Article in press - uncorrected proof Horm Mol Biol Clin Invest 2011;6(2):211–214 2011 by Walter de Gruyter • Berlin • New York. DOI 10.1515/HMBCI.2011.008
Effect of drospirenone on proliferation of human benign and cancerous epithelial breast cells Harald Seeger1, Xiangjan Ruan2, Hans Neubauer1 and Alfred O. Mueck1,*
Introduction
1
Two recent studies, the Women’s Health Initiative (WHI) and the Million Women Study (MWS), have above all raised concerns about the relationship between progestogens and increased risk of breast cancer in the climacteric and postmenopause w1, 2x. The WHI study was terminated early after five years, owing to an increased incidence of breast cancer in the group treated with combined estrogen and progestogen therapy. The MWS concluded that breast cancer risk was increased two-fold in current users of combined hormone replacement therapy (HRT) compared with a factor of 1.3 for estrogen-only therapy. A crucial role of progestogens in increasing breast cancer risk was supported by the WHI estrogen mono-arm showing no increase but rather a reduction of breast cancer risk, which was significant for patients with more than 80% adherence to study medication w3x. However, in the French E3N-EPIC trial of over 80,000 postmenopausal women it was reported that hormone therapy containing the progestin medroxyprogesterone acetate (MPA) or norethisterone (NET) was associated with a significant increase in risk of breast cancer, whereas hormone therapy including progesterone and certain other progestins did not induce an increased risk w4x. By stimulating the production of survival factors, estradiol (E2) and other steroid hormones can influence cell proliferation. These survival factors include growth factors and cytokines. Epithelial and stromal cell-derived growth factors are understood to be significant in the regulation of breast epithelial cells directly via autocrine, paracrine, juxtacrine or intracrine pathways. Further responses stimulated by growth factors can activate signaling pathways which support the growth of cancer cells w5x. To further explore these findings in vitro, we investigated the effect of natural progesterone (P), drospirenone (DRSP), levonorgestrel (LNG) and MPA on growth factor treated primary normal breast epithelial cells and estradiol-treated estrogen receptor positive breast cancer cells in terms of proliferation.
University Women’s Hospital, Tu¨bingen, Germany Department of Gynecological Endocrinology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China 2
Abstract Background: Proliferation of human breast epithelial cells is regulated by sex hormones. Epidemiological studies indicate that progestogen addition to estrogen therapy can increase breast cancer risk. However, it remains unclear if all progestogens react in a similar manner. Here, the new progestogen drospirenone (DRSP) was compared to progesterone and other synthetic progestins. Design and methods: Human benign epithelial breast cells (HMECs) were incubated for 7 days with DRSP, progesterone (P), medroxyprogesterone acetate (MPA) and levonorgestrel (LNG) in the presence of a growth factor mixture (GF). HCC1500 and T-47D cells (human estrogen and progesterone receptor-positive primary breast cancer cells) were also incubated with the progestogens, but in the presence of estradiol (E2). The proliferation rate was measured by the MTT assay. Results: DRSP and P elicited a similar significant inhibition of proliferation of HMECs in combination with GFs. LNG and MPA had no effect. DRSP, P, MPA and LNG were able to significantly inhibit the proliferation of HCC1500 and T47D cells in combination with E2. No significant difference between the progestogens was observed in HCC1500 cells, whereas in T-47D cells both DRSP and P were significantly more effective at 10 mM than LNG and MPA. Conclusion: Because different results were found in the same experimental model, it appears that progestogens do not react similarly on the proliferation of human breast epithelial cells. However, for assessment of breast cancer risk different models should be used, because various mechanism(s) might be involved. It is also important to use benign as well as cancerous cell lines. The choice of progestogen could be of significance in terms of breast cancer risk under hormone therapy. Keywords: drospirenone; human breast epithelial cells; proliferation. *Corresponding author: Professor Alfred O. Mueck, MD, PharmD, PhD, University Women’s Hospital, Department of Endocrinology and Menopause, Head, Center of Women’s Health BW, Head, Calwer Strasse 7, D-72076 Tuebingen, Germany Phone: q49/7071-29 84801, Fax: q49/7071-29 4801, E-mail: [email protected] Received October 15, 2010; accepted January 26, 2011; previously published online March 8, 2011
Materials and methods P, LNG, MPA and E2 were purchased from Sigma Chemicals (Munich, Germany). DRSP was kindly provided by Bayer Health Care, Berlin, Germany. The compounds were dissolved in ethanol and were stored as concentrated stock solutions at –208C. Epidermal growth factor (EGF), basic-fibroblast growth factor (bFGF) and insulin-like growth factor (IGF-I) were purchased from
2010/040
Article in press - uncorrected proof 212 Seeger et al.: Drospirenone and proliferation of human epithelial cells
Sigma Chemicals. The compounds were reconstituted according to the manufacturer’s instructions stated on the package insert and were stored in aliquots at –208C.
Cells Normal human mammary epithelial cells (HMECs) isolated from reduction mammoplasty tissue, were purchased from Invitrogen, Heidelberg, Germany. Cells were maintained in serum-free mammary epithelial cell medium purchased from PromoCell, Heidelberg, Germany, supplemented with 100 U/mL penicillin plus 100 mg/mL streptomycin. HCC1500, a human estrogen and progesterone receptor-positive primary breast cancer cell line was purchased from ATCC (Middlesex, UK). Cells were maintained in RPMI-1640 medium (without phenol red) purchased from Sigma, which was modified to contain 1 mM sodium pyruvate, 2 mM L-glutamine, 4.5 g/L glucose, 10% (v/v) heat inactivated fetal bovine serum and 100 U/mL penicillin plus 100 mg/mL streptomycin. T-47D, a human estrogen and progesterone receptor-positive primary breast cancer cell line was purchased from ATCC. Cells were maintained in RPMI-1640 medium (without phenol red) purchased from Sigma, which was modified to contain 1 mM sodium pyruvate, 2 mM L-glutamine, 4.5 g/L glucose, 10% (v/v) heat inactivated fetal bovine serum and 100 U/mL penicillin plus 100 mg/mL streptomycin.
Human cancerous breast epithelial cells
In HCC1500 cells all four progestogens tested elicited a significant inhibitory effect at both concentrations. The inhibition was in the range of 13%–35%. There was no significant difference between the progestogens (Figure 2). The same results was observed for T47-D cells (Figure 3). The inhibition of the four progestogens was in the range of
Figure 1 Changes of proliferation of human mammary epithelial cells after addition of drospirenone (DRSP), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) or progesterone (P) in combination with a growth factor mixture (S). (Means"SD; *p-0.05 vs. S, **p-0.01 vs. S.)
Proliferation For proliferation, 96-well plates were seeded with approximately 1000 cells per well. The cells were incubated for 3 days at 378C in the appropriate culture medium. Progestogens in the concentrations of 0.01 mM, 0.1 mM, 1 mM and 10 mM in combination with growth factors (EGF, FGF, IGF) 0.001 mM or E2 0.001 mM were then freshly added over the next 4 or 7 days at 48- or 72-h intervals. Cell proliferation was measured by the MTT assay. This assay has been validated in the routine laboratory of our hospital, where it has been in use for several years.
Statistical analysis Proliferation was measured versus controls and performed in triplicate. Statistical analysis was done by analysis of variance with the logarithmized values followed by Dunnett’s procedure from triplicates of two independent experiments. The overall a-level was set at 0.05.
Figure 2 Changes of proliferation of human primary cancerous epithelial cells (HCC1500) after addition of drospirenone (DRSP), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) or progesterone (P) in combination with estradiol (S). (Means"SD; **p-0.01 vs. S.)
Results Human normal breast epithelial cells (HMECs)
The proliferation rate of HMECs was increased by approximately 2000% by the addition of the growth factor mixture (Figure 1). The addition of DRSP and P resulted in a significant inhibition of this proliferation rate at both concentrations tested, the reduction values were 16% and 25% for DRSP and 25% and 40% for P, respectively. The inhibition was significantly different between these progestogens at the concentration of 10 mM. No significant inhibitory effect was found for LNG and MPA.
Figure 3 Changes of proliferation of human primary cancerous epithelial cells (T-47D) after addition of drospirenone (DRSP), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) or progesterone (P) in combination with estradiol (S). (Means"SD; *p-0.05; **p-0.01 vs. S.)
Article in press - uncorrected proof Seeger et al.: Drospirenone and proliferation of human epithelial cells 213
20%–60%. Here, DRSP and P showed a significant greater inhibition at 10 mM than LNG and MPA.
Discussion The proliferation of normal and malignant cells is under the control of both estrogen and growth factors. In normal epithelial cells, estrogen-receptor expressing cells represent only a minority of the total cells and do not proliferate w6x. Current opinion is that estrogens act proliferatively in a paracrine manner by inducing the production of stromal-derived growth factors and cytokines or their receptors via the activation of epithelial or stromal estrogen receptors. Growth factors can play an important role in the promotion of receptor-positive breast cancer by crosstalk with the steroid receptor and are mainly responsible for the progression of estrogen-receptor negative breast cancer. Among the growth factors which are important for cell growth are the EGF family, IGF-I and IGF-II, FGFs, transforming growth factor-a (TGFa) and platelet-derived growth factors. It is important to differentiate between normal and malignant estrogen-receptor positive breast cells. In normal human epithelial breast cells the stimulation occurred by addition of a growth factor mixture. DRSP had a significant stronger inhibitory effect than LNG and MPA. The effect of DRSP was comparable to that of P. The proliferation of the human cancerous epithelial breast cells (HCC1500) and T-47D was stimulated by the addition of estradiol. P and the synthetic progestins DRSP, LNG and MPA could significantly reduce the estradiol-induced proliferation of these cells at the two highest concentrations. There was no significant difference between the various synthetic progestins in HCC1500 cells. However in T-47D cells both DRSP and P had a stronger inhibitory effect than LNG and MPA at 10 mM. Thus in both models, i.e., benign and cancerous breast epithelial cells, P and DRSP behave in a similar manner with an antiproliferative effect, whereas LNG and MPA differed from P and DRSP at least when using benign breast epithelial cells. Regarding P, other groups have published supporting results, where E2-induced stimulation of MCF-7 cells has been shown to be inhibited by P w7–11x. It has been proposed that progesterone inhibits the mitogenic effects of IGFs in breast cancer cells w12x. Breast cancer cells express IGF-I receptors but not IGF, which comes from nearby stromal cells. Varying results regarding the effects of MPA on cancerous breast cells have been published by other research groups in agreement or in contrast to our inhibitory results in the presence of growth factors and/or E2. MPA alone has been shown to induce a modest, but statistically significant cell growth at 10 –6 M in a specific subgroup of MCF-7 cells w8x, and inhibition of E2-stimulated proliferation at the higher MPA concentrations has been illustrated by others w7–9x. However, MPA combined with E2 has also been found to stimulate proliferation of MCF-7 cells w13x. Cappelletti et al.
w7x showed that MPA was able to effectively counteract cell growth induced by the growth factor TGF-a in MCF-7 cells. LNG alone has been shown to stimulate proliferation of MCF-7 cells at high pharmacological doses of 0.1 mM and 1 mM, but to reduce E2-stimulated growth of only a specific subgroup of malignant MCF-7 cells by approximately 60% over the concentration range of 1 mM–1 nM w8, 14x. Another research group showed no suppression of E2-induced stimulation of MCF-7 cells w9x. Van der Burg et al. w15x demonstrated that LNG was able to inhibit the mitogenic effect of E2 on MCF-7 cells but was not able to inhibit the synergistic combination of E2 with insulin where, in combination with insulin, LNG stimulated proliferation at 1 mM. Our results are in agreement with other groups’ findings where LNG has been found to have an inhibitory effect in the presence of E2 alone. To date, there is a paucity of data available regarding the effects of LNG on the proliferation of normal and malignant epithelial breast cells. There are also conflicting epidemiological data concerning this progestogen w16x. Despite their widespread use, in vitro models have certain limitations: 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 might not occur in vivo. Limitations of this in vitro study might be the high concentrations needed for an effective antiproliferative effect. The clinically relevant blood concentrations for the progestogens most commonly used for HRT, MPA and norethisterone (NET) are in the range of 0.005–0.01 mM for MPA w17x and around 0.01 mM for NET w18x. The effective concentrations in our experiments are in the range of 1–10 mM, and thus are much higher than serum levels achieved during therapy. However, higher concentrations could be required in vitro in short-time tests in which the reaction threshold can only be achieved with supraphysiological dosages. Higher concentrations can also be reached in vivo in the vessel wall or organs compared with the concentrations usually measured in the blood. The antiproliferative effects of the progestogens tested are partially rather low. Thus, it seems to be difficult to assess the in vivo relevance of the observed effects. However, the proliferative effect of estradiol on the cancerous cell lines is also rather low compared with the effect of growth factors on the benign epithelial cells. A further limitation of our work is the short incubation period of the cells with the substrates under investigation, in comparison with the longer time period for which hormone therapy is usually prescribed. That duration of therapy could indeed be an important factor for breast cancer risk is emphasized by the results of the WHI study, where breast cancer risk was significantly higher compared with placebo only in women given combined HRT for 10 years or more, but not in those treated only for the duration of the study period, i.e., 5.2 years w1x. In vitro experiments can support but not replace clinical trials and therefore further clinical studies are needed to determine which progestogens, if any, have the lowest breast cancer risk. In conclusion, the results of these in vitro experiments clearly show that differences exist between the effect of pro-
Article in press - uncorrected proof 214 Seeger et al.: Drospirenone and proliferation of human epithelial cells
gestogens on normal and cancerous human epithelial cells, respectively. In addition, the results also demonstrate that within the progestogen class differences can be seen in terms of the reaction on a specific cell type. This fact should be considered when choosing a progestogen for hormone therapy in postmenopausal women.
9.
10.
Acknowledgement Fund project: Beijing Municipality health technology High-level Talent (Number 2009-3-25).
References 1. Writing Group for the Women’s Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women. J Am Med Assoc 2002;288:321–33. 2. Million Women Study Collaborators. Breast cancer and hormone replacement therapy in the Million Women Study. Lancet 2003; 362:419–27. 3. The Women’s Health Initiative Steering Committee. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy. J Am Med Assoc 2004;291:1701–12. 4. Fournier A, Berrino F, Clavel-Chapelon F. Unequal risks for breast cancer associated with different hormone replacement therapies: results from the E3N cohort study. Breast Cancer Res Treat 2008;107:103–11. 5. Rahmean KM, Sarkar FH. Steroid hormone mimics: molecular mechanisms of cell growth and apoptosis in normal and malignant mammary epithelial cells. J Steroid Biochem Mol Biol 2002;80:191–201. 6. Ali S, Coombes RC. Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer 2002;2: 101–15. 7. Cappelletti V, Miodini P, Fioravanti L, DiFronzo G. Effect of progestin treatment on estradiol- and growth factor-stimulated breast cancer cell lines. Anticancer Res 1995;15:2551–6. 8. Schoonen WG, Joosten JW, Kloosterboer HJ. Effects of two classes of progestagens, pregnane and 19-nortestosterone derivatives,
11.
12.
13. 14.
15.
16.
17.
18.
on cell growth of human breast tumor cells: I. MCF-7 cell lines. J Steroid Biochem Mol Biol 1995;55:423–37. Seeger H, Wallwiener D, Mueck AO. The effect of progesterone and synthetic progestins on serum- and estradiol-stimulated proliferation of human breast cancer cells. Horm Metab Res 2003;35:76–80. Mueck AO, Seeger H, Wallwiener D. Comparison of the proliferative effects of estradiol and conjugated equine estrogens on human breast cancer cells and impact of continuous combined progestogen addition. Climacteric 2004;6:221–7. Kra¨mer EA, Seeger H, Kra¨mer B, Wallwiener D, Mueck AO. The effect of progesterone, testosterone and synthetic progestogens on growth factor- and estradiol-treated human cancerous and benign breast cells. Eur J Obstet Gynecol Reprod 2006;27: 139–41. Speroff L. Postmenopausal estrogen-progestin therapy and breast cancer: a clinical response to epidemiological reports. Climacteric 2000;3:3–12. Franke HR, Vermes I. Differential effects of progestagens on breast cancer cell lines. Maturitas 2003;46:S55–8. Jeng MH, Parker CJ, Jordan VC. Estrogenic potential of progestins in oral contraceptives to stimulate human breast cancer cell proliferation. Cancer Res 1992;52:6539–56. Van der Burg B, Kalkhoven E, Isbru¨cker L, De Laat SW. Effects of progestins on the proliferation of estrogen-dependent human breast cancer cells under growth factor-defined conditions. J Steroid Biochem Mol Biol 1992;42:457–65. Persson I, Yuan J, Bergkvist L, Schairer C. Cancer incidence and mortality in women receiving estrogen and estrogen-progestin replacement therapy – long-term follow-up of a Swedish cohort. Int J Cancer 1996;67:327–32. Svensson LO, Johnson SH, Olsson SE. 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 1994;18: 229–38. Stanczyk FZ, Brenner PF, Mishell DR, Ortiz A, Gentzschein EK, Goebelsmann U. A radioimmunoassay for norethindrone (NET): measurement of serum net concentrations following ingestion of NET-containing oral contraceptive steroids. Contraception 1978;18:615–33.
Copyright of Hormone Molecular Biology & Clinical Investigation is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Copyright of Hormone Molecular Biology & Clinical Investigation is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Effect of drospirenone on proliferation of human benign and cancerous epithelial breast cells Harald Seeger1, Xiangjan Ruan2, Hans Neubauer1 and Alfred O. Mueck1,*
Introduction
1
Two recent studies, the Women’s Health Initiative (WHI) and the Million Women Study (MWS), have above all raised concerns about the relationship between progestogens and increased risk of breast cancer in the climacteric and postmenopause w1, 2x. The WHI study was terminated early after five years, owing to an increased incidence of breast cancer in the group treated with combined estrogen and progestogen therapy. The MWS concluded that breast cancer risk was increased two-fold in current users of combined hormone replacement therapy (HRT) compared with a factor of 1.3 for estrogen-only therapy. A crucial role of progestogens in increasing breast cancer risk was supported by the WHI estrogen mono-arm showing no increase but rather a reduction of breast cancer risk, which was significant for patients with more than 80% adherence to study medication w3x. However, in the French E3N-EPIC trial of over 80,000 postmenopausal women it was reported that hormone therapy containing the progestin medroxyprogesterone acetate (MPA) or norethisterone (NET) was associated with a significant increase in risk of breast cancer, whereas hormone therapy including progesterone and certain other progestins did not induce an increased risk w4x. By stimulating the production of survival factors, estradiol (E2) and other steroid hormones can influence cell proliferation. These survival factors include growth factors and cytokines. Epithelial and stromal cell-derived growth factors are understood to be significant in the regulation of breast epithelial cells directly via autocrine, paracrine, juxtacrine or intracrine pathways. Further responses stimulated by growth factors can activate signaling pathways which support the growth of cancer cells w5x. To further explore these findings in vitro, we investigated the effect of natural progesterone (P), drospirenone (DRSP), levonorgestrel (LNG) and MPA on growth factor treated primary normal breast epithelial cells and estradiol-treated estrogen receptor positive breast cancer cells in terms of proliferation.
University Women’s Hospital, Tu¨bingen, Germany Department of Gynecological Endocrinology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China 2
Abstract Background: Proliferation of human breast epithelial cells is regulated by sex hormones. Epidemiological studies indicate that progestogen addition to estrogen therapy can increase breast cancer risk. However, it remains unclear if all progestogens react in a similar manner. Here, the new progestogen drospirenone (DRSP) was compared to progesterone and other synthetic progestins. Design and methods: Human benign epithelial breast cells (HMECs) were incubated for 7 days with DRSP, progesterone (P), medroxyprogesterone acetate (MPA) and levonorgestrel (LNG) in the presence of a growth factor mixture (GF). HCC1500 and T-47D cells (human estrogen and progesterone receptor-positive primary breast cancer cells) were also incubated with the progestogens, but in the presence of estradiol (E2). The proliferation rate was measured by the MTT assay. Results: DRSP and P elicited a similar significant inhibition of proliferation of HMECs in combination with GFs. LNG and MPA had no effect. DRSP, P, MPA and LNG were able to significantly inhibit the proliferation of HCC1500 and T47D cells in combination with E2. No significant difference between the progestogens was observed in HCC1500 cells, whereas in T-47D cells both DRSP and P were significantly more effective at 10 mM than LNG and MPA. Conclusion: Because different results were found in the same experimental model, it appears that progestogens do not react similarly on the proliferation of human breast epithelial cells. However, for assessment of breast cancer risk different models should be used, because various mechanism(s) might be involved. It is also important to use benign as well as cancerous cell lines. The choice of progestogen could be of significance in terms of breast cancer risk under hormone therapy. Keywords: drospirenone; human breast epithelial cells; proliferation. *Corresponding author: Professor Alfred O. Mueck, MD, PharmD, PhD, University Women’s Hospital, Department of Endocrinology and Menopause, Head, Center of Women’s Health BW, Head, Calwer Strasse 7, D-72076 Tuebingen, Germany Phone: q49/7071-29 84801, Fax: q49/7071-29 4801, E-mail: [email protected] Received October 15, 2010; accepted January 26, 2011; previously published online March 8, 2011
Materials and methods P, LNG, MPA and E2 were purchased from Sigma Chemicals (Munich, Germany). DRSP was kindly provided by Bayer Health Care, Berlin, Germany. The compounds were dissolved in ethanol and were stored as concentrated stock solutions at –208C. Epidermal growth factor (EGF), basic-fibroblast growth factor (bFGF) and insulin-like growth factor (IGF-I) were purchased from
2010/040
Article in press - uncorrected proof 212 Seeger et al.: Drospirenone and proliferation of human epithelial cells
Sigma Chemicals. The compounds were reconstituted according to the manufacturer’s instructions stated on the package insert and were stored in aliquots at –208C.
Cells Normal human mammary epithelial cells (HMECs) isolated from reduction mammoplasty tissue, were purchased from Invitrogen, Heidelberg, Germany. Cells were maintained in serum-free mammary epithelial cell medium purchased from PromoCell, Heidelberg, Germany, supplemented with 100 U/mL penicillin plus 100 mg/mL streptomycin. HCC1500, a human estrogen and progesterone receptor-positive primary breast cancer cell line was purchased from ATCC (Middlesex, UK). Cells were maintained in RPMI-1640 medium (without phenol red) purchased from Sigma, which was modified to contain 1 mM sodium pyruvate, 2 mM L-glutamine, 4.5 g/L glucose, 10% (v/v) heat inactivated fetal bovine serum and 100 U/mL penicillin plus 100 mg/mL streptomycin. T-47D, a human estrogen and progesterone receptor-positive primary breast cancer cell line was purchased from ATCC. Cells were maintained in RPMI-1640 medium (without phenol red) purchased from Sigma, which was modified to contain 1 mM sodium pyruvate, 2 mM L-glutamine, 4.5 g/L glucose, 10% (v/v) heat inactivated fetal bovine serum and 100 U/mL penicillin plus 100 mg/mL streptomycin.
Human cancerous breast epithelial cells
In HCC1500 cells all four progestogens tested elicited a significant inhibitory effect at both concentrations. The inhibition was in the range of 13%–35%. There was no significant difference between the progestogens (Figure 2). The same results was observed for T47-D cells (Figure 3). The inhibition of the four progestogens was in the range of
Figure 1 Changes of proliferation of human mammary epithelial cells after addition of drospirenone (DRSP), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) or progesterone (P) in combination with a growth factor mixture (S). (Means"SD; *p-0.05 vs. S, **p-0.01 vs. S.)
Proliferation For proliferation, 96-well plates were seeded with approximately 1000 cells per well. The cells were incubated for 3 days at 378C in the appropriate culture medium. Progestogens in the concentrations of 0.01 mM, 0.1 mM, 1 mM and 10 mM in combination with growth factors (EGF, FGF, IGF) 0.001 mM or E2 0.001 mM were then freshly added over the next 4 or 7 days at 48- or 72-h intervals. Cell proliferation was measured by the MTT assay. This assay has been validated in the routine laboratory of our hospital, where it has been in use for several years.
Statistical analysis Proliferation was measured versus controls and performed in triplicate. Statistical analysis was done by analysis of variance with the logarithmized values followed by Dunnett’s procedure from triplicates of two independent experiments. The overall a-level was set at 0.05.
Figure 2 Changes of proliferation of human primary cancerous epithelial cells (HCC1500) after addition of drospirenone (DRSP), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) or progesterone (P) in combination with estradiol (S). (Means"SD; **p-0.01 vs. S.)
Results Human normal breast epithelial cells (HMECs)
The proliferation rate of HMECs was increased by approximately 2000% by the addition of the growth factor mixture (Figure 1). The addition of DRSP and P resulted in a significant inhibition of this proliferation rate at both concentrations tested, the reduction values were 16% and 25% for DRSP and 25% and 40% for P, respectively. The inhibition was significantly different between these progestogens at the concentration of 10 mM. No significant inhibitory effect was found for LNG and MPA.
Figure 3 Changes of proliferation of human primary cancerous epithelial cells (T-47D) after addition of drospirenone (DRSP), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) or progesterone (P) in combination with estradiol (S). (Means"SD; *p-0.05; **p-0.01 vs. S.)
Article in press - uncorrected proof Seeger et al.: Drospirenone and proliferation of human epithelial cells 213
20%–60%. Here, DRSP and P showed a significant greater inhibition at 10 mM than LNG and MPA.
Discussion The proliferation of normal and malignant cells is under the control of both estrogen and growth factors. In normal epithelial cells, estrogen-receptor expressing cells represent only a minority of the total cells and do not proliferate w6x. Current opinion is that estrogens act proliferatively in a paracrine manner by inducing the production of stromal-derived growth factors and cytokines or their receptors via the activation of epithelial or stromal estrogen receptors. Growth factors can play an important role in the promotion of receptor-positive breast cancer by crosstalk with the steroid receptor and are mainly responsible for the progression of estrogen-receptor negative breast cancer. Among the growth factors which are important for cell growth are the EGF family, IGF-I and IGF-II, FGFs, transforming growth factor-a (TGFa) and platelet-derived growth factors. It is important to differentiate between normal and malignant estrogen-receptor positive breast cells. In normal human epithelial breast cells the stimulation occurred by addition of a growth factor mixture. DRSP had a significant stronger inhibitory effect than LNG and MPA. The effect of DRSP was comparable to that of P. The proliferation of the human cancerous epithelial breast cells (HCC1500) and T-47D was stimulated by the addition of estradiol. P and the synthetic progestins DRSP, LNG and MPA could significantly reduce the estradiol-induced proliferation of these cells at the two highest concentrations. There was no significant difference between the various synthetic progestins in HCC1500 cells. However in T-47D cells both DRSP and P had a stronger inhibitory effect than LNG and MPA at 10 mM. Thus in both models, i.e., benign and cancerous breast epithelial cells, P and DRSP behave in a similar manner with an antiproliferative effect, whereas LNG and MPA differed from P and DRSP at least when using benign breast epithelial cells. Regarding P, other groups have published supporting results, where E2-induced stimulation of MCF-7 cells has been shown to be inhibited by P w7–11x. It has been proposed that progesterone inhibits the mitogenic effects of IGFs in breast cancer cells w12x. Breast cancer cells express IGF-I receptors but not IGF, which comes from nearby stromal cells. Varying results regarding the effects of MPA on cancerous breast cells have been published by other research groups in agreement or in contrast to our inhibitory results in the presence of growth factors and/or E2. MPA alone has been shown to induce a modest, but statistically significant cell growth at 10 –6 M in a specific subgroup of MCF-7 cells w8x, and inhibition of E2-stimulated proliferation at the higher MPA concentrations has been illustrated by others w7–9x. However, MPA combined with E2 has also been found to stimulate proliferation of MCF-7 cells w13x. Cappelletti et al.
w7x showed that MPA was able to effectively counteract cell growth induced by the growth factor TGF-a in MCF-7 cells. LNG alone has been shown to stimulate proliferation of MCF-7 cells at high pharmacological doses of 0.1 mM and 1 mM, but to reduce E2-stimulated growth of only a specific subgroup of malignant MCF-7 cells by approximately 60% over the concentration range of 1 mM–1 nM w8, 14x. Another research group showed no suppression of E2-induced stimulation of MCF-7 cells w9x. Van der Burg et al. w15x demonstrated that LNG was able to inhibit the mitogenic effect of E2 on MCF-7 cells but was not able to inhibit the synergistic combination of E2 with insulin where, in combination with insulin, LNG stimulated proliferation at 1 mM. Our results are in agreement with other groups’ findings where LNG has been found to have an inhibitory effect in the presence of E2 alone. To date, there is a paucity of data available regarding the effects of LNG on the proliferation of normal and malignant epithelial breast cells. There are also conflicting epidemiological data concerning this progestogen w16x. Despite their widespread use, in vitro models have certain limitations: 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 might not occur in vivo. Limitations of this in vitro study might be the high concentrations needed for an effective antiproliferative effect. The clinically relevant blood concentrations for the progestogens most commonly used for HRT, MPA and norethisterone (NET) are in the range of 0.005–0.01 mM for MPA w17x and around 0.01 mM for NET w18x. The effective concentrations in our experiments are in the range of 1–10 mM, and thus are much higher than serum levels achieved during therapy. However, higher concentrations could be required in vitro in short-time tests in which the reaction threshold can only be achieved with supraphysiological dosages. Higher concentrations can also be reached in vivo in the vessel wall or organs compared with the concentrations usually measured in the blood. The antiproliferative effects of the progestogens tested are partially rather low. Thus, it seems to be difficult to assess the in vivo relevance of the observed effects. However, the proliferative effect of estradiol on the cancerous cell lines is also rather low compared with the effect of growth factors on the benign epithelial cells. A further limitation of our work is the short incubation period of the cells with the substrates under investigation, in comparison with the longer time period for which hormone therapy is usually prescribed. That duration of therapy could indeed be an important factor for breast cancer risk is emphasized by the results of the WHI study, where breast cancer risk was significantly higher compared with placebo only in women given combined HRT for 10 years or more, but not in those treated only for the duration of the study period, i.e., 5.2 years w1x. In vitro experiments can support but not replace clinical trials and therefore further clinical studies are needed to determine which progestogens, if any, have the lowest breast cancer risk. In conclusion, the results of these in vitro experiments clearly show that differences exist between the effect of pro-
Article in press - uncorrected proof 214 Seeger et al.: Drospirenone and proliferation of human epithelial cells
gestogens on normal and cancerous human epithelial cells, respectively. In addition, the results also demonstrate that within the progestogen class differences can be seen in terms of the reaction on a specific cell type. This fact should be considered when choosing a progestogen for hormone therapy in postmenopausal women.
9.
10.
Acknowledgement Fund project: Beijing Municipality health technology High-level Talent (Number 2009-3-25).
References 1. Writing Group for the Women’s Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women. J Am Med Assoc 2002;288:321–33. 2. Million Women Study Collaborators. Breast cancer and hormone replacement therapy in the Million Women Study. Lancet 2003; 362:419–27. 3. The Women’s Health Initiative Steering Committee. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy. J Am Med Assoc 2004;291:1701–12. 4. Fournier A, Berrino F, Clavel-Chapelon F. Unequal risks for breast cancer associated with different hormone replacement therapies: results from the E3N cohort study. Breast Cancer Res Treat 2008;107:103–11. 5. Rahmean KM, Sarkar FH. Steroid hormone mimics: molecular mechanisms of cell growth and apoptosis in normal and malignant mammary epithelial cells. J Steroid Biochem Mol Biol 2002;80:191–201. 6. Ali S, Coombes RC. Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer 2002;2: 101–15. 7. Cappelletti V, Miodini P, Fioravanti L, DiFronzo G. Effect of progestin treatment on estradiol- and growth factor-stimulated breast cancer cell lines. Anticancer Res 1995;15:2551–6. 8. Schoonen WG, Joosten JW, Kloosterboer HJ. Effects of two classes of progestagens, pregnane and 19-nortestosterone derivatives,
11.
12.
13. 14.
15.
16.
17.
18.
on cell growth of human breast tumor cells: I. MCF-7 cell lines. J Steroid Biochem Mol Biol 1995;55:423–37. Seeger H, Wallwiener D, Mueck AO. The effect of progesterone and synthetic progestins on serum- and estradiol-stimulated proliferation of human breast cancer cells. Horm Metab Res 2003;35:76–80. Mueck AO, Seeger H, Wallwiener D. Comparison of the proliferative effects of estradiol and conjugated equine estrogens on human breast cancer cells and impact of continuous combined progestogen addition. Climacteric 2004;6:221–7. Kra¨mer EA, Seeger H, Kra¨mer B, Wallwiener D, Mueck AO. The effect of progesterone, testosterone and synthetic progestogens on growth factor- and estradiol-treated human cancerous and benign breast cells. Eur J Obstet Gynecol Reprod 2006;27: 139–41. Speroff L. Postmenopausal estrogen-progestin therapy and breast cancer: a clinical response to epidemiological reports. Climacteric 2000;3:3–12. Franke HR, Vermes I. Differential effects of progestagens on breast cancer cell lines. Maturitas 2003;46:S55–8. Jeng MH, Parker CJ, Jordan VC. Estrogenic potential of progestins in oral contraceptives to stimulate human breast cancer cell proliferation. Cancer Res 1992;52:6539–56. Van der Burg B, Kalkhoven E, Isbru¨cker L, De Laat SW. Effects of progestins on the proliferation of estrogen-dependent human breast cancer cells under growth factor-defined conditions. J Steroid Biochem Mol Biol 1992;42:457–65. Persson I, Yuan J, Bergkvist L, Schairer C. Cancer incidence and mortality in women receiving estrogen and estrogen-progestin replacement therapy – long-term follow-up of a Swedish cohort. Int J Cancer 1996;67:327–32. Svensson LO, Johnson SH, Olsson SE. 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 1994;18: 229–38. Stanczyk FZ, Brenner PF, Mishell DR, Ortiz A, Gentzschein EK, Goebelsmann U. A radioimmunoassay for norethindrone (NET): measurement of serum net concentrations following ingestion of NET-containing oral contraceptive steroids. Contraception 1978;18:615–33.
Copyright of Hormone Molecular Biology & Clinical Investigation is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Copyright of Hormone Molecular Biology & Clinical Investigation is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
