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Characterization of the stimulatory effect of medroxyprogesterone acetate and chlormadinone acetate on growth factor treated normal human breast epithelial cells
Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 174–178
Characterization of the stimulatory effect of medroxyprogesterone acetate and chlormadinone acetate on growth factor treated normal human breast epithelial cells Elizabeth A. Kr¨amer, Harald Seeger, Bernhard Kr¨amer, Diethelm Wallwiener, Alfred O. Mueck ∗ Section of Endocrinology and Menopause, University Womens’ Hospital Tuebingen, Calwerstrasse 7, 72 076 Tuebingen, Germany Received 18 July 2005; accepted 9 September 2005
Abstract Objective: Evidence is increasing that adding progestogens to hormone replacement therapy may be more harmful than beneficial, however it is debatable whether all progestogens act equally on breast cells. Mitogenic growth factors from stromal breast tissue are important in growthregulation of breast cells, and may modify responses to progestogens. We investigated the effect of two C-21 derivatives, medroxyprogesterone acetate (MPA) and chlormadinone acetate (CMA) on growth-factor treated normal breast epithelial cells and tried to explore the underlying mechanisms of proliferation. Method: MCF10A (human epithelial, estrogen- and progesterone-receptor negative normal breast cells) were incubated with MPA or CMA at 0.1 and 1 M for 7 days with the growth factors (GFs) EGF, bFGF and IGF-I at 1pM. The same combinations, as well as growth factors alone, were also incubated with the proliferation inhibitors PD98059 and LY294002 at 1 M for 4 days. Cell proliferation rate was measured by the ATP-assay. Results: MPA 0.1 and 1 M, and CMA 1 M in combination with GFs both significantly increased cell proliferation rate, with MPA having the greatest effect. MPA- and CMA-induced proliferation of GF stimulated cells was blocked by both PD98059 (selective inhibitor of MAP kinases) and LY294002 (phosphatidylinositol 3-kinase inhibitor); GF stimulated cells could not be significantly reduced by any of the inhibitors used. Conclusion: MPA and CMA have a stimulatory effect on benign growth factor stimulated MCF10A cells, possibly via activation of MAP kinase and subsequent substrates and activation of PI3-kinase. GF induced proliferation appear to be mediated by pathways other than those investigated here. Growth factors and progestogens therefore have an additive, synergistic effect on cell proliferation, eliciting their effects via different pathways. © 2005 Elsevier Ltd. All rights reserved. Keywords: Medroxyprogesterone acetate; Chlormadinone acetate; Growth factors; Breast epithelial cells; Proliferation
1. Introduction The proliferation of human breast cells is evidently controlled by sex steroid hormones, and there is considerable data available substantiating the role of both endogenous and exogenous estrogens as mitogens in the pathogenesis of breast cancer [1]. Progestogens have now also come under the spotlight, and after publication of the results of the Women’s Health ∗
Corresponding author. Fax: +49 7071 29 4801. E-mail address: [email protected] (A.O. Mueck).
0960-0760/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.11.002
Initiative (WHI) and the Million Women Study (MWS), concerns have been raised over the relationship between them and increased risk of breast cancer in the climacteric and postmenopause [2,3]. The recent publication of the estrogen-only arm of the WHI, which showed a possible reduction in breast cancer risk [4], has added further suspicion over the in vivo effects of progestogens. It has been suggested that steroid hormones may act via mechanisms which include production and release of growth factors and/or their receptors. Epithelial and stromal cellderived growth factors are understood to be significant in
E.A. Kr¨amer et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 174–178
the regulation of breast epithelial cells directly via autocrine, paracrine, juxtacrine or intracrine pathways. Furthermore, growth factors are involved in the activation of signalling pathways which support the growth of cancer cells [5]. In a previous work, comparing the effect of progesterone and various synthetic progestins on the apoptosis/proliferation ratio of normal and cancerous breast cells, we found that only medroxyprogesterone acetate (MPA), a C-21 progestin, but not progesterone and no C-19 progestin stimulated the growth factor induced enhancement of proliferation of normal breast epithelial cells [Menopause, in press]. To further explore these findings in vitro, we investigated the effect of MPA and another C-21 derivative currently under investigations for its use in HRT, i.e. chlormadinone acetate (CMA), on growth-factor treated normal breast epithelial cells and tried to explore the underlying mechanisms.
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first incubated with the growth inhibitor alone at 1 M for 1 h at 37 ◦ C, which was then removed and replaced by a combination of MPA or CMA 1 and 0.1 M in combination with growth factors (EGF, bFGF, IGF-I) 1pM and PD98059 or LY294002 in turn at 1 M. This procedure was carried out twice, with a 48 h interval in between, followed by a further 48 h interval before measurement of cell proliferation. Cell proliferation was measured by the ATP chemosensitivity test, where proliferation is quantified by measuring light emitted during the bioluminescence reaction of luciferene in the presence of ATP and luciferase [6]. 2.1. Statistical analysis Proliferation was measured versus controls and performed in quadruplicate. Statistical analysis was done by ANOVA with the logarithmated values followed by Dunnett’s procedure. The overall alpha level was set at 0.05.
2. Material and methods Medroxyprogesterone acetate (MPA) and chlormadinone acetate (CMA) were purchased from Sigma Chemicals. The compounds were dissolved in ethanol to give a concentration of 10−2 M and were stored as concentrated stock solutions at −20 ◦ C. Epidermal growth factor (EGF), fibroblast growth factorbasic (bFGF) and insulin-like growth factor (IGF-I) were purchased from Sigma Chemicals. The compounds were reconstituted according to the manufacturer’s instructions stated on the package insert and were stored in aliquots at −20 ◦ C. The proliferation inhibitors PD98059 and LY294002 were purchased from Calbiochem, Germany. The compounds were dissolved in dimethyl sulphoxide (DMSO) according to the manufacturer’s instructions stated on the package insert, and were stored at −20 ◦ C. MCF10A, a human, non-tumorigenic, estrogen and progesterone receptor negative breast epithelial cell line was purchased from American Type Culture Collection (ATCC), USA. Cells were maintained in serum-free Mammary Epithelial Cell Medium purchased from PromoCell, Germany, supplemented with 100 ng/ml cholera toxin purchased from List Biological Laboratories Inc., California, USA and 100 U/ml penicillin plus 100 g/ml streptomycin. All assays were conducted using AIM-V serum-free medium for MCF10A cells, and stock concentrations of progestogens and growth factors were further diluted with this assay medium during working experiments to give a final ethanol concentration of <0.01% per well, as were the proliferation inhibitors to give a final DMSO concentration of <0.01% per well. Ninety-six well plates were seeded with approximately 1000 cells per well in AIM-V serum-free medium. The cells were incubated for 3 days at 37 ◦ C. The cells on the 96well plate to be incubated with the growth inhibitors were
3. Results The results for the effect of the progestogens in combination with growth factors on the MCF10A cell line are illustrated in Fig. 1. The combination of the growth factors EGF, FGF and IGF-I (GFs) alone confirmed a significant proliferative response compared to control. MPA and CMA alone had no significant effect on cell proliferation. However, MPA in combination with growth factors induced a significant 73 and 86% increase in cell proliferation at 0.1 and 1 M concentrations respectively in comparison to GFs alone (p = <0.01). CMA in combination with growth factors also induced a proliferative response, but less than that of MPA, with a significant effect at 1 M only (61% increase in cell proliferation) in comparison to GFs alone. Further assays were conducted with MPA and CMA at a concentration of 1 M in combination with GFs at 1 pM, as significant proliferative effects were seen with both progestogens at these concentrations.
Fig. 1. Proliferative responses of MCF10A to MPA and CMA in concentrations of 0.1 and 1 M alone and in combination with growth factors (GFs) 1pM (Cont = medium—only control, GFs = EGF + bFGF + IGFI, MPA = medroxyprogesterone acetate, CMA = chlormadinone acetate). ** p < 0.01 vs. control.
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Fig. 2. Proliferative responses of MCF10A to growth factors (GFs) 1pM alone and in the presence of PD98059 and LY294002 (Cont = medium—only control, GFs = EGF + bFGF + IGF-I).
Fig. 3. Proliferative responses of MCF10A to MPA 1 M in combination with growth factors (GFs) 1pM and in the presence of PD98059 and LY294002 (Contr = medium—only control, GFs = EGF + bFGF + IGFI, MPA = medroxyprogesterone acetate). ** p < 0.01 vs. MPA + GF.
Fig. 4. Proliferative responses of MCF10A to CMA 1 M in combination with growth factors (GFs) 1 pM and in the presence of PD98059 and LY294002 (Contr = medium—only control, GFs = EGF + bFGF + IGFI, CMA = chlormadinone acetate). ** p < 0.01 vs. CMA + GF.
The effects of the two proliferation inhibitors on the growth factors alone were tested, and none of the two inhibitors had a significant effect on increased cell proliferation (Fig. 2). The results of combining the two progestogens and growth factors in the presence of growth inhibitors are shown in Figs. 3 and 4. PD98059 and LY294002 significantly inhibited cell growth induced by MPA in combination with growth factors, with PD98059 exerting the greatest blockade. The same was true for CMA as shown in Fig. 4.
4. Discussion Hormonal regulation of the normal breast and the influence of exogenous hormones on breast cancer risk remain controversial subjects. Among the growth factors which are
important for cell growth are the epidermal growth factor (EGF) family, insulin-like growth factors I and II (IGF-I and IGF-II), fibroblast growth factors (FGFs), transforming growth factor-␣ (TGF-␣) and platelet-derived growth factors (PDGFs). The effects of growth factors are mainly mediated by specific receptors which activate signal transduction pathways downstream. The most important pathways affected by growth factors are mitogen activated kinases (MAP kinases) and the phosphoinositol-3-kinase (PI3 K) pathway. To investigate the mechanism of growth factor and progestogens induced proliferation we used specific inhibitors of the different pathways, i.e. PD98059, a selective inhibitor of MAP kinases and LY294002, a specific phosphatidylinositol 3kinase inhibitor. A mixture of the three growth factors EGF, bFGF and IGF-I was used in this work. Work completed in-house (not shown) demonstrated a proliferative response to each individual agent, but a maximal additive proliferative response when used in combination. According to our results, the stimulation of MCF-10A cells by growth factors appears to be mainly mediated by a pathway other than those mentioned above, since no specific inhibitor used was able to significantly block the stimulation. In contrast to our results, Makarevich et al. [7] showed that addition of EGF to rabbit granulosa cells activated ERK-related MAP kinase. The reasons for this discrepancy are currently unclear, and effects may differ with varying cell lines. MPA and CMA alone had no significant effect on cell proliferation, however, for the first time, we found that chlormadinone acetate, a synthetic C-21 progesterone derivative, is able to further enhance the mitotic effects of growth factors on estrogen receptor negative normal epithelial breast cells. However, this effect was lower than that of medroxyprogesterone acetate, another C-21 derivative, which showed a similar proliferation rate as previously. To further investigate the pathway by which MPA and CMA increase the proliferation of normal breast cells, we incubated MCF10A with growth factors alone, then MPA and CMA in turn together with a combination of the growth factors used previously and one of two proliferation inhibitors. PD98059 and LY294002 were both able to inhibit growth factor stimulated cells in the presence of MPA. This indicates that MPA may elicit its proliferative effects via mixed pathways, including activation of MAP kinase and subsequent substrates and activation of PI3K. Cell proliferation induced by a combination of CMA and growth factors was also significantly reduced by the two inhibitors, indicating that CMA may exert its proliferative effect via the same pathways as MPA. We present work completed using a receptor-negative cell line, however, other published literature details results which are most likely receptor-mediated. Due to a lack of other work with receptor-negative cells, we have discussed our results with findings from currently available literature. Clinical and animal trials have already demonstrated a possible negative effect of MPA on the proliferation of normal breast epithelial
E.A. Kr¨amer et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 174–178
cells. Hofseth et al. [8] showed that HRT with estrogen alone or estrogen plus MPA was associated with increased epithelial proliferation in the normal postmenopausal breast, which is consistent with our in vitro findings. Combination therapy caused more proliferation than estrogen alone [8]. Postmenopausal adult female cynomolgus macaques treated with conjugated equine estrogens alone or with medroxyprogesterone acetate, exhibited a greater proliferative response of healthy mammary gland epithelium with combined therapy than with estrogens alone [9]. These and our results indicate that MPA may enhance the mitotic rate of normal epithelial breast cells in the presence of growth factors and therefore long-term use may increase the probability of faults in DNA-replication. Furthermore, the results of the WHI indicated that patients who were not using hormones prior to the start of the study had no increased hazard ratio for breast cancer, however subjects with prior hormone use for up to 5, 5–10 and more than 10 years showed an increasing risk [3]. These data suggest that long-term use of MPA may increase breast cancer risk by enhancing the mitotic rate of normal epithelial cells. Chlormadinone acetate was shown to have no influence on breast cancer risk in a case–control study involving combined oral contraceptives containing CMA. Risk was not elevated in ever-users, did not increase with duration of use and did not change with time since initial exposure or with time since most recent use [10]. In contrast to this, a study of the use of oral contraceptives containing CMA found an elevated relative risk for breast cancer of 1.3. The authors state that although not statistically significant, the positive correlation observed for breast cancer may be real [11], which is consistent with our stimulatory results. Up to now, there is a paucity of data available regarding the effects of CMA on the proliferation of normal epithelial breast cells. As the MCF10A cell line is both ER and PR negative, there may be other unidentified primary membrane receptors present in this cell line. Lange et al. [12] suggested that progesterone selectively increases the sensitivity of key kinase cascades to growth factors in breast cancer cells, thereby priming cells for stimulation by latent growth signals. Our work was carried out using a benign breast epithelial cell line, which in in-house work (not shown) did not have any proliferative response to progesterone in the presence of growth factors above that of growth factors alone. However, it is feasible that a similar mechanism may exist in the MCF10A cell line in the presence of growth factors and other progestogens. Regarding the limitations of our study, despite their widespread use, in vitro models have boundaries: the choice of culture conditions can unintentionally affect the experimental outcome, and cultured cells are adapted to grow in vitro; the changes which have allowed this ability may not occur in vivo. The homogenicity of cell lines can be viewed as an advantage or disadvantage, on the one hand allowing the study of cells which represent a tissue population, but on the other hand, responses may not fully mimic those of the complex in vivo situation. Limitations of this in vitro study might
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be the high concentrations needed for an effective effect. We only present results of rather high progestogen concentrations of 0.1 and 1 M, since lower in vitro concentrations did not show any relevant effect. The clinically relevant blood concentrations for MPA are in the range of 4 × 10−9 M to 10−8 M [13]. However, higher concentrations may be required in vitro in short-time tests in which the reaction threshold can only be achieved with supraphysiological dosages. Higher concentrations may also be reached in vivo in the vessel wall or organs compared to the concentrations usually measured in the blood.
5. Conclusion Mitogenic growth factors from stromal breast tissue are important in the growth-regulation of breast cells and may modify responses to progestogens. We have shown that both MPA and CMA have a stimulatory effect on growth factor stimulated benign MCF10A cells via mixed pathways, possibly including activation of MAP kinase and subsequent substrates and by activation of PI3-kinase. Growth factors may exert its proliferative effects on MCF-10A cells via pathways other than blocked by PD98059 and LY294002. Growth factors and progestogens in combination therefore have an additive, synergistic effect on cell proliferation, eliciting their effects over different pathways. In conclusion, despite the experimental limitations described, we suggest our results indicate that certain progestogens are able to induce proliferation of benign human breast epithelial cells, independent of the effects of growth factors, and therefore the choice of progestogen may be important in terms of influencing a possible breast cancer risk.
References [1] M. Clemons, P. Goss, Estrogen and the risk of breast cancer, New. Engl. J. Med. 344 (2001) 276–285. [2] Writing Group for the Women’s Health Initiative Investigators, Risks and benefits of estrogen plus progestin in healthy postmenopausal women, JAMA 288 (2002) 321–333. [3] Million Women Study Collaborators, Breast Cancer and hormone replacement therapy in the Million Women Study, Lancet, 362 (2003) 419–427. [4] The Women’s Health Initiative Steering Committee, Effects of Conjugated Equine Estrogen in Postmenopausal Women with Hysterectomy, JAMA 291 (2004) 1701–1712. [5] K.M.W. Rahmean, F.H. Sarkar, Steroid hormone mimics: molecular mechanisms of cell growth and apoptosis in normal and malignant mammary epithelial cells, J. Steroid Biochem. Mol. Biol. 80 (2002) 191–201. [6] P.E. Andreotti, J.T. Thornthwaite, I.S. Morse, ATP tumor chemosensitivity assay, in: P.E. Stanley, L.J. Kricka (Eds.), Bioluminescence and Chemoluminescence: Current Status, John Wiley & Sons, Chichester, 1991, pp. 417–420. [7] A.V. Makarevich, A.V. Sirotkin, P. Chrenek, J. Bulla, Effect of epidermal growth factor (EGF) on steroid and cyclic nucleotide
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secretion, proliferation and ERK-related MAP-kinase in cultured rabbit granulosa cells, Exp. Clin. Endocrinol. Diabetes 110 (2002) 124–129. [8] L.J. Hosfeth, A.M. Raafat, J.R. Osuch, D.R. Pathak, C.A. Slomski, S.Z. Haslam, Hormone replacement therapy with estrogen or estrogen plus medroxyprogesterone acetate is associated with increased epithelial proliferation in the normal postmenopausal breast, J. Clin. Endocrinol. Metab. 84 (1999) 4559–4565. [9] J.M. Cline, G.S. Soderqvist, E. von Schoultz, L. Skoog, B. von Schoulz, Effects of hormone replacement therapy on the mammary gland of surgically postmenopausal cynomolgus macaques, Am. J. Obstet. Gynecol. 174 (1996) 93–100. [10] K. Ebeling, R. Ray, P. Nischan, D.B. Thomas, D. Kunde, H. Stalsberg, Combined oral contraceptives containing chlormadinone
acetate and breast cancer: results of a case–control study, Br. J. Cancer 63 (1991) 804–808. [11] P. Nischan, K. Ebeling, Oral contraceptives containing chlormadinone acetate and cancer incidence at selected sites in the German Democratic Republic—a correlation analysis, Int. J. Cancer 34 (1984) 671–674. [12] C.A. Lange, J.K. Richer, T. Shen, K.B. Horwitz, Convergence of progesterone and epidermal growth factor signaling in breast cancer, J. Biol. Chem. 273 (1998) 31308–31316. [13] L.O. Svensson, S.H. Johnson, S.E. Olsson, Plasma concentrations of medroxyprogesterone acetate, estradiol and estrone following oral administration of Klimaxil® , Trisequence® /Provera® , and Divina® . A randomised, single-blind, triple cross-over bioavailability study in menopausal women, Maturitas 18 (1994) 229–238.
Characterization of the stimulatory effect of medroxyprogesterone acetate and chlormadinone acetate on growth factor treated normal human breast epithelial cells Elizabeth A. Kr¨amer, Harald Seeger, Bernhard Kr¨amer, Diethelm Wallwiener, Alfred O. Mueck ∗ Section of Endocrinology and Menopause, University Womens’ Hospital Tuebingen, Calwerstrasse 7, 72 076 Tuebingen, Germany Received 18 July 2005; accepted 9 September 2005
Abstract Objective: Evidence is increasing that adding progestogens to hormone replacement therapy may be more harmful than beneficial, however it is debatable whether all progestogens act equally on breast cells. Mitogenic growth factors from stromal breast tissue are important in growthregulation of breast cells, and may modify responses to progestogens. We investigated the effect of two C-21 derivatives, medroxyprogesterone acetate (MPA) and chlormadinone acetate (CMA) on growth-factor treated normal breast epithelial cells and tried to explore the underlying mechanisms of proliferation. Method: MCF10A (human epithelial, estrogen- and progesterone-receptor negative normal breast cells) were incubated with MPA or CMA at 0.1 and 1 M for 7 days with the growth factors (GFs) EGF, bFGF and IGF-I at 1pM. The same combinations, as well as growth factors alone, were also incubated with the proliferation inhibitors PD98059 and LY294002 at 1 M for 4 days. Cell proliferation rate was measured by the ATP-assay. Results: MPA 0.1 and 1 M, and CMA 1 M in combination with GFs both significantly increased cell proliferation rate, with MPA having the greatest effect. MPA- and CMA-induced proliferation of GF stimulated cells was blocked by both PD98059 (selective inhibitor of MAP kinases) and LY294002 (phosphatidylinositol 3-kinase inhibitor); GF stimulated cells could not be significantly reduced by any of the inhibitors used. Conclusion: MPA and CMA have a stimulatory effect on benign growth factor stimulated MCF10A cells, possibly via activation of MAP kinase and subsequent substrates and activation of PI3-kinase. GF induced proliferation appear to be mediated by pathways other than those investigated here. Growth factors and progestogens therefore have an additive, synergistic effect on cell proliferation, eliciting their effects via different pathways. © 2005 Elsevier Ltd. All rights reserved. Keywords: Medroxyprogesterone acetate; Chlormadinone acetate; Growth factors; Breast epithelial cells; Proliferation
1. Introduction The proliferation of human breast cells is evidently controlled by sex steroid hormones, and there is considerable data available substantiating the role of both endogenous and exogenous estrogens as mitogens in the pathogenesis of breast cancer [1]. Progestogens have now also come under the spotlight, and after publication of the results of the Women’s Health ∗
Corresponding author. Fax: +49 7071 29 4801. E-mail address: [email protected] (A.O. Mueck).
0960-0760/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.11.002
Initiative (WHI) and the Million Women Study (MWS), concerns have been raised over the relationship between them and increased risk of breast cancer in the climacteric and postmenopause [2,3]. The recent publication of the estrogen-only arm of the WHI, which showed a possible reduction in breast cancer risk [4], has added further suspicion over the in vivo effects of progestogens. It has been suggested that steroid hormones may act via mechanisms which include production and release of growth factors and/or their receptors. Epithelial and stromal cellderived growth factors are understood to be significant in
E.A. Kr¨amer et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 174–178
the regulation of breast epithelial cells directly via autocrine, paracrine, juxtacrine or intracrine pathways. Furthermore, growth factors are involved in the activation of signalling pathways which support the growth of cancer cells [5]. In a previous work, comparing the effect of progesterone and various synthetic progestins on the apoptosis/proliferation ratio of normal and cancerous breast cells, we found that only medroxyprogesterone acetate (MPA), a C-21 progestin, but not progesterone and no C-19 progestin stimulated the growth factor induced enhancement of proliferation of normal breast epithelial cells [Menopause, in press]. To further explore these findings in vitro, we investigated the effect of MPA and another C-21 derivative currently under investigations for its use in HRT, i.e. chlormadinone acetate (CMA), on growth-factor treated normal breast epithelial cells and tried to explore the underlying mechanisms.
175
first incubated with the growth inhibitor alone at 1 M for 1 h at 37 ◦ C, which was then removed and replaced by a combination of MPA or CMA 1 and 0.1 M in combination with growth factors (EGF, bFGF, IGF-I) 1pM and PD98059 or LY294002 in turn at 1 M. This procedure was carried out twice, with a 48 h interval in between, followed by a further 48 h interval before measurement of cell proliferation. Cell proliferation was measured by the ATP chemosensitivity test, where proliferation is quantified by measuring light emitted during the bioluminescence reaction of luciferene in the presence of ATP and luciferase [6]. 2.1. Statistical analysis Proliferation was measured versus controls and performed in quadruplicate. Statistical analysis was done by ANOVA with the logarithmated values followed by Dunnett’s procedure. The overall alpha level was set at 0.05.
2. Material and methods Medroxyprogesterone acetate (MPA) and chlormadinone acetate (CMA) were purchased from Sigma Chemicals. The compounds were dissolved in ethanol to give a concentration of 10−2 M and were stored as concentrated stock solutions at −20 ◦ C. Epidermal growth factor (EGF), fibroblast growth factorbasic (bFGF) and insulin-like growth factor (IGF-I) were purchased from Sigma Chemicals. The compounds were reconstituted according to the manufacturer’s instructions stated on the package insert and were stored in aliquots at −20 ◦ C. The proliferation inhibitors PD98059 and LY294002 were purchased from Calbiochem, Germany. The compounds were dissolved in dimethyl sulphoxide (DMSO) according to the manufacturer’s instructions stated on the package insert, and were stored at −20 ◦ C. MCF10A, a human, non-tumorigenic, estrogen and progesterone receptor negative breast epithelial cell line was purchased from American Type Culture Collection (ATCC), USA. Cells were maintained in serum-free Mammary Epithelial Cell Medium purchased from PromoCell, Germany, supplemented with 100 ng/ml cholera toxin purchased from List Biological Laboratories Inc., California, USA and 100 U/ml penicillin plus 100 g/ml streptomycin. All assays were conducted using AIM-V serum-free medium for MCF10A cells, and stock concentrations of progestogens and growth factors were further diluted with this assay medium during working experiments to give a final ethanol concentration of <0.01% per well, as were the proliferation inhibitors to give a final DMSO concentration of <0.01% per well. Ninety-six well plates were seeded with approximately 1000 cells per well in AIM-V serum-free medium. The cells were incubated for 3 days at 37 ◦ C. The cells on the 96well plate to be incubated with the growth inhibitors were
3. Results The results for the effect of the progestogens in combination with growth factors on the MCF10A cell line are illustrated in Fig. 1. The combination of the growth factors EGF, FGF and IGF-I (GFs) alone confirmed a significant proliferative response compared to control. MPA and CMA alone had no significant effect on cell proliferation. However, MPA in combination with growth factors induced a significant 73 and 86% increase in cell proliferation at 0.1 and 1 M concentrations respectively in comparison to GFs alone (p = <0.01). CMA in combination with growth factors also induced a proliferative response, but less than that of MPA, with a significant effect at 1 M only (61% increase in cell proliferation) in comparison to GFs alone. Further assays were conducted with MPA and CMA at a concentration of 1 M in combination with GFs at 1 pM, as significant proliferative effects were seen with both progestogens at these concentrations.
Fig. 1. Proliferative responses of MCF10A to MPA and CMA in concentrations of 0.1 and 1 M alone and in combination with growth factors (GFs) 1pM (Cont = medium—only control, GFs = EGF + bFGF + IGFI, MPA = medroxyprogesterone acetate, CMA = chlormadinone acetate). ** p < 0.01 vs. control.
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Fig. 2. Proliferative responses of MCF10A to growth factors (GFs) 1pM alone and in the presence of PD98059 and LY294002 (Cont = medium—only control, GFs = EGF + bFGF + IGF-I).
Fig. 3. Proliferative responses of MCF10A to MPA 1 M in combination with growth factors (GFs) 1pM and in the presence of PD98059 and LY294002 (Contr = medium—only control, GFs = EGF + bFGF + IGFI, MPA = medroxyprogesterone acetate). ** p < 0.01 vs. MPA + GF.
Fig. 4. Proliferative responses of MCF10A to CMA 1 M in combination with growth factors (GFs) 1 pM and in the presence of PD98059 and LY294002 (Contr = medium—only control, GFs = EGF + bFGF + IGFI, CMA = chlormadinone acetate). ** p < 0.01 vs. CMA + GF.
The effects of the two proliferation inhibitors on the growth factors alone were tested, and none of the two inhibitors had a significant effect on increased cell proliferation (Fig. 2). The results of combining the two progestogens and growth factors in the presence of growth inhibitors are shown in Figs. 3 and 4. PD98059 and LY294002 significantly inhibited cell growth induced by MPA in combination with growth factors, with PD98059 exerting the greatest blockade. The same was true for CMA as shown in Fig. 4.
4. Discussion Hormonal regulation of the normal breast and the influence of exogenous hormones on breast cancer risk remain controversial subjects. Among the growth factors which are
important for cell growth are the epidermal growth factor (EGF) family, insulin-like growth factors I and II (IGF-I and IGF-II), fibroblast growth factors (FGFs), transforming growth factor-␣ (TGF-␣) and platelet-derived growth factors (PDGFs). The effects of growth factors are mainly mediated by specific receptors which activate signal transduction pathways downstream. The most important pathways affected by growth factors are mitogen activated kinases (MAP kinases) and the phosphoinositol-3-kinase (PI3 K) pathway. To investigate the mechanism of growth factor and progestogens induced proliferation we used specific inhibitors of the different pathways, i.e. PD98059, a selective inhibitor of MAP kinases and LY294002, a specific phosphatidylinositol 3kinase inhibitor. A mixture of the three growth factors EGF, bFGF and IGF-I was used in this work. Work completed in-house (not shown) demonstrated a proliferative response to each individual agent, but a maximal additive proliferative response when used in combination. According to our results, the stimulation of MCF-10A cells by growth factors appears to be mainly mediated by a pathway other than those mentioned above, since no specific inhibitor used was able to significantly block the stimulation. In contrast to our results, Makarevich et al. [7] showed that addition of EGF to rabbit granulosa cells activated ERK-related MAP kinase. The reasons for this discrepancy are currently unclear, and effects may differ with varying cell lines. MPA and CMA alone had no significant effect on cell proliferation, however, for the first time, we found that chlormadinone acetate, a synthetic C-21 progesterone derivative, is able to further enhance the mitotic effects of growth factors on estrogen receptor negative normal epithelial breast cells. However, this effect was lower than that of medroxyprogesterone acetate, another C-21 derivative, which showed a similar proliferation rate as previously. To further investigate the pathway by which MPA and CMA increase the proliferation of normal breast cells, we incubated MCF10A with growth factors alone, then MPA and CMA in turn together with a combination of the growth factors used previously and one of two proliferation inhibitors. PD98059 and LY294002 were both able to inhibit growth factor stimulated cells in the presence of MPA. This indicates that MPA may elicit its proliferative effects via mixed pathways, including activation of MAP kinase and subsequent substrates and activation of PI3K. Cell proliferation induced by a combination of CMA and growth factors was also significantly reduced by the two inhibitors, indicating that CMA may exert its proliferative effect via the same pathways as MPA. We present work completed using a receptor-negative cell line, however, other published literature details results which are most likely receptor-mediated. Due to a lack of other work with receptor-negative cells, we have discussed our results with findings from currently available literature. Clinical and animal trials have already demonstrated a possible negative effect of MPA on the proliferation of normal breast epithelial
E.A. Kr¨amer et al. / Journal of Steroid Biochemistry & Molecular Biology 98 (2006) 174–178
cells. Hofseth et al. [8] showed that HRT with estrogen alone or estrogen plus MPA was associated with increased epithelial proliferation in the normal postmenopausal breast, which is consistent with our in vitro findings. Combination therapy caused more proliferation than estrogen alone [8]. Postmenopausal adult female cynomolgus macaques treated with conjugated equine estrogens alone or with medroxyprogesterone acetate, exhibited a greater proliferative response of healthy mammary gland epithelium with combined therapy than with estrogens alone [9]. These and our results indicate that MPA may enhance the mitotic rate of normal epithelial breast cells in the presence of growth factors and therefore long-term use may increase the probability of faults in DNA-replication. Furthermore, the results of the WHI indicated that patients who were not using hormones prior to the start of the study had no increased hazard ratio for breast cancer, however subjects with prior hormone use for up to 5, 5–10 and more than 10 years showed an increasing risk [3]. These data suggest that long-term use of MPA may increase breast cancer risk by enhancing the mitotic rate of normal epithelial cells. Chlormadinone acetate was shown to have no influence on breast cancer risk in a case–control study involving combined oral contraceptives containing CMA. Risk was not elevated in ever-users, did not increase with duration of use and did not change with time since initial exposure or with time since most recent use [10]. In contrast to this, a study of the use of oral contraceptives containing CMA found an elevated relative risk for breast cancer of 1.3. The authors state that although not statistically significant, the positive correlation observed for breast cancer may be real [11], which is consistent with our stimulatory results. Up to now, there is a paucity of data available regarding the effects of CMA on the proliferation of normal epithelial breast cells. As the MCF10A cell line is both ER and PR negative, there may be other unidentified primary membrane receptors present in this cell line. Lange et al. [12] suggested that progesterone selectively increases the sensitivity of key kinase cascades to growth factors in breast cancer cells, thereby priming cells for stimulation by latent growth signals. Our work was carried out using a benign breast epithelial cell line, which in in-house work (not shown) did not have any proliferative response to progesterone in the presence of growth factors above that of growth factors alone. However, it is feasible that a similar mechanism may exist in the MCF10A cell line in the presence of growth factors and other progestogens. Regarding the limitations of our study, despite their widespread use, in vitro models have boundaries: the choice of culture conditions can unintentionally affect the experimental outcome, and cultured cells are adapted to grow in vitro; the changes which have allowed this ability may not occur in vivo. The homogenicity of cell lines can be viewed as an advantage or disadvantage, on the one hand allowing the study of cells which represent a tissue population, but on the other hand, responses may not fully mimic those of the complex in vivo situation. Limitations of this in vitro study might
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be the high concentrations needed for an effective effect. We only present results of rather high progestogen concentrations of 0.1 and 1 M, since lower in vitro concentrations did not show any relevant effect. The clinically relevant blood concentrations for MPA are in the range of 4 × 10−9 M to 10−8 M [13]. However, higher concentrations may be required in vitro in short-time tests in which the reaction threshold can only be achieved with supraphysiological dosages. Higher concentrations may also be reached in vivo in the vessel wall or organs compared to the concentrations usually measured in the blood.
5. Conclusion Mitogenic growth factors from stromal breast tissue are important in the growth-regulation of breast cells and may modify responses to progestogens. We have shown that both MPA and CMA have a stimulatory effect on growth factor stimulated benign MCF10A cells via mixed pathways, possibly including activation of MAP kinase and subsequent substrates and by activation of PI3-kinase. Growth factors may exert its proliferative effects on MCF-10A cells via pathways other than blocked by PD98059 and LY294002. Growth factors and progestogens in combination therefore have an additive, synergistic effect on cell proliferation, eliciting their effects over different pathways. In conclusion, despite the experimental limitations described, we suggest our results indicate that certain progestogens are able to induce proliferation of benign human breast epithelial cells, independent of the effects of growth factors, and therefore the choice of progestogen may be important in terms of influencing a possible breast cancer risk.
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