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Effects of levonorgestrel-releasing IUS and progesterone receptor modulator PRM CDB-2914 on uterine leiomyomas
Contraception 75 (2007) S99 – S103
Review article
Effects of levonorgestrel-releasing IUS and progesterone receptor modulator PRM CDB-2914 on uterine leiomyomas Takeshi Maruoa,4, Noriyuki Oharaa, Hiroya Matsuoa, Qin Xua, Wei Chena, Regine Sitruk-Wareb, Elof D.B. Johanssonb a
Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan b Center for Biomedical Research, The Population Council, New York, NY 10021, USA Received 5 January 2007; accepted 12 January 2007
Abstract We have found that the use of levonorgestrel-releasing IUS results in a remarkable decrease in endometrial proliferation and a remarkable increase in apoptosis in the endometrium; therefore, it is effective for long-term management of menorrhagic women with uterine myomas because of the striking reduction in menorrhagia. This prompted us to characterize the effects of progesterone (P4) and progesterone receptor modulator (PRM) CDB2914 on uterine myoma growth. In vitro studies with cultured uterine leiomyoma cells and normal myometrial cells revealed that P4 stimulated the proliferative activity in leiomyoma cells, but not in normal myometrial cells. P4 increased EGF expression, whereas E2 augmented EGF-R expression in leiomyoma cells, indicating that P4 and E2 act in combination to stimulate leiomyoma cell growth. P4 also increased Bcl-2 expression and decreased TNF-a expression in those cells. Unlike the EGF expression, IGF-I expression in leiomyoma cells was inhibited by P4. These results suggest that P4 has dual actions on leiomyoma growth: one is to stimulate the growth through up-regulating EGF and Bcl-2 expression, and the other is to inhibit the growth through downregulating IGF-I expression in the cells. By contrast, CDB2914 inhibited proliferation and stimulated apoptosis of leiomyoma cells without affecting normal myometrial cells. Furthermore, CDB2914 inhibited vascular endothelial growth factor and adrenomedullin expression in leiomyoma cells, but not in normal myometrial cells. The cell type-specific action of CDB2914 on leiomyoma cells, without affecting the surrounding normal myometrial cells, is meaningful for understanding the usefulness of CDB2914 in the medical treatment of uterine myomas. D 2007 Elsevier Inc. All rights reserved. Keywords: Progesterone; Progesterone receptor modulator; Human leiomyoma; Apoptosis
1. Effects of levonorgestrel-releasing IUS on menorrhagia in women with uterine myomas Uterine leiomyoma is benign smooth muscle cell tumor of the myometrium, occurring in as many as 30% of women over 35 years of age. Leiomyoma has been thought to be an estrogen-dependent tumor because of its frequent occurrence during reproductive age and its regression after menopause. Homeostatic control of the net growth of tumors is thought to be the result of the dynamic balance between cell proliferation and cell death; too much growth can come from too little death as well as from too much proliferation. Actually, apoptosis, or programmed cell death,
4 Corresponding author. Tel.: +81 78 382 6000; fax: +81 78 382 6019. E-mail address: [email protected] (T. Maruo). 0010-7824/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2007.01.025
is known to occur in tumors either spontaneously or in response to treatment. Recently, we have found that the use of the levonorgestrel-releasing intrauterine system (IUS) (LNg-IUS) is effective in long-term contraception and management of menorrhagic women with uterine myomas, because of a striking reduction in menorrhagia [1–4]. Although some women with large intramural myomas had spontaneous expulsion of LNg-IUS at various intervals, they wanted reinsertion of the device because of remarkable reduction in menorrhagia. Significant increases in hemoglobin levels in blood were obtained after insertion of the devices (Fig. 1). No significant differences were noted in myoma volume and uterine volume, as assessed by MRI examination between pretreatment and 12 months of use. LNg-IUS was proven to be an effective modality for the long-term management of menorrhagia due to uterine myoma and adenomyosis. These
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Fig. 1. Hemoglobin levels in blood before and after insertion of LNg-IUS in menorrhagic women with intramural myomas. Bars represent meanFSD.
clinical experiences prompted us to characterize the effects of progesterone on the proliferation and apoptosis of leiomyoma cells and normal myometrial cells. 2. Effects of progesterone on the proliferative activity and apoptosis of leiomyoma cells and myometrial cells Estrogen has received much attention as the major factor responsible for leiomyoma development. To investigate the mitogenic effects of progesterone (P4) on uterine leiomyoma cells and normal myometrial cells, an in vitro culture system of leiomyoma cells and normal myometrial cells was established. Western immunoblot analysis showed that the 36-kDa PCNA expression in leiomyoma cells was more abundant than that in normal myometrial cells (Fig. 2). In normal myometrial cells, E2 increased the 36-kDa PCNA protein expression, but P4 did not. In leiomyoma cells, not only E2 but also P4 increased the PCNA protein expression in the cells. This demonstrates that in leiomyoma cells both E2 and P4 up-regulate the cell-proliferating activity, whereas in normal myometrial smooth muscle cells, only E2 upregulates the cell proliferating activity [5]. Furthermore, we have provided the evidence that EGF expression in the
Fig. 2. Effects of E2 and P4 on PCNA protein expression in cultured normal myometrial cells and leiomyoma cells as assessed by Western immunoblot analysis.
cultured leiomyoma cells is up-regulated by P4, whereas EGF-R expression in those cells is up-regulated by E2 [5]. As EGF is known to play a crucial role as a local factor in the autocrine/paracrine regulation of leiomyoma growth, it is conceivable that P4 and E2 act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF and EGF-R expression in uterine leiomyoma. We also have demonstrated greater abundance of Bcl-2 protein in leiomyomas relative to the normal myometrium of the same individual uterus. The abundant expression of Bcl-2 protein in leiomyoma may be one of the molecular bases for the enhanced growth of leiomyoma relative to that of normal myometrium in the uterus. Bcl-2 protein expression in leiomyoma cells was up-regulated by P4 [6]. It seems likely therefore that P4 may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells. We showed that IGF-I plays crucial roles in leiomyoma cell growth not only in promoting the proliferative potential through up-regulation of PCNA expression but also in inhibiting apoptosis through up-regulation of Bcl-2 protein expression [7]. Treatment with P4 significantly decreases IGF-I mRNA expression in cultured leiomyoma cells, whereas treatment with E2 does not affect IGF-I mRNA expression in those cells (Fig. 3). No significant
Fig. 3. Effect of sex steroids on IGF-I mRNA expression in cultured leiomyoma cells, as assessed by quantitative RT-PCR analysis with Southern blot analysis. Lane 1, untreated control cultures; Lane 2, E2 (10 ng/mL)-treated; Lane 3, P4 (100 ng/mL)-treated; Lane 4, combined treatment with E2 (10 ng/mL) and progesterone (100 ng/mL); Lane 5, negative control. Data are presented as the fold increases over the untreated control value and as the meanFSD. *pb0.01.
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differences are noted in IGF-I receptor mRNA expression between untreated cultures and cultures treated with either E2 or P4. These results provide the evidence that progesterone down-regulates IGF-I expression in cultured leiomyoma cells without affecting IGF-I receptor expression in those cells. P4 therefore may have dual actins on uterine leiomyoma growth: one is to stimulate and the other is to inhibit uterine leiomyoma growth, which may explain, at least in part, why the size of uterine leiomyomas during the use of LNg-IUS decreases in some cases but increases in other cases. Whether uterine leiomyoma either decreases or increases in size during LNg-IUS use may be dependent on the local autocrine/ paracrine growth factor conditions around each leiomyoma. Furthermore, the dual actions of P4 on uterine leiomyoma growth may also explain in part why it is rare to find the increase in the size of uterine leiomyomas over the course of pregnancy despite the overwhelming increase in circulating concentrations of sex steroid hormones [8–11]. 3. Effects of progesterone receptor modulator (PRM) CDB-2914 on cell proliferation and apoptosis in leiomyoma cells and normal myometrial cells The effects of P4 on target tissues are mediated by P4 receptor (PR), which belongs to the nuclear receptor family. PR functions as a ligand-activated transcription factor to regulate the expression of specific sets of target genes. PR-A regresses the biological actions of P4 by inhibiting PR activation. In this context, RU486 (mifepristone) has been shown to cause a significant regression of the size of leiomyomas, but undesirable side effects related to the antiglucocorticoid effect of RU486 are reported to occur during the treatment, as evidenced by the rise in serum testosterone, androstenedione and dehydroepiandrosterone sulfate. This antiglucocorticoid effect of RU486 is considered neither necessary nor even useful in the long-term treatment of leiomyoma. CDB-2914 (17a-acetoxy-11h-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione) is a novel PRM that binds competitively to PR with high affinity and has little or no antiglucocorticoid activity. The lesser antiglucocorticoid activity of CDB-2914 compared with that of RU486 may provide considerable advantage for the long-term treatment of leiomyomas. Actually, we have demonstrated that CDB-2914 exerts an inhibitory effect on the number of viable cultured leiomyoma cells in a dosedependent manner. Treatment with CDB-2914 resulted in a decrease in PCNA expression in cultured leiomyoma cells in a dose-dependent manner and reversed the stimulatory effect of P4 on PCNA expression in those cells [12]. These results indicate that CDB-2914 exerts an antiproliferative activity in cultured leiomyoma cells. Because the expression of PCNA is known to be elevated at the late G1 and S phases of proliferating cells, CDB-
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2914 may cause cell cycle arrest at the G0/G1 phase in cultured leiomyoma cells. In this context, our recent study has demonstrated that CD-B2914 augments apoptosis of cultured leiomyoma cells through up-regulating cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) expression and down-regulating Bcl-2 protein expression in those cells [12]. The time-course study demonstrated that cleaved caspase-3 expression was augmented by CDB-2914 with a peak at 12 h of treatment, whereas cleaved PARP expression was augmented by CDB-2914 with a peak at 24 h of treatment. By contrast, Bcl-2 protein expression in those cells was attenuated by CDB-2914 in a timedependent manner. It is now evident that CDB-2914 treatment inhibits the proliferation of cultured leiomyoma cells by down-regulating PCNA expression and induces apoptosis by up-regulating cleaved caspase-3 and cleaved PARP expression and down-regulating Bcl-2 protein expression in those cells. 4. Effects of PRM CDB-2914 on the expression of angiogenic factors and their receptors in uterine leiomyoma cells and normal myometrial cells Uterine leiomyomas are associated with irregular vascular networks. Angiogenic factors, including vascular endothelial growth factor (VEGF) and adrenomedullin (ADM), have been speculated to be involved in the angiogenesis of uterine leiomyomas. We have demonstrated that VEGF-A, VEGF-B and ADM protein are expressed in cultured human uterine leiomyoma cells and normal myometrial cells together with the presence of VEGFR-1, VEGFR-2 and ADMR protein. The physiological tissue level of P4 (100 ng/mL) was found to augment VEGF-A, VEGF-B and ADM expression in both cultured leiomyoma cells and normal myometrial cells. This suggests that P4 may promote the physiological actions of VEGF and ADM such as cell growth and/or angiogenesis in uterine leiomyomas and normal myometrium. In contrast, a novel PRM CDB-2914 reversed the P4-induced up-regulation of VEGF-A, VEGF-B and ADM expression in cultured leiomyoma cells. However, in normal myometrial cells, CDB-2914 had no inhibitory effects on VEGF-A, VEGF-B and ADM expression. Furthermore, the inhibitory effects of CDB2914 on VEGFR-1, VEGFR-2 and ADMR expression in cultured leiomyoma cells were dose dependent, whereas graded concentrations of CDB-2914 did not affect VEGFR-1, VEGFR-2 and ADMR expression in normal myometrial cells. These results suggest that the action of CDB-2914 may be cell-type specific and that CDB-2914 treatment may attenuate VEGF- and ADM-mediated multiple intracellular signaling leading to cell proliferation, cell survival and angiogenesis in leiomyomas by disrupting the VEGF/VEGFR system and ADM/ADMR system, but not in normal myometrium [13].
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5. Effects of PRM CDB-2914 on PR-A and PR-B expression in uterine leiomyoma cells and normal myometrial cells To elucidate the mechanism underlying the cell typespecific action of CDB-2914, we examined the changes in PR-A and PR-B expression in response to graded concentrations of CDB-2914 in cultured leiomyoma cells and normal myometrial cells. We have demonstrated that CDB2914 treatment increased PR-A and decreased PR-B expression in cultured leiomyoma cells in a dose-dependent manner, resulting in the increase in the PR-A/PR-B ratio in these cells. Moreover, the increase in the PR-A/PR-B ratio correlated with the decrease in VEGF-A, VEGF-B, VEGFR-1, VEGFR-2, ADM and ADMR expression in cultured leiomyoma cells treated with CDB-2914 [13]. Unlike in cultured leiomyoma cells, in cultured normal myometrial cells, CDB-2914 treatment had no effects on PR-A and PR-B expression. The relative levels of PR-A and PR-B expression in the cells are reported to be critical for appropriate cellular response to P4. Two PR isoforms have different transcriptional activities. PR-B functions as a transcriptional activator of P4-responsive genes, whereas PR-A functions as a ligand-dependent repressor of PR-B transcriptional activity. It is evident that PR-B is the dominant inducer of VEGF mRNA in breast cancer cells and that PR-A may suppress PR-B-dependent induction of VEGF. It can be postulated that decreased PR-B expression may lead to decreased gene transcription of VEGF and ADM, and that increased PR-A expression in cultured leiomyoma cells treated with CDB2914 may act to suppress the transcriptional activities of PR-B, resulting in the decrease in VEGF and ADM gene transcription in those cells. On the other hand, unlike cultured leiomyoma cells, no changes in PR isoform expression were found in cultured normal myometrial cells in response to CDB-2914 treatment.
6. Conclusion Although P4 contributes to the promotion of leiomyoma cell growth and survival through up-regulating EGF and Bcl-2 expression as well as down-regulating TNFa expression in the cells, P4 also exerts an inhibitory effect on leiomyoma growth and survival through down-regulating IGF-I expression in those cells. It is likely that P4 may have dual actions on leiomyoma growth depending on the local growth factor conditions around each leiomyoma. Nevertheless, the net effect of P4 on leiomyoma growth seems to favor the mitogenic activity of leiomyoma cells. Consistent with the above findings, it is now evident that treatment with a PRM CDB-2914 inhibits the proliferation of cultured leiomyoma cells by down-regulating PCNA expression and induces apoptosis by up-regulating cleaved caspase3 and cleaved PARP expression and down-regulating Bcl-2 protein expression in those cells. Furthermore, we have
demonstrated that P4 up-regulates VEGF-A, VEGF-B and ADM expression in both cultured human uterine leiomyoma cells and normal myometrial cells, but that CDB-2914 treatment selectively down-regulates VEGF-A, VEGF-B and ADM expression together with VEGFR-1, VEGFR-2 and ADMR expression in cultured leiomyoma cells without affecting the expression of these angiogenic factors and their receptors in cultured normal myometrial cells. In addition, we have shown that PR-B expression is elevated in cultured leiomyoma cells compared with cultured normal myometrial cells, whereas there are no differences in PR-A expression between the two types of cells. The increase in the PR-A/PRB ratio correlates with the decrease in VEGF, VEGFR, ADM and ADMR expression in cultured leiomyoma cells treated with CDB-2914, but not in normal myometrial cells. These findings suggest that CDB-2914 may inhibit growth and angiogenesis of uterine leiomyomas in a cell type-specific manner through disrupting the VEGF/VEGFR system and ADM/ADMR system without affecting the surrounding normal myometrial cells in the uterus. This is meaningful for understanding the usefulness of CDB-2914 in the medical treatment of uterine myomas. Acknowledgments The authors wish to thank Dr. Andre´ Ulmann and Ms. Erin E Gainer of HRA Pharma (Paris, France) for kindly providing us with CDB-2914 and supporting our research. This work was supported in part by Grants-in-Aid for Scientific Research 1437053 from the Japanese Ministry of Education, Science and Culture and by the OgyaaDonation Foundation of the Japan Association of Obstetricians and Gynecologists. References [1] Maruo T, Matsuo H, Samoto T, Gao Z, Spitz IM, Johansson E. Effects of progesterone on uterine leiomyoma growth and apoptosis. Steroids 2000;65:585 – 92. [2] Maruo T, Laoag-Fernandez JB, Matsuo H, et al. Effects of levonorgestrel-releasing intrauterine system on the endometrium and the relevance to the management of menorrhagia caused by uterine myoma and adenomyosis. In: Maruo T, Barlow D, Mardon H, Kennedy S, editors. Cell and molecular biology of endometrium in health and disease. Osaka7 Soeisha; 2002. p. 193 – 207. [3] Maruo T, Laoag-Fernandez JB, Pakarinen P, Murakoshi H, Spitz IM, Johansson E. Effects of the levonorgestrel-releasing intrauterine system on proliferation and apoptosis in the endometrium. Hum Reprod 2001;16:2103 – 8. [4] Maruo T. Endometrial effects of levonorgestrel intrauterine delivery. Gynecol Forum 2006;11:27 – 30. [5] Shimomura Y, Matsuo H, Samoto T, Maruo T. Up-regulation by progesterone of proliferating cell number antigen and epidermal growth factor expression in human uterine leiomyoma. J Clin Endocrinol Metab 1998;83:2192 – 8. [6] Matsuo H, Maruo T, Samoto T. Increased expression of Bcl-2 protein in human uterine leiomyoma and its up-regulation by progesterone. J Clin Endocrinol Metab 1997;82:293 – 9. [7] Gao Z, Matsuo H, Wang Y, Nakago S, Maruo T. Up-regulation by IGF-I of proliferating cell nuclear antigen and Bcl-2 protein expression in
T. Maruo et al. / Contraception 75 (2007) S99 – S103 human uterine leiomyoma cells. J Clin Endocrinol Metab 2001; 86:5593 – 9. [8] Gao Z, Matsuo H, Nakago S, Kurachi O, Maruo T. p53 tumor suppressor protein content in human uterine leiomyomas and its down-regulation by 17 beta-estradiol. J Clin Endocrinol Metab 2002;87:3915 – 20. [9] Yamada T, Nakago S, Kurachi O, et al. Progesterone down-regulates insulin-like growth factor-I expression in cultured human uterine leiomyoma cells. Hum Reprod 2004;19:1 – 7. [10] Maruo T, Ohara N, Wang J, Matsuo H. Sex steroidal regulation of uterine leiomyoma growth and apoptosis. Hum Reprod Update 2004;10:207 – 20.
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[11] Maruo T, Matsuo H, Yamada T, et al. Effects of progesterone on growth factor expression in human uterine leiomyoma. Steroids 2003;68:817 – 24. [12] Xu Q, Takekida S, Ohara N, et al. Progesterone receptor modulator CDB-2914 down-regulates proliferative cell nuclear antigen and Bcl-2 protein expression and up-regulates caspase-3 and (adenosine 5Vdiphosphate-ribose) polymerase expression in cultured human uterine leiomyoma cells. J Clin Endocrinol Metab 2005;90:953 – 61. [13] Xu Q, Ohara N, Maruo T, et al. Progesterone receptor modulator CDB2914 down-regulates VEGF, adrenomedullin and their receptors and modulates progesterone receptor content in cultured human uterine leiomyoma cells. Hum Reprod 2006;21:2408 – 16.
Review article
Effects of levonorgestrel-releasing IUS and progesterone receptor modulator PRM CDB-2914 on uterine leiomyomas Takeshi Maruoa,4, Noriyuki Oharaa, Hiroya Matsuoa, Qin Xua, Wei Chena, Regine Sitruk-Wareb, Elof D.B. Johanssonb a
Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan b Center for Biomedical Research, The Population Council, New York, NY 10021, USA Received 5 January 2007; accepted 12 January 2007
Abstract We have found that the use of levonorgestrel-releasing IUS results in a remarkable decrease in endometrial proliferation and a remarkable increase in apoptosis in the endometrium; therefore, it is effective for long-term management of menorrhagic women with uterine myomas because of the striking reduction in menorrhagia. This prompted us to characterize the effects of progesterone (P4) and progesterone receptor modulator (PRM) CDB2914 on uterine myoma growth. In vitro studies with cultured uterine leiomyoma cells and normal myometrial cells revealed that P4 stimulated the proliferative activity in leiomyoma cells, but not in normal myometrial cells. P4 increased EGF expression, whereas E2 augmented EGF-R expression in leiomyoma cells, indicating that P4 and E2 act in combination to stimulate leiomyoma cell growth. P4 also increased Bcl-2 expression and decreased TNF-a expression in those cells. Unlike the EGF expression, IGF-I expression in leiomyoma cells was inhibited by P4. These results suggest that P4 has dual actions on leiomyoma growth: one is to stimulate the growth through up-regulating EGF and Bcl-2 expression, and the other is to inhibit the growth through downregulating IGF-I expression in the cells. By contrast, CDB2914 inhibited proliferation and stimulated apoptosis of leiomyoma cells without affecting normal myometrial cells. Furthermore, CDB2914 inhibited vascular endothelial growth factor and adrenomedullin expression in leiomyoma cells, but not in normal myometrial cells. The cell type-specific action of CDB2914 on leiomyoma cells, without affecting the surrounding normal myometrial cells, is meaningful for understanding the usefulness of CDB2914 in the medical treatment of uterine myomas. D 2007 Elsevier Inc. All rights reserved. Keywords: Progesterone; Progesterone receptor modulator; Human leiomyoma; Apoptosis
1. Effects of levonorgestrel-releasing IUS on menorrhagia in women with uterine myomas Uterine leiomyoma is benign smooth muscle cell tumor of the myometrium, occurring in as many as 30% of women over 35 years of age. Leiomyoma has been thought to be an estrogen-dependent tumor because of its frequent occurrence during reproductive age and its regression after menopause. Homeostatic control of the net growth of tumors is thought to be the result of the dynamic balance between cell proliferation and cell death; too much growth can come from too little death as well as from too much proliferation. Actually, apoptosis, or programmed cell death,
4 Corresponding author. Tel.: +81 78 382 6000; fax: +81 78 382 6019. E-mail address: [email protected] (T. Maruo). 0010-7824/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2007.01.025
is known to occur in tumors either spontaneously or in response to treatment. Recently, we have found that the use of the levonorgestrel-releasing intrauterine system (IUS) (LNg-IUS) is effective in long-term contraception and management of menorrhagic women with uterine myomas, because of a striking reduction in menorrhagia [1–4]. Although some women with large intramural myomas had spontaneous expulsion of LNg-IUS at various intervals, they wanted reinsertion of the device because of remarkable reduction in menorrhagia. Significant increases in hemoglobin levels in blood were obtained after insertion of the devices (Fig. 1). No significant differences were noted in myoma volume and uterine volume, as assessed by MRI examination between pretreatment and 12 months of use. LNg-IUS was proven to be an effective modality for the long-term management of menorrhagia due to uterine myoma and adenomyosis. These
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Fig. 1. Hemoglobin levels in blood before and after insertion of LNg-IUS in menorrhagic women with intramural myomas. Bars represent meanFSD.
clinical experiences prompted us to characterize the effects of progesterone on the proliferation and apoptosis of leiomyoma cells and normal myometrial cells. 2. Effects of progesterone on the proliferative activity and apoptosis of leiomyoma cells and myometrial cells Estrogen has received much attention as the major factor responsible for leiomyoma development. To investigate the mitogenic effects of progesterone (P4) on uterine leiomyoma cells and normal myometrial cells, an in vitro culture system of leiomyoma cells and normal myometrial cells was established. Western immunoblot analysis showed that the 36-kDa PCNA expression in leiomyoma cells was more abundant than that in normal myometrial cells (Fig. 2). In normal myometrial cells, E2 increased the 36-kDa PCNA protein expression, but P4 did not. In leiomyoma cells, not only E2 but also P4 increased the PCNA protein expression in the cells. This demonstrates that in leiomyoma cells both E2 and P4 up-regulate the cell-proliferating activity, whereas in normal myometrial smooth muscle cells, only E2 upregulates the cell proliferating activity [5]. Furthermore, we have provided the evidence that EGF expression in the
Fig. 2. Effects of E2 and P4 on PCNA protein expression in cultured normal myometrial cells and leiomyoma cells as assessed by Western immunoblot analysis.
cultured leiomyoma cells is up-regulated by P4, whereas EGF-R expression in those cells is up-regulated by E2 [5]. As EGF is known to play a crucial role as a local factor in the autocrine/paracrine regulation of leiomyoma growth, it is conceivable that P4 and E2 act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF and EGF-R expression in uterine leiomyoma. We also have demonstrated greater abundance of Bcl-2 protein in leiomyomas relative to the normal myometrium of the same individual uterus. The abundant expression of Bcl-2 protein in leiomyoma may be one of the molecular bases for the enhanced growth of leiomyoma relative to that of normal myometrium in the uterus. Bcl-2 protein expression in leiomyoma cells was up-regulated by P4 [6]. It seems likely therefore that P4 may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells. We showed that IGF-I plays crucial roles in leiomyoma cell growth not only in promoting the proliferative potential through up-regulation of PCNA expression but also in inhibiting apoptosis through up-regulation of Bcl-2 protein expression [7]. Treatment with P4 significantly decreases IGF-I mRNA expression in cultured leiomyoma cells, whereas treatment with E2 does not affect IGF-I mRNA expression in those cells (Fig. 3). No significant
Fig. 3. Effect of sex steroids on IGF-I mRNA expression in cultured leiomyoma cells, as assessed by quantitative RT-PCR analysis with Southern blot analysis. Lane 1, untreated control cultures; Lane 2, E2 (10 ng/mL)-treated; Lane 3, P4 (100 ng/mL)-treated; Lane 4, combined treatment with E2 (10 ng/mL) and progesterone (100 ng/mL); Lane 5, negative control. Data are presented as the fold increases over the untreated control value and as the meanFSD. *pb0.01.
T. Maruo et al. / Contraception 75 (2007) S99 – S103
differences are noted in IGF-I receptor mRNA expression between untreated cultures and cultures treated with either E2 or P4. These results provide the evidence that progesterone down-regulates IGF-I expression in cultured leiomyoma cells without affecting IGF-I receptor expression in those cells. P4 therefore may have dual actins on uterine leiomyoma growth: one is to stimulate and the other is to inhibit uterine leiomyoma growth, which may explain, at least in part, why the size of uterine leiomyomas during the use of LNg-IUS decreases in some cases but increases in other cases. Whether uterine leiomyoma either decreases or increases in size during LNg-IUS use may be dependent on the local autocrine/ paracrine growth factor conditions around each leiomyoma. Furthermore, the dual actions of P4 on uterine leiomyoma growth may also explain in part why it is rare to find the increase in the size of uterine leiomyomas over the course of pregnancy despite the overwhelming increase in circulating concentrations of sex steroid hormones [8–11]. 3. Effects of progesterone receptor modulator (PRM) CDB-2914 on cell proliferation and apoptosis in leiomyoma cells and normal myometrial cells The effects of P4 on target tissues are mediated by P4 receptor (PR), which belongs to the nuclear receptor family. PR functions as a ligand-activated transcription factor to regulate the expression of specific sets of target genes. PR-A regresses the biological actions of P4 by inhibiting PR activation. In this context, RU486 (mifepristone) has been shown to cause a significant regression of the size of leiomyomas, but undesirable side effects related to the antiglucocorticoid effect of RU486 are reported to occur during the treatment, as evidenced by the rise in serum testosterone, androstenedione and dehydroepiandrosterone sulfate. This antiglucocorticoid effect of RU486 is considered neither necessary nor even useful in the long-term treatment of leiomyoma. CDB-2914 (17a-acetoxy-11h-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione) is a novel PRM that binds competitively to PR with high affinity and has little or no antiglucocorticoid activity. The lesser antiglucocorticoid activity of CDB-2914 compared with that of RU486 may provide considerable advantage for the long-term treatment of leiomyomas. Actually, we have demonstrated that CDB-2914 exerts an inhibitory effect on the number of viable cultured leiomyoma cells in a dosedependent manner. Treatment with CDB-2914 resulted in a decrease in PCNA expression in cultured leiomyoma cells in a dose-dependent manner and reversed the stimulatory effect of P4 on PCNA expression in those cells [12]. These results indicate that CDB-2914 exerts an antiproliferative activity in cultured leiomyoma cells. Because the expression of PCNA is known to be elevated at the late G1 and S phases of proliferating cells, CDB-
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2914 may cause cell cycle arrest at the G0/G1 phase in cultured leiomyoma cells. In this context, our recent study has demonstrated that CD-B2914 augments apoptosis of cultured leiomyoma cells through up-regulating cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) expression and down-regulating Bcl-2 protein expression in those cells [12]. The time-course study demonstrated that cleaved caspase-3 expression was augmented by CDB-2914 with a peak at 12 h of treatment, whereas cleaved PARP expression was augmented by CDB-2914 with a peak at 24 h of treatment. By contrast, Bcl-2 protein expression in those cells was attenuated by CDB-2914 in a timedependent manner. It is now evident that CDB-2914 treatment inhibits the proliferation of cultured leiomyoma cells by down-regulating PCNA expression and induces apoptosis by up-regulating cleaved caspase-3 and cleaved PARP expression and down-regulating Bcl-2 protein expression in those cells. 4. Effects of PRM CDB-2914 on the expression of angiogenic factors and their receptors in uterine leiomyoma cells and normal myometrial cells Uterine leiomyomas are associated with irregular vascular networks. Angiogenic factors, including vascular endothelial growth factor (VEGF) and adrenomedullin (ADM), have been speculated to be involved in the angiogenesis of uterine leiomyomas. We have demonstrated that VEGF-A, VEGF-B and ADM protein are expressed in cultured human uterine leiomyoma cells and normal myometrial cells together with the presence of VEGFR-1, VEGFR-2 and ADMR protein. The physiological tissue level of P4 (100 ng/mL) was found to augment VEGF-A, VEGF-B and ADM expression in both cultured leiomyoma cells and normal myometrial cells. This suggests that P4 may promote the physiological actions of VEGF and ADM such as cell growth and/or angiogenesis in uterine leiomyomas and normal myometrium. In contrast, a novel PRM CDB-2914 reversed the P4-induced up-regulation of VEGF-A, VEGF-B and ADM expression in cultured leiomyoma cells. However, in normal myometrial cells, CDB-2914 had no inhibitory effects on VEGF-A, VEGF-B and ADM expression. Furthermore, the inhibitory effects of CDB2914 on VEGFR-1, VEGFR-2 and ADMR expression in cultured leiomyoma cells were dose dependent, whereas graded concentrations of CDB-2914 did not affect VEGFR-1, VEGFR-2 and ADMR expression in normal myometrial cells. These results suggest that the action of CDB-2914 may be cell-type specific and that CDB-2914 treatment may attenuate VEGF- and ADM-mediated multiple intracellular signaling leading to cell proliferation, cell survival and angiogenesis in leiomyomas by disrupting the VEGF/VEGFR system and ADM/ADMR system, but not in normal myometrium [13].
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5. Effects of PRM CDB-2914 on PR-A and PR-B expression in uterine leiomyoma cells and normal myometrial cells To elucidate the mechanism underlying the cell typespecific action of CDB-2914, we examined the changes in PR-A and PR-B expression in response to graded concentrations of CDB-2914 in cultured leiomyoma cells and normal myometrial cells. We have demonstrated that CDB2914 treatment increased PR-A and decreased PR-B expression in cultured leiomyoma cells in a dose-dependent manner, resulting in the increase in the PR-A/PR-B ratio in these cells. Moreover, the increase in the PR-A/PR-B ratio correlated with the decrease in VEGF-A, VEGF-B, VEGFR-1, VEGFR-2, ADM and ADMR expression in cultured leiomyoma cells treated with CDB-2914 [13]. Unlike in cultured leiomyoma cells, in cultured normal myometrial cells, CDB-2914 treatment had no effects on PR-A and PR-B expression. The relative levels of PR-A and PR-B expression in the cells are reported to be critical for appropriate cellular response to P4. Two PR isoforms have different transcriptional activities. PR-B functions as a transcriptional activator of P4-responsive genes, whereas PR-A functions as a ligand-dependent repressor of PR-B transcriptional activity. It is evident that PR-B is the dominant inducer of VEGF mRNA in breast cancer cells and that PR-A may suppress PR-B-dependent induction of VEGF. It can be postulated that decreased PR-B expression may lead to decreased gene transcription of VEGF and ADM, and that increased PR-A expression in cultured leiomyoma cells treated with CDB2914 may act to suppress the transcriptional activities of PR-B, resulting in the decrease in VEGF and ADM gene transcription in those cells. On the other hand, unlike cultured leiomyoma cells, no changes in PR isoform expression were found in cultured normal myometrial cells in response to CDB-2914 treatment.
6. Conclusion Although P4 contributes to the promotion of leiomyoma cell growth and survival through up-regulating EGF and Bcl-2 expression as well as down-regulating TNFa expression in the cells, P4 also exerts an inhibitory effect on leiomyoma growth and survival through down-regulating IGF-I expression in those cells. It is likely that P4 may have dual actions on leiomyoma growth depending on the local growth factor conditions around each leiomyoma. Nevertheless, the net effect of P4 on leiomyoma growth seems to favor the mitogenic activity of leiomyoma cells. Consistent with the above findings, it is now evident that treatment with a PRM CDB-2914 inhibits the proliferation of cultured leiomyoma cells by down-regulating PCNA expression and induces apoptosis by up-regulating cleaved caspase3 and cleaved PARP expression and down-regulating Bcl-2 protein expression in those cells. Furthermore, we have
demonstrated that P4 up-regulates VEGF-A, VEGF-B and ADM expression in both cultured human uterine leiomyoma cells and normal myometrial cells, but that CDB-2914 treatment selectively down-regulates VEGF-A, VEGF-B and ADM expression together with VEGFR-1, VEGFR-2 and ADMR expression in cultured leiomyoma cells without affecting the expression of these angiogenic factors and their receptors in cultured normal myometrial cells. In addition, we have shown that PR-B expression is elevated in cultured leiomyoma cells compared with cultured normal myometrial cells, whereas there are no differences in PR-A expression between the two types of cells. The increase in the PR-A/PRB ratio correlates with the decrease in VEGF, VEGFR, ADM and ADMR expression in cultured leiomyoma cells treated with CDB-2914, but not in normal myometrial cells. These findings suggest that CDB-2914 may inhibit growth and angiogenesis of uterine leiomyomas in a cell type-specific manner through disrupting the VEGF/VEGFR system and ADM/ADMR system without affecting the surrounding normal myometrial cells in the uterus. This is meaningful for understanding the usefulness of CDB-2914 in the medical treatment of uterine myomas. Acknowledgments The authors wish to thank Dr. Andre´ Ulmann and Ms. Erin E Gainer of HRA Pharma (Paris, France) for kindly providing us with CDB-2914 and supporting our research. This work was supported in part by Grants-in-Aid for Scientific Research 1437053 from the Japanese Ministry of Education, Science and Culture and by the OgyaaDonation Foundation of the Japan Association of Obstetricians and Gynecologists. References [1] Maruo T, Matsuo H, Samoto T, Gao Z, Spitz IM, Johansson E. Effects of progesterone on uterine leiomyoma growth and apoptosis. Steroids 2000;65:585 – 92. [2] Maruo T, Laoag-Fernandez JB, Matsuo H, et al. Effects of levonorgestrel-releasing intrauterine system on the endometrium and the relevance to the management of menorrhagia caused by uterine myoma and adenomyosis. In: Maruo T, Barlow D, Mardon H, Kennedy S, editors. Cell and molecular biology of endometrium in health and disease. Osaka7 Soeisha; 2002. p. 193 – 207. [3] Maruo T, Laoag-Fernandez JB, Pakarinen P, Murakoshi H, Spitz IM, Johansson E. Effects of the levonorgestrel-releasing intrauterine system on proliferation and apoptosis in the endometrium. Hum Reprod 2001;16:2103 – 8. [4] Maruo T. Endometrial effects of levonorgestrel intrauterine delivery. Gynecol Forum 2006;11:27 – 30. [5] Shimomura Y, Matsuo H, Samoto T, Maruo T. Up-regulation by progesterone of proliferating cell number antigen and epidermal growth factor expression in human uterine leiomyoma. J Clin Endocrinol Metab 1998;83:2192 – 8. [6] Matsuo H, Maruo T, Samoto T. Increased expression of Bcl-2 protein in human uterine leiomyoma and its up-regulation by progesterone. J Clin Endocrinol Metab 1997;82:293 – 9. [7] Gao Z, Matsuo H, Wang Y, Nakago S, Maruo T. Up-regulation by IGF-I of proliferating cell nuclear antigen and Bcl-2 protein expression in
T. Maruo et al. / Contraception 75 (2007) S99 – S103 human uterine leiomyoma cells. J Clin Endocrinol Metab 2001; 86:5593 – 9. [8] Gao Z, Matsuo H, Nakago S, Kurachi O, Maruo T. p53 tumor suppressor protein content in human uterine leiomyomas and its down-regulation by 17 beta-estradiol. J Clin Endocrinol Metab 2002;87:3915 – 20. [9] Yamada T, Nakago S, Kurachi O, et al. Progesterone down-regulates insulin-like growth factor-I expression in cultured human uterine leiomyoma cells. Hum Reprod 2004;19:1 – 7. [10] Maruo T, Ohara N, Wang J, Matsuo H. Sex steroidal regulation of uterine leiomyoma growth and apoptosis. Hum Reprod Update 2004;10:207 – 20.
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[11] Maruo T, Matsuo H, Yamada T, et al. Effects of progesterone on growth factor expression in human uterine leiomyoma. Steroids 2003;68:817 – 24. [12] Xu Q, Takekida S, Ohara N, et al. Progesterone receptor modulator CDB-2914 down-regulates proliferative cell nuclear antigen and Bcl-2 protein expression and up-regulates caspase-3 and (adenosine 5Vdiphosphate-ribose) polymerase expression in cultured human uterine leiomyoma cells. J Clin Endocrinol Metab 2005;90:953 – 61. [13] Xu Q, Ohara N, Maruo T, et al. Progesterone receptor modulator CDB2914 down-regulates VEGF, adrenomedullin and their receptors and modulates progesterone receptor content in cultured human uterine leiomyoma cells. Hum Reprod 2006;21:2408 – 16.