They were then treated with 10 nM R5020 or vehicle in serum-free, phenol red-free medium for 5 days with no medium change or additional hormone added. Cells floating in the medium and still attached to the flask were then combined as follows. Cells still attached to the flask were harvested by incubating 10 min in Hank’s Balanced Salt Solution without calcium or magnesium, but with 1 mM EDTA, and aspirated well to separate them into individual cells. These cells were then combined with those that had been floating, washed twice with 0.9% NaCl, and then suspended in the saline at >3 × 106 cells/ml. Next 2 l of 0.1 mg/ml acridine orange, 0.1 mg/ml ethidium bromide, in 0.9% NaCl were added. Approximately 10 l of dyed cells from each flask were then examined by placing on a microscope slide, covering with a 10 mm × 10 mm no. 1 cover slip, and observing with a Nikon Microphot SA microscope equipped for fluorescence imaging. At least 100 cells from each flask were then scored for apoptosis [31] by a scorer “blinded” as to the samples’ identity. R5020 is statistically different from control at p < 0.05 by Student’s unpaired t-test. Representative of more than 5 separate experiments done in triplicate flasks for each sample. (C) Progestin inhibition of caspase 3 activation. Cells were grown to confluency in fourteen 150 cm2 flasks in whole medium, described in Fig. 1A. Then seven flasks each were treated with 10 nM R5020 or vehicle in serum-free, phenol red-free medium for 6 days with no medium change
These in vitro findings are certainly far removed from the much more complex situation in breast cancer patients. However, it is tempting to speculate that progestin protection of breast cancer cells in culture may point to potential clinical relevance. Progestin inhibition of cell death could help explain the above-cited studies showing greater risk of contracting breast cancer conferred by hormone replacement therapy (HRT) including combined estrogen and progestin as compared with estrogen alone [21–25,27]. In addition, it may point to progestin promotion of undiagnosed, occult breast cancer in the absence of therapy. However, there would be additional significance if progestins also protect breast cancer cells against cell death induced by drugs used in breast cancer treatment. In order to determine if a progestin would inhibit cell death caused by chemotherapeutic agents, we tested doxorubicin and 5-fluorouracil, both of which are commonly used to treat breast cancer. Fig. 6A and B, respectively, show that the progestin R5020, at physiological progestin concentration, does indeed protect human breast cancer cells from cell death caused by doxorubicin and 5-fluorouracil. In these experiments, the cells were treated with the agents in serumfree medium for only 1 or 2 days. The reason for the short treatment is that, as can be seen in the samples without fluorouracil or doxorubicin, progestin inhibition of cell death from serum starvation is not apparent after these short times. Therefore, the progestin protection from cell death at these early times is only from the chemotherapy-induced cell death, and is not complicated by simultaneous progestin inhibition of serum-starvation-induced cell death. Although the in vitro conditions used here are much simpler than the complex situation in breast cancer patients, these data make it tempting to or additional hormone added. Following this, floating cells were harvested separately from cells still attached to the flask and pelleted by centrifugation. After removal of the supernatant, cell pellets were washed twice in 25 ml of ice cold phosphate buffered saline. Cytoplasmic extracts were isolated as described by Dignam et al. [32] with the inclusion of protease inhibitors [pepstatin, aprotinin and leupeptin] at 1 g/ml in the extraction buffer. The phosphatase inhibitors NaF [5 mM] and Na3 VO4 [1 mM] were included. Protein concentration was determined by colorimetric assay [Bio-Rad Laboratories, Hercules, CA, USA]; 125 g of control [C] and R5020-treated [R] and 200 g of positive control [+] protein were separated on a 12.5% SDSPAGE gel and transferred to nitrocellulose. Transfer efficiency was assessed by incubation with 0.1% Ponceau. Western analysis was performed using a polyclonal antibody raised against a synthetic peptide corresponding to residues surrounding the cleavage site of human caspase 3 [1:1000, Cell Signaling Technology; Beverly, MA]. A human neuroblastoma cell line, SKN-SH, treated with 5 M thimerosal served as positive control. Data shown are from extracts of floating cells; attached cells and combined floating and attached cells gave similar results.
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Fig. 6. Progestin inhibition of cell death caused by the chemotherapeutic agents doxorubicin and 5-fluorouracil. (A) T47D cells were grown to confluency in triplicate flasks for each treatment as in Fig. 1A. They were then switched to serum-free, phenol red-free medium with either vehicle [control], 100 nM R5020, 10 M doxorubicin, or both doxorubicin and R5020 for 24 h. Vehicles were H2 O for doxorubicin and 0.1% [final concentration in medium] ethanol for R5020. Cells were then analyzed for cell death as in Fig. 1. All differences are significant at the p < 0.003 level by the Student–Newman–Keuls multiple comparison procedure except that between control and R5020, which are statistically the same. Representative of at least three independent experiments. (B) T47D cells were grown to confluency in triplicate flasks for each treatment as in Fig. 1A. They were then switched to serum-free, phenol red-free medium with either vehicle [control], 10 nM R5020, 10 mM 5-fluorouracil, or both fluorouracil and R5020 for 48 h. Vehicles were dimethyl sulfoxide for fluorouracil and 0.1% [final concentration in medium] ethanol for R5020. Cells were then analyzed for cell death as in Fig. 1A. All differences are significant at the p < 0.04 level by the Student–Newman–Keuls multiple comparison procedure except that between control and R5020, which are statistically the same. Representative of at least three independent experiments. Single asterisk denotes statistically the same as each other, different from both others; double and triple asterisks denote statistically different from all others.
speculate that progestins may make chemotherapy less effective.
4. Discussion This paper confirms and extends our initial report of progestin inhibition of breast cancer cell death [19], showing that progestins protect from apoptosis in T47D cells. It is probably apoptosis also in the other lines, as serum withdrawal is known to generally cause cell death by apoptosis [20,40]. Additionally, the data show that it is not restricted to cell lines with high levels of progesterone receptor, but occurs in human breast cancer cell lines with vastly different progesterone receptor content, ranging from high to none at all. The data of Fig. 1C suggest that this phenomenon is occurring at least partly through the progesterone receptor in cells with
substantial levels of PR. In the line MDA-MB-231, which has no PR, it is unclear how progestin protection occurs. It is possible that it is occurring through the glucocorticoid receptor, as dexamethasone also inhibits cell death in this line (J.L. Dillon, C.M. Guetzloff, B.M. McCloud, C.M. Hoshibata, M.R. Moore, unpublished observations), which has a high level of GR, but not in T47D cells, which have very low levels of GR. Still, Peluso has reported that progestin inhibition of cell death in granulosa cells with no classical nuclear PR can occur through a 60 kDa progesterone binding protein found in the cell membrane [41]. Our findings in this and other reports support a generally stimulatory role of progestins in breast cancer, in which progestins increase the number of breast cancer cells by both stimulating the rate of proliferation [9,14–19] and serving as survival factors [19 and this report], inhibiting apoptosis. While several authors agree with the notion that progestins in general stimulate breast cancer [10–13], many reports express the view that progestins generally inhibit the progression of breast cancer [4–7] and some have reported that progestins cause cell death, rather than inhibiting it [42,43]. In fact, progestins are sometimes used to treat breast cancer after tamoxifen failure [44]. How can one reconcile the above discrepancies concerning the role of progestins in breast cancer? One way to search for an answer may be to consider the results of the many recent HRT studies [21–25,27] and relate them to the previous in vivo (animal) and in vitro studies. The HRT studies, probably the best “bottom line” currently available, suggest that progestins in general stimulate breast cancer. They have repeatedly shown that combined estrogen–progestin therapy increases the risk of breast cancer over that with estrogen alone. In general, the in vitro studies reporting progestin inhibition of human breast cancer cell accumulation have been done under conditions in which the control cells (without progestin) were growing rapidly, with doubling times of 24–36 h. In many cases these studies were done in the presence of estrogen, either in the serum used or added to the medium in the phenol red [45] or as estradiol-17. We have previously reported that under these conditions, progestins will not make the cells accumulate faster, but inhibit cell accumulation, at least for a short time (1–2 weeks) [14,15]. Progestins in these cases seem to be inhibiting growth stimulation by the estrogen, acting in an antiestrogenic capacity. Since it is now known that growth factors such as EGF [46] and IGF-1 [insulin-like growth factor] [47] can activate the estrogen receptor even in the absence of estrogen, it is possible therefore that progestins might act in an “antiestrogenic” fashion even in the absence of estrogen, resulting in short term inhibition of cell accumulation as compared to the control cells growing in the presence of ER-activating growth factors. Our previous in vitro studies were done in conditions in which the control cells grew very slowly, if at all [9,14–19], and showed progestin stimulation of breast cancer cell accu-
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mulation. It is under these in vitro conditions, in which the estrogen has been removed from the serum by charcoalstripping, and the control cells are growing very slowly, that progestins lead to increased cell numbers. We find that estrogen alone stimulates proliferation much more than progestin, and that when both hormones are present progestin acts in an antiestrogenic fashion, so that the cells proliferate at an intermediate rate [9]. Then why are progestins such as medroxyprogesterone acetate effective treatments, at least for a while, in some breast cancer patients, if progestins in general increase breast cancer cell accumulation? One possible answer consistent with the available data is that progestins initially inhibit cancer cell accumulation in patients because of their antiestrogenic properties, but that eventually their anti-cell death action becomes the predominant characteristic, eventually resulting in a greater accumulation of cancer cells than there would be without progestin. It is unclear how to explain the discrepancy between our finding of progestin inhibition of breast cancer cell death and other reports indicating the opposite [42,43]. Formby and Wiley found that 10 M progesterone, which they describe as a high physiological concentration found in plasma during the third trimester of pregnancy, promoted apoptosis in both T47D and MCF-7 cells in 5% charcoal-stripped fetal bovine serum-containing medium without phenol red. However, they do not say how long the cells were in this medium before they began treatment with progestin. In our experiments showing progestin stimulation of proliferation and inhibition of cell death in charcoal-stripped serum-containing medium [19], we grew the cells in this medium for 5 days with one medium change before beginning progestin treatment, to help insure that endogenous progestins and estrogens left over from our routine whole serum-containing growth medium would not influence the results. In addition, we used 10 nM R5020 and 100 nM progesterone, fairly low physiological concentrations of progestin compared to those found in breast duct fluid during the menstrual cycle [48]. As shown in Fig. 1D, we have shown that progestin inhibition of cell death from serum starvation occurs in a dose–responsive manner, from 0.1 nM through 1 M. Although we have observed progestin inhibition of cell death in all passages of T47D cells we have tried, it is possible that Formby and Wiley’s T47D cells were different from ours, even though both theirs and ours came from the ATCC. In any case, our data are consistent with the recent clinical studies of HRT [21–25,27], showing that progestins in general stimulate breast cancer in patients. Estrogens, like progestins, have been reported to both inhibit [43] and promote [49,50] breast cancer cell death, depending on the conditions. Gompel et al. [43], however, using reduced serum and reduced growth factor to kill cells, i.e. in conditions somewhat similar to ours, found that estradiol inhibited cell death caused by the progestin ORG2058, but that estradiol did not inhibit cell death when used alone in human breast cancer cell lines, just as we report here. However, they used only one concentration of estradiol, 10 nM.
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In their conditions, estradiol inhibited only cell death caused by the progestin ORG2058. These authors reported that the progestin ORG2058, at 100 nM, enhanced breast cancer cell death. This, of course, is contrary to our findings that both the synthetic progestin R5020 and the naturally occurring hormone progesterone inhibit cell death. Perhaps the difference is in the particular progestin used and/or the strain of cell line used. However, as we stated in the introduction, and in support of our findings, Ory et al. have shown that the clinically used medroxyprogesterone acetate inhibits cell death in T47D cells [20]. Gompel et al. did not report on progesterone effects in their article [43]. As shown above, we saw no inhibition by estradiol-17 of serum-starvation-induced cell death at any physiological concentration in T47D cells, and none at 10 nM in any of the three human breast cancer cell lines we tested, two with high levels of estrogen receptor ␣ and one with none. Since we saw progestin inhibition of cell death at this same concentration in all three lines, it appears that under our conditions progestins are better inhibitors of cell death than estrogens. Although the situation in breast cancer patients is much more complex than the relatively simple in vitro environment described here, it is tempting to speculate that this may partly explain why HRT with combined progestin and estrogen gives greater breast cancer risk than estrogen alone [21–25,27]. That is, with estrogen alone there may be less inhibition of cell death than with the progestin added, leading to decreased tumor burden with estrogen alone. This would be consistent with our findings in the current report that combined estrogen and progestin inhibit cell death in T47D cells to the same extent as progestin alone, whereas estrogen alone does not inhibit cell death. As shown in Fig. 6A, progestins protect breast cancer cells from the killing effects of the S-phase specific chemotherapeutic drug doxorubicin, a drug often used to treat breast cancer and known to kill tumor cells by apoptosis [51]. Although doxorubicin’s exact mechanism of antineoplastic activity is not known, it is thought to involve intercalation between DNA base pairs, inhibiting DNA replication and transcription, in part through inhibition of DNA topoisomerase II. It also may bind to cell membrane lipids, affecting signal transduction, and generate oxygen free radicals [52]. Fig. 6B shows progestin inhibition of the breast cancer cell killing effects of the widely used chemotherapeutic agent 5fluorouracil, another S-phase specific drug, which acts by inhibition of the enzyme thymidylate synthase, which normally converts dUMP to dTMP. Thus, we have shown that progestins inhibit the in vitro action of at least two chemotherapeutic drugs, with different mechanisms of action, used to treat breast cancer. It is tempting to speculate that endogenous progestins may be acting in a similar fashion in breast cancer patients being treated with chemotherapy, lessening the chemotherapeutic efficacy of treatment. Even though reduced apoptosis is known to play an important role in the progression of cancer in general [53] and breast cancer in particular [54], our data showing progestin
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inhibition of cell death caused by both serum starvation and chemotherapeutic drugs should be interpreted with caution, due to the relatively simple in vitro system used. Still, they are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for patients with undiagnosed disease and those undergoing treatment. Further, they make it tempting to speculate that breast cancer treatment with combined antiprogestin and antiestrogen may be better than antiestrogen alone.
Acknowledgments This research was supported by a grant from Papa John’s Pizza through the V Foundation for Cancer Research and by a Research Challenge Grant from the State of West Virginia. We would like to thank Dr. Laura Jenski for use of the flow cytometer, Dr. William Rhoten for use of the fluorescence microscope, Courtney M Hoshibata for the estrogen dose–response experiments and Dr. Dean P. Edwards of Baylor University College of Medicine for his generous gift of the PR monoclonal antibody 1294.
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Progestin inhibition of cell death in human breast cancer cell lines Michael R. Moore a,∗ , James B. Spence a , Kelley K. Kiningham b , Joshua L. Dillon a a
Department of Biochemistry and Molecular Biology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, United States b Department of Pharmacology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, United States Received 29 March 2005; accepted 26 September 2005
Abstract Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17 surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 M. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment. © 2006 Elsevier Ltd. All rights reserved. Keywords: Progesterone; Apoptosis; Breast cancer; Chemotherapy; Estrogen
1. Introduction While estrogens have convincingly been shown to stimulate breast cancer [1,2], and antiestrogens are one of the main forms of adjuvant therapy [3], the role of progestins has been more controversial. Various laboratories have reported that progestins inhibit [4,5], stimulate for one turn of the cell cycle and then inhibit [6,7], have no effect [8], and stimulate breast cancer [9–13]. We were the first to report, in 1987, progestin stimulation of proliferation in the human breast cancer cell line T47D and have since confirmed and extended this observation in several other reports [9,14–19]. We have found that progestins not only stimulate proliferation, but also inhibit cell death [19]. Ory et al. later confirmed our observation of progestin inhi∗ Corresponding author at: Department of Biochemistry and Molecular Biology, Joan C. Edwards School of Medicine, Marshall University, 1542 Spring Valley Drive, Huntington, WV 25755-9320, United States. Tel.: +1 304 696 7324; fax: +1 304 696 7253. E-mail address: [email protected] (M.R. Moore).
0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.09.008
bition of cell death in human breast cancer cells, reporting its mediation by medroxyprogesterone acetate in T47D, MCF-7 and H466-B cells [20]. Many recent large-scale clinical studies have strongly strengthened the idea that progestins in general stimulate breast cancer. Schairer et al., in a study involving 46,355 postmenopausal women on hormone replacement therapy [HRT], comparing HRT involving combined estrogen–progestin to that with estrogen alone, found that the risk of breast cancer increased 8% per year of combined HRT as compared to 1% per year with estrogen alone [21]. Persson et al. drew a similar conclusion, that combined estrogen–progestin HRT gave a greater risk of breast cancer than estrogen alone, in their study of 11,231 Swedish women [22]. In still another study of 3534 women, Ross et al. concluded that combined progestin–estrogen HRT increased breast cancer risk significantly more than estrogen alone [23]. Yet another report by Li et al. describes their study of 1029 women, finding that combined progestin–estrogen HRT increases breast cancer risk substantially more than estrogen alone [24]. More recently, the Women’s Health Initiative study [25] stopped the arm
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of the study involving 16,608 postmenopausal women taking either both estrogen and progestin or placebo because “the test statistic for invasive breast cancer exceeded the stopping boundary for this adverse effect”. Further, Lydon et al. reported, using PR knockout mice, that mammary gland carcinogenesis is critically dependent on PR function [26]. Finally, the “Million Women Study”, involving 1,084,110 women aged 50–64 in the United Kingdom, found an increased risk of incident and fatal breast cancer for those women on HRT using combined estrogen–progestin than for those on estrogen only [27]. Progestins have been shown to inhibit cell death also in normal mammary epithelial cells in vivo. Berg et al. showed that both progestins and glucocorticoids (separately) prevent apoptosis in the lactating rat mammary gland [28]. Feng et al. showed this also in postweaning (mother’s) mouse mammary gland [29]. Moran et al. found that glucocorticoid inhibits apoptosis in the immortalized human mammary epithelial cell line MCF10A [30]. Hence, it appears that progestin inhibition of breast cancer cell death is an unfortunate holdover from its function in normal breast cells. Based on our previous report [19], we have begun to further characterize progestin inhibition of breast cancer cell death, both from serum withdrawal and from chemotherapeutic agents.
2. Materials and methods 2.1. Reagents R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20dione) was purchased from NEN Life Science (Boston, MA). RU486 (17-hydroxy-11-[4-dimethyl-aminophenyl1]-17␣-[prop-1-ynil]-estra-4,9-dien-3-one) was a gift from Dr. R. Deraedt of Roussel-Uclaf (Romainville, France). Progesterone, testosterone, dexamethasone, aldosterone, estradiol-17, doxorubicin and 5-fluorouracil were from Sigma–Aldrich. Antibody to activated caspase 3 was from Cell Signaling Technology, Beverly, MA. 2.2. Cell culture All human breast cancer cell lines (T47D, MCF-7, and MDA-MB-231), obtained from the American Type Culture Collection, were grown in Corning plastic flasks (Corning, NY) in 5% CO2 in air at 37 ◦ C. Routine growth medium was minimum essential medium, powdered (autoclavable) plus non-essential amino acids, 2 mM l-glutamine, 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin (Gibco) and 6 ng/ml insulin (Sigma). This medium contains phenol red. Cells were harvested by replacing the growth medium with Hank’s Balanced Salt Solution without calcium and magnesium but with 1 mM EDTA, incubating 10 min at 37 ◦ C, aspirating the cells and centrifuging. Cells were then frozen at −80 ◦ C for storage prior to making extracts. The
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human neuroblastoma cell line SK-N-SH was obtained from the American Type Culture Collection and grown at 37 ◦ C in a humidified atmosphere with 5% CO2 . Growth medium was minimum essential medium plus 10% fetal calf serum, 1 mM sodium pyruvate, 1% nonessential amino acids and 1% penicillin–streptomycin–neomycin (Gibco). 2.3. Cell death assays Routine cell death assays were performed by trypan blue exclusion manually, using a hemocytometer to determine the total number of live and dead cells in each flask, extrapolated from an actual count of around 1500 cells per flask. In most experiments, cell death was assessed after 6 days of serum withdrawal, although in some experiments after 5–8 days. This was simply a matter of convenience, as the phenomenon is visible to the naked eye by the greater turbidity in control cell medium due to floating dead cells from day 5 on. Apoptosis was confirmed by three different methods: fluorescence microscopy, immunoblotting for activated caspase 3, and fluorescence activated cell sorting. 2.4. Fluorescence microscopy Cells were grown to confluency in 25 cm2 flasks in triplicate for each treatment in routine growth medium. They were then treated with 10 nM R5020 or vehicle in serum-free, phenol red-free medium for 5 days with no medium change or additional hormone added. Cells floating in the medium and still attached to the flask were then combined as follows. Cells still attached to the flask were harvested by incubating 10 min in Hank’s Balanced Salt Solution without calcium or magnesium, but with 1 mM EDTA, and aspirated well to separate them into individual cells. These cells were then combined with those that had been floating, washed twice with 0.9% NaCl, and then suspended in the saline at >3 × 106 cells/ml. Next 2 l of 0.1 mg/ml acridine orange, 0.1 mg/ml ethidium bromide, in 0.9% NaCl were added. Approximately 10 l of dyed cells from each flask were then examined by placing on a microscope slide, covering with a 10 mm × 10 mm no. 1 cover slip, and observing with a Nikon Microphot SA microscope equipped for fluorescence imaging. At least 100 cells from each flask were then scored for apoptosis [31] by a scorer “blinded” as to the samples’ identity. 2.5. Immunoblotting for activated caspase 3 Cells were grown to confluency in fourteen 150 cm2 flasks in routine growth medium. After washing with serumfree, phenol red-free medium, 7 flasks each were treated with 10 nM R5020 or vehicle in serum-free, phenol red-free medium for 6 days with neither medium change nor additional hormone added. Following this, floating cells were harvested separately from cells still attached to the flask and pelleted by centrifugation. After removal of the supernatant, cell pellets were washed twice in 25 ml of ice cold phos-
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phate buffered saline. Cytoplasmic extracts were isolated as described by Dignam et al. [32] with the inclusion of protease inhibitors [pepstatin, aprotinin and leupeptin] at 1 g/ml in the extraction buffer. The phosphatase inhibitors NaF (5 mM) and Na3 VO4 (1 mM) were included. Protein concentration was determined by colorimetric assay (Bio-Rad Laboratories, Hercules, CA, USA); 125 g of control and R5020-treated and 200 g of positive control (+) protein were separated on a 12.5% SDS-PAGE gel and transferred to nitrocellulose. Transfer efficiency was assessed by incubation with 0.1% Ponceau. Western analysis was performed using a polyclonal antibody raised against a synthetic peptide corresponding to residues surrounding the cleavage site of human caspase-3 (1:1000, Cell Signaling Technology; Beverly, MA). 2.6. Fluorescence activated cell sorting Cells were grown to confluency in 25 cm2 flasks in triplicate for each treatment in routine growth medium. They were then treated with 10 nM R5020 or vehicle in serum-free, phenol red-free medium for 5 days with no medium change or additional hormone added. The cells floating in the supernatant were then pelleted at 300 × g for 5 min, washed once with phosphate buffered saline without calcium and magnesium and resuspended at about 107 cells/ml. To 100 l of this suspension in a 12 mm × 75 mm round bottom tube, 1 ml of ice-cold ethanol was then added dropwise, while vortexing slowly. The tubes were then capped and allowed to fix at 4 ◦ C for 18–24 h. They were then stored like this or stained with propidium iodide staining solution (50 g/ml propidium iodide, 1000 units/ml RNase A, 1 mg/ml glucose in phosphate buffered saline [without calcium and magnesium]). To stain the cells, they were first vortexed, and centrifuged at 1200 × g for 5 min. After discarding the supernatant, the cells were gently vortexed, 1 ml of the above staining solution was added, and the cells were again gently vortexed. The tubes were then capped and incubated with rocking at room temperature for at least 30 min. The cells were then analyzed in a Becton–Dickinson flow cytometer.
3. Results 3.1. Progestin inhibition of cell death in the progesterone receptor-rich human breast cancer cell line T47D We showed in earlier work [19] that under our conditions progestins increase the numbers of T47D human breast cancer cells in culture both by stimulating the rate of proliferation for many, probably unlimited numbers of doublings, and by simultaneously inhibiting cell death, acting as survival factors. This earlier work was done in charcoal-stripped serum-containing medium. In order to further characterize the phenomenon of progestin inhibition of cell death, we decided to use serum starvation as a reproducible way of
killing the cells and determine whether progestins and estrogens would serve as survival factors. Serum starvation has been used by many laboratories to examine the effects of various substances on cell death, including, among numerous others, studies on hepatoma cells [33], MCF-7 human breast cancer cells [34] and vascular endothelial cells [35]. The data of Fig. 1A show that the progestin R5020, at a physiological concentration, 10 nM, does indeed protect from cell death. However, much to our surprise, estradiol-17, at the same concentration, does not. This is in spite of the fact that we have previously shown that estradiol dramatically stimulates proliferation at this concentration [19]. In addition, the combination of estradiol and R5020 at these same concentrations inhibits cell death to the same extent as progestin alone (data not shown). The time point of 6 days mentioned in the legend was selected because after 6 days of serum starvation, the protective effect of progestin from cell death is readily apparent. That is, the medium of the control cells is turbid due to the floating dead cells, whereas that of the progestintreated cells is much clearer. Fig. 1B shows that the naturally occurring hormone progesterone, at the physiological concentration 100 nM, also protects from cell death. 3.2. Effect of the antiprogestin RU486 We have previously reported that RU486 has antiprogestin, progestin, and antiestrogenic effects on proliferation in T47D cells [15,16]. Fig. 1C shows that RU486, as expected, both attenuates progestin inhibition of cell death and slightly inhibits cell death on its own. This recapitulates its antagonistic/agonistic properties for proliferation we reported on earlier. We used 1 nM R5020 and 10 nM RU486 in these experiments so that RU486 could inhibit R5020s effect without its (RU486’s) concentration being so high that its progestin-like properties became too apparent. 3.3. Progestin concentration dependency In a previous report, we showed that progestin stimulation of T47D human breast cancer cell proliferation occurs in a dose–responsive manner in the physiological range of progestin concentration [15]. Fig. 1D shows that the same is true for progestin inhibition of cell death. The shape of the curves are very similar for stimulation of proliferation and cell death inhibition, both beginning at around 0.01 nM, becoming maximal somewhere around the nM range and then diminishing as the concentration goes through the M range. 3.4. Estradiol-17β does not inhibit serum-starvation-induced cell death at any physiological concentration Because of the surprising data above indicating that estradiol did not inhibit cell death at 10 nM, we tested the possibility that higher physiological concentrations would be effective. However, the data of Fig. 2 show that while the
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Fig. 1. Progestin inhibition of cell death. (A) Progestin inhibits cell death; estrogen does not. T47D human breast cancer cells were grown to confluency in triplicate flasks for each treatment. Growth medium was minimum essential medium [autoclavable] [powdered] with Earle’s salts, plus non-essential amino acids, 2 mM l-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin [GIBCO], 6 ng/ml insulin, and 10% fetal bovine serum. All other media described in this report are variations of this maintenance medium. The cells were then washed three times with serum-free, phenol red-free medium, and incubated in this for 6 days with 10 nM R5020, 10 nM estradiol-17, or vehicle [ethanol]. Fresh hormone or ethanol was added every 2 days without changing the medium; final ethanol concentration at day 6 was 0.03%. Cells in the supernatant and those still attached to the flask were then counted and assayed for viability with trypan blue. In this and all other figures error bars indicate S.E.M. unless otherwise indicated. By the Student–Newman–Keuls multiple comparison procedure, R5020 is different from control and estradiol at p < 0.003; control and estradiol are statistically the same. Representative of at least three independent experiments. (B) Progesterone inhibits T47D cell death. Cells were grown and treated in triplicate as in (A), except that progesterone [100 nM final concentration] was added each day, due to the short half-life of progesterone. The difference is significant at p < 0.01 by Student’s t-test. (C) RU486 antagonism of progestin inhibition of T47D cell death. Cells were grown and treated in triplicate as in (A). Ethanol [vehicle] concentration was the same in all flasks and was 0.06% at the end of the experiment. All differences are statistically significant at p < 0.02 by the Student-Newman-Keuls multiple comparison procedure except between RU486 and [R5020 + RU486], which are the same. Representative of several experiments. (D) Concentration dependence of progestin inhibition of cell death in T47D human breast cancer cells. T47D cells were grown to confluency in the same whole medium described in (A). Next, they were switched to serum-free, phenol red-free medium with either vehicle [0.1% ethanol, control], or the indicated concentration of the progestin R5020 for 6 days without medium change or further hormone addition. Cells were then analyzed for cell death as in (A). Average ± S.E.M. of three independent experiments. Single asterisk denotes statistically different from control by Student’s t-test, p < 0.05; double asterisks denote statistically different from each other by Student’s t-test, p < 0.01.
progestin R5020 at 10 nM dramatically inhibits, estradiol17 does not inhibit T47D human breast cancer cell death at any physiological concentration, from 0.001 nM to 1 M. This is inspite of the fact that we and others have reported that it significantly stimulates proliferation of these cells. In addition, as we stated above, combining estradiol with R5020 has no effect on the ability of the progestin to inhibit cell death. 3.5. Inhibition of cell death shows specificity for progestins in T47D cells
Fig. 2. Estradiol-17 does not inhibit T47D cell death at any physiological concentration. T47D cells were grown to confluency in whole medium as described in Fig. 1A. They were then serum starved by switching to serumfree, phenol red-free medium and incubated for 6 days, with fresh hormone at the indicated concentration or vehicle added every other day, as described in Fig. 1A. In the sample treated with R5020, the R5020 concentration was 10 nM. Representative of three independent experiments.
In order to further test the steroid hormonal specificity of this phenomenon, we treated the cells with 10 nM testosterone, dexamethasone, R5020 and aldosterone, and observed their effects on cell death. As shown in Fig. 3, only the progestin was effective. Thus, in T47D cells, only progestins, among the five major classes of steroid hormones, are protective from cell death at this concentration.
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Fig. 3. Hormonal specificity in T47D cells. Cells were grown and treated as in Fig. 1A, with or without 10 nM hormone, duplicate flasks for each treatment. Error bars indicate range. Representative of two independent experiments.
3.6. Progestin inhibition of cell death in human breast cancer cell lines with widely differing progesterone receptor contents We next wanted to determine if progestin inhibition of human breast cancer cell death was restricted to the progesterone receptor-rich cell line T47D or occurred in other lines as well. Fig. 4A shows that the phenomenon occurs in MCF-7 cells, which have an intermediate level of progesterone receptor. Once again, however, estradiol does not protect from cell death, at a concentration which has been shown to stimulate proliferation of this cell line [2]. The human breast cancer cell line MDA-MB-231 is progesterone receptor and estrogen receptor negative [36] and so we expected to find no progestin inhibition of cell death in this cell line. However, Fig. 4B shows progestin protection from cell death even in these cells, although once again estradiol had no effect. Because of this surprising result, we attempted to detect PR in these cells using the highly sensitive monoclonal antibody 1294 [37]. Although we could easily detect PR by immunoblot in T47D and MCF-7 cells, it was undetectable in the MDA-MB-231 cells (data not shown), as previously reported. Since progestins are known to bind to and to be capable of activating the glucocorticoid receptor, although with lower affinity than to the progesterone receptor [38], we reasoned that perhaps progestins were acting through the GR in the MDA-MB-231 cells, which are rich in glucocorticoid receptor [36]. We have since found glucocorticoid inhibition of cell death in these GR-rich cells (J.L. Dillon, C.M. Guetzloff, B.M. McCloud, C.M. Hoshibata, M.R. Moore, unpublished observations), although as shown in Fig. 3, it does not occur in T47D cells, which have a very low level of GR. Although this raises the possibility that progestin action in PR-negative MDA-MB-231 cells may be occurring through the GR, further experiments will be necessary to determine how progestins are acting in this PR-negative cell line.
Fig. 4. (A) Progestin inhibits cell death in MCF-7 human breast cancer cells; estrogen does not. MCF-7 cells, rich in ER and PR-positive, were grown to confluency in maintenance medium and then treated 7 days in serumfree, phenol red-free medium in triplicate with 10 nM estrogen, progestin or vehicle as described in Fig. 1A. R5020 is different from both control and estradiol at p < 0.006 by Student–Newman–Keuls multiple comparison procedure. Control and estradiol are the same. Representative of two experiments. (B) Progestin inhibits cell death in PR-negative, ER-negative MDA-MB-231 human breast cancer cells; estrogen does not. MDA-MB-231 cells were grown to confluency in maintenance medium and then treated 8 days in serum-free, phenol red-free medium as described in Fig. 1A. R5020 is different from control and estradiol at p < 0.007 by Student–Newman–Keuls multiple comparison procedure. Control and estradiol are the same. Representative of three experiments.
3.7. Progestin inhibition of apoptosis In a previous report, we showed that progestins protect from cell death, but did not determine if they were protecting from apoptosis [19]. In order to determine this, we used several methods. Fig. 5 A shows T47D cells stained with ethidium bromide and acridine orange to differentiate among live cells, dead apoptotic cells, and dead non-apoptotic cells by fluorescence microscopy. Fig. 5B shows quantitatively that this method reveals progestin protection from apoptosis. As a further test for progestin protection from apoptosis, we determined levels of activated caspase 3. Fig. 5C shows that there is much more activated caspase 3 in control cells than in progestin-treated cells. Once again, these data indicate that progestins are inhibiting apoptosis [39]. Finally, we used fluorescence activated cell sorting of propidium iodide-stained cells to test for cells with a subG1 content of DNA, another indicator of apoptosis. This assay also showed more cells with sub-G1 DNA content in
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the control cells than in the progestin-treated cells (data not shown). From all of the above data, we conclude that progestins protect T47D human breast cancer cells in culture from apoptosis. 3.8. Progestin protection from chemotherapy-induced cell death
Fig. 5. (A) Representative T47D cells scored for apoptosis by the method described in (B) [31]. Acridine orange stains live cell nuclei green. Ethidium bromide penetrates the membranes of only dead cells, staining the nuclei orangish red. Apoptotic cells are identified mainly by their condensed nuclei. L: live cell; D/NA: dead non-apoptotic cell; D/A: dead apoptotic. Pictured are one live cell and one dead non-apoptotic cell. All four of the other cells are dead apoptotic. (B) Progestin inhibition of apoptosis in T47D human breast cancer cells. Cells were grown to confluency in 25 cm2 flasks in triplicate for each treatment in whole medium as in Fig. 1A. They were then treated with 10 nM R5020 or vehicle in serum-free, phenol red-free medium for 5 days with no medium change or additional hormone added. Cells floating in the medium and still attached to the flask were then combined as follows. Cells still attached to the flask were harvested by incubating 10 min in Hank’s Balanced Salt Solution without calcium or magnesium, but with 1 mM EDTA, and aspirated well to separate them into individual cells. These cells were then combined with those that had been floating, washed twice with 0.9% NaCl, and then suspended in the saline at >3 × 106 cells/ml. Next 2 l of 0.1 mg/ml acridine orange, 0.1 mg/ml ethidium bromide, in 0.9% NaCl were added. Approximately 10 l of dyed cells from each flask were then examined by placing on a microscope slide, covering with a 10 mm × 10 mm no. 1 cover slip, and observing with a Nikon Microphot SA microscope equipped for fluorescence imaging. At least 100 cells from each flask were then scored for apoptosis [31] by a scorer “blinded” as to the samples’ identity. R5020 is statistically different from control at p < 0.05 by Student’s unpaired t-test. Representative of more than 5 separate experiments done in triplicate flasks for each sample. (C) Progestin inhibition of caspase 3 activation. Cells were grown to confluency in fourteen 150 cm2 flasks in whole medium, described in Fig. 1A. Then seven flasks each were treated with 10 nM R5020 or vehicle in serum-free, phenol red-free medium for 6 days with no medium change
These in vitro findings are certainly far removed from the much more complex situation in breast cancer patients. However, it is tempting to speculate that progestin protection of breast cancer cells in culture may point to potential clinical relevance. Progestin inhibition of cell death could help explain the above-cited studies showing greater risk of contracting breast cancer conferred by hormone replacement therapy (HRT) including combined estrogen and progestin as compared with estrogen alone [21–25,27]. In addition, it may point to progestin promotion of undiagnosed, occult breast cancer in the absence of therapy. However, there would be additional significance if progestins also protect breast cancer cells against cell death induced by drugs used in breast cancer treatment. In order to determine if a progestin would inhibit cell death caused by chemotherapeutic agents, we tested doxorubicin and 5-fluorouracil, both of which are commonly used to treat breast cancer. Fig. 6A and B, respectively, show that the progestin R5020, at physiological progestin concentration, does indeed protect human breast cancer cells from cell death caused by doxorubicin and 5-fluorouracil. In these experiments, the cells were treated with the agents in serumfree medium for only 1 or 2 days. The reason for the short treatment is that, as can be seen in the samples without fluorouracil or doxorubicin, progestin inhibition of cell death from serum starvation is not apparent after these short times. Therefore, the progestin protection from cell death at these early times is only from the chemotherapy-induced cell death, and is not complicated by simultaneous progestin inhibition of serum-starvation-induced cell death. Although the in vitro conditions used here are much simpler than the complex situation in breast cancer patients, these data make it tempting to or additional hormone added. Following this, floating cells were harvested separately from cells still attached to the flask and pelleted by centrifugation. After removal of the supernatant, cell pellets were washed twice in 25 ml of ice cold phosphate buffered saline. Cytoplasmic extracts were isolated as described by Dignam et al. [32] with the inclusion of protease inhibitors [pepstatin, aprotinin and leupeptin] at 1 g/ml in the extraction buffer. The phosphatase inhibitors NaF [5 mM] and Na3 VO4 [1 mM] were included. Protein concentration was determined by colorimetric assay [Bio-Rad Laboratories, Hercules, CA, USA]; 125 g of control [C] and R5020-treated [R] and 200 g of positive control [+] protein were separated on a 12.5% SDSPAGE gel and transferred to nitrocellulose. Transfer efficiency was assessed by incubation with 0.1% Ponceau. Western analysis was performed using a polyclonal antibody raised against a synthetic peptide corresponding to residues surrounding the cleavage site of human caspase 3 [1:1000, Cell Signaling Technology; Beverly, MA]. A human neuroblastoma cell line, SKN-SH, treated with 5 M thimerosal served as positive control. Data shown are from extracts of floating cells; attached cells and combined floating and attached cells gave similar results.
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Fig. 6. Progestin inhibition of cell death caused by the chemotherapeutic agents doxorubicin and 5-fluorouracil. (A) T47D cells were grown to confluency in triplicate flasks for each treatment as in Fig. 1A. They were then switched to serum-free, phenol red-free medium with either vehicle [control], 100 nM R5020, 10 M doxorubicin, or both doxorubicin and R5020 for 24 h. Vehicles were H2 O for doxorubicin and 0.1% [final concentration in medium] ethanol for R5020. Cells were then analyzed for cell death as in Fig. 1. All differences are significant at the p < 0.003 level by the Student–Newman–Keuls multiple comparison procedure except that between control and R5020, which are statistically the same. Representative of at least three independent experiments. (B) T47D cells were grown to confluency in triplicate flasks for each treatment as in Fig. 1A. They were then switched to serum-free, phenol red-free medium with either vehicle [control], 10 nM R5020, 10 mM 5-fluorouracil, or both fluorouracil and R5020 for 48 h. Vehicles were dimethyl sulfoxide for fluorouracil and 0.1% [final concentration in medium] ethanol for R5020. Cells were then analyzed for cell death as in Fig. 1A. All differences are significant at the p < 0.04 level by the Student–Newman–Keuls multiple comparison procedure except that between control and R5020, which are statistically the same. Representative of at least three independent experiments. Single asterisk denotes statistically the same as each other, different from both others; double and triple asterisks denote statistically different from all others.
speculate that progestins may make chemotherapy less effective.
4. Discussion This paper confirms and extends our initial report of progestin inhibition of breast cancer cell death [19], showing that progestins protect from apoptosis in T47D cells. It is probably apoptosis also in the other lines, as serum withdrawal is known to generally cause cell death by apoptosis [20,40]. Additionally, the data show that it is not restricted to cell lines with high levels of progesterone receptor, but occurs in human breast cancer cell lines with vastly different progesterone receptor content, ranging from high to none at all. The data of Fig. 1C suggest that this phenomenon is occurring at least partly through the progesterone receptor in cells with
substantial levels of PR. In the line MDA-MB-231, which has no PR, it is unclear how progestin protection occurs. It is possible that it is occurring through the glucocorticoid receptor, as dexamethasone also inhibits cell death in this line (J.L. Dillon, C.M. Guetzloff, B.M. McCloud, C.M. Hoshibata, M.R. Moore, unpublished observations), which has a high level of GR, but not in T47D cells, which have very low levels of GR. Still, Peluso has reported that progestin inhibition of cell death in granulosa cells with no classical nuclear PR can occur through a 60 kDa progesterone binding protein found in the cell membrane [41]. Our findings in this and other reports support a generally stimulatory role of progestins in breast cancer, in which progestins increase the number of breast cancer cells by both stimulating the rate of proliferation [9,14–19] and serving as survival factors [19 and this report], inhibiting apoptosis. While several authors agree with the notion that progestins in general stimulate breast cancer [10–13], many reports express the view that progestins generally inhibit the progression of breast cancer [4–7] and some have reported that progestins cause cell death, rather than inhibiting it [42,43]. In fact, progestins are sometimes used to treat breast cancer after tamoxifen failure [44]. How can one reconcile the above discrepancies concerning the role of progestins in breast cancer? One way to search for an answer may be to consider the results of the many recent HRT studies [21–25,27] and relate them to the previous in vivo (animal) and in vitro studies. The HRT studies, probably the best “bottom line” currently available, suggest that progestins in general stimulate breast cancer. They have repeatedly shown that combined estrogen–progestin therapy increases the risk of breast cancer over that with estrogen alone. In general, the in vitro studies reporting progestin inhibition of human breast cancer cell accumulation have been done under conditions in which the control cells (without progestin) were growing rapidly, with doubling times of 24–36 h. In many cases these studies were done in the presence of estrogen, either in the serum used or added to the medium in the phenol red [45] or as estradiol-17. We have previously reported that under these conditions, progestins will not make the cells accumulate faster, but inhibit cell accumulation, at least for a short time (1–2 weeks) [14,15]. Progestins in these cases seem to be inhibiting growth stimulation by the estrogen, acting in an antiestrogenic capacity. Since it is now known that growth factors such as EGF [46] and IGF-1 [insulin-like growth factor] [47] can activate the estrogen receptor even in the absence of estrogen, it is possible therefore that progestins might act in an “antiestrogenic” fashion even in the absence of estrogen, resulting in short term inhibition of cell accumulation as compared to the control cells growing in the presence of ER-activating growth factors. Our previous in vitro studies were done in conditions in which the control cells grew very slowly, if at all [9,14–19], and showed progestin stimulation of breast cancer cell accu-
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mulation. It is under these in vitro conditions, in which the estrogen has been removed from the serum by charcoalstripping, and the control cells are growing very slowly, that progestins lead to increased cell numbers. We find that estrogen alone stimulates proliferation much more than progestin, and that when both hormones are present progestin acts in an antiestrogenic fashion, so that the cells proliferate at an intermediate rate [9]. Then why are progestins such as medroxyprogesterone acetate effective treatments, at least for a while, in some breast cancer patients, if progestins in general increase breast cancer cell accumulation? One possible answer consistent with the available data is that progestins initially inhibit cancer cell accumulation in patients because of their antiestrogenic properties, but that eventually their anti-cell death action becomes the predominant characteristic, eventually resulting in a greater accumulation of cancer cells than there would be without progestin. It is unclear how to explain the discrepancy between our finding of progestin inhibition of breast cancer cell death and other reports indicating the opposite [42,43]. Formby and Wiley found that 10 M progesterone, which they describe as a high physiological concentration found in plasma during the third trimester of pregnancy, promoted apoptosis in both T47D and MCF-7 cells in 5% charcoal-stripped fetal bovine serum-containing medium without phenol red. However, they do not say how long the cells were in this medium before they began treatment with progestin. In our experiments showing progestin stimulation of proliferation and inhibition of cell death in charcoal-stripped serum-containing medium [19], we grew the cells in this medium for 5 days with one medium change before beginning progestin treatment, to help insure that endogenous progestins and estrogens left over from our routine whole serum-containing growth medium would not influence the results. In addition, we used 10 nM R5020 and 100 nM progesterone, fairly low physiological concentrations of progestin compared to those found in breast duct fluid during the menstrual cycle [48]. As shown in Fig. 1D, we have shown that progestin inhibition of cell death from serum starvation occurs in a dose–responsive manner, from 0.1 nM through 1 M. Although we have observed progestin inhibition of cell death in all passages of T47D cells we have tried, it is possible that Formby and Wiley’s T47D cells were different from ours, even though both theirs and ours came from the ATCC. In any case, our data are consistent with the recent clinical studies of HRT [21–25,27], showing that progestins in general stimulate breast cancer in patients. Estrogens, like progestins, have been reported to both inhibit [43] and promote [49,50] breast cancer cell death, depending on the conditions. Gompel et al. [43], however, using reduced serum and reduced growth factor to kill cells, i.e. in conditions somewhat similar to ours, found that estradiol inhibited cell death caused by the progestin ORG2058, but that estradiol did not inhibit cell death when used alone in human breast cancer cell lines, just as we report here. However, they used only one concentration of estradiol, 10 nM.
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In their conditions, estradiol inhibited only cell death caused by the progestin ORG2058. These authors reported that the progestin ORG2058, at 100 nM, enhanced breast cancer cell death. This, of course, is contrary to our findings that both the synthetic progestin R5020 and the naturally occurring hormone progesterone inhibit cell death. Perhaps the difference is in the particular progestin used and/or the strain of cell line used. However, as we stated in the introduction, and in support of our findings, Ory et al. have shown that the clinically used medroxyprogesterone acetate inhibits cell death in T47D cells [20]. Gompel et al. did not report on progesterone effects in their article [43]. As shown above, we saw no inhibition by estradiol-17 of serum-starvation-induced cell death at any physiological concentration in T47D cells, and none at 10 nM in any of the three human breast cancer cell lines we tested, two with high levels of estrogen receptor ␣ and one with none. Since we saw progestin inhibition of cell death at this same concentration in all three lines, it appears that under our conditions progestins are better inhibitors of cell death than estrogens. Although the situation in breast cancer patients is much more complex than the relatively simple in vitro environment described here, it is tempting to speculate that this may partly explain why HRT with combined progestin and estrogen gives greater breast cancer risk than estrogen alone [21–25,27]. That is, with estrogen alone there may be less inhibition of cell death than with the progestin added, leading to decreased tumor burden with estrogen alone. This would be consistent with our findings in the current report that combined estrogen and progestin inhibit cell death in T47D cells to the same extent as progestin alone, whereas estrogen alone does not inhibit cell death. As shown in Fig. 6A, progestins protect breast cancer cells from the killing effects of the S-phase specific chemotherapeutic drug doxorubicin, a drug often used to treat breast cancer and known to kill tumor cells by apoptosis [51]. Although doxorubicin’s exact mechanism of antineoplastic activity is not known, it is thought to involve intercalation between DNA base pairs, inhibiting DNA replication and transcription, in part through inhibition of DNA topoisomerase II. It also may bind to cell membrane lipids, affecting signal transduction, and generate oxygen free radicals [52]. Fig. 6B shows progestin inhibition of the breast cancer cell killing effects of the widely used chemotherapeutic agent 5fluorouracil, another S-phase specific drug, which acts by inhibition of the enzyme thymidylate synthase, which normally converts dUMP to dTMP. Thus, we have shown that progestins inhibit the in vitro action of at least two chemotherapeutic drugs, with different mechanisms of action, used to treat breast cancer. It is tempting to speculate that endogenous progestins may be acting in a similar fashion in breast cancer patients being treated with chemotherapy, lessening the chemotherapeutic efficacy of treatment. Even though reduced apoptosis is known to play an important role in the progression of cancer in general [53] and breast cancer in particular [54], our data showing progestin
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inhibition of cell death caused by both serum starvation and chemotherapeutic drugs should be interpreted with caution, due to the relatively simple in vitro system used. Still, they are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for patients with undiagnosed disease and those undergoing treatment. Further, they make it tempting to speculate that breast cancer treatment with combined antiprogestin and antiestrogen may be better than antiestrogen alone.
Acknowledgments This research was supported by a grant from Papa John’s Pizza through the V Foundation for Cancer Research and by a Research Challenge Grant from the State of West Virginia. We would like to thank Dr. Laura Jenski for use of the flow cytometer, Dr. William Rhoten for use of the fluorescence microscope, Courtney M Hoshibata for the estrogen dose–response experiments and Dr. Dean P. Edwards of Baylor University College of Medicine for his generous gift of the PR monoclonal antibody 1294.
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