Restricted accessResearch articleFirst published online 2006-12
Estrogen Metabolite 2-Methoxyestradiol Induces Apoptosis and Inhibits Cell Proliferation and Collagen Production in Rat and Human Leiomyoma Cells: A Potential Medicinal Treatment for Uterine Fibroids
Objective:The current study sought to investigate the effect of the estrogen metabolite 2-methoxyestradiol (2-MeOHE2) on apoptosis, cell proliferation, and collagen synthesis in human and rat leiomyoma cells.
Methods:[3H] thymidine and [3H] proline incorporation studies were conducted. The expression of vascular endothelial growth factor (VEGF), cyclin D1, Bcl-2, and Bax were evaluated by Western blot. Flow cytometry analysis was used to study the effect of 2-MeOHE2 on apoptosis and the cell cycle.
Results:Compared with untreated controls, treatment of rat leiomyoma (ELT3) cells with 2-MeOHE2 (0.1, 1, 2, 5, or 10 μM) reduced cell proliferation by 17%, 52%, 61%, 73%, and 79%, respectively (P <.05). Similarly, in human uterine leiomyoma cell line (huLM) cells, proliferation was reduced by 4%, 18%, 37%, 41%, and 51%, respectively. 2-MeOHE2 also caused a concentration-dependent inhibition of collagen synthesis by 4%, 16%, 23%, 51%, and 70%, respectively, in huLM cells (P <.05). Cell cycle analysis indicated that 2-MeOHE2 treatment (1 to 5 μM) in huLM cells resulted in G2/M cell cycle arrest and a 45% increase in apoptosis compared with untreated control (P <.05). Western immunoblotting analysis indicated that 2-MeOHE2 induces a concentration-dependent reduction in the expression of cyclin D1, Bcl-2, and VEGF proteins in both rat and human leiomyoma cell lines.
Conclusions:This study provides the first evidence that 2-MeOHE2 is a potent antiproliferative/apoptotic and collagen synthesis inhibiting agent in human and rat leiomyoma cells. To the best of our knowledge, this is the first report showing the potential use of 2-methoxyestradiol as a nonsurgical alternative therapy for uterine leiomyomas.
1. Al-Hendy A, Lee EJ, Wang HQ, Copland JA. Gene therapy of uterine leiomyomas: Adenovirus-mediated expression of dominant negative estrogen receptor inhibits tumor growth in nude mice. Am J Obstet Gynecol2004;191:1621-1631.
2.
2. Walker CL, Stewart EA. Uterine fibroids: The elephant in the room. Science2005;308:1589-1592.
3.
3. Cook JD, Davis BJ, Cai SL, et al. Interaction between genetic susceptibility and early-life environmental exposure determines tumor-suppressor-gene penetrance. Proc Natl Acad Sci U S A2005;102:8644-8649.
4.
4. Maruo T, Ohara N, Wang J, Matsuo H. Sex steroidal regulation of uterine leiomyoma growth and apoptosis. Hum Reprod Update2004;10:207-220.
5.
5. Cook JD, Walker CL. Treatment strategies for uterine leiomyoma: The role of hormonal modulation. Semin Reprod Med2004;22:105-111.
6.
6. Martel KM, Ko AC, Christman GM, Stribley JM. Apoptosis in human uterine leiomyomas. Semin Reprod Med2004;22:91-103.
7.
7. Strober S, Lowsky RJ, Shizuru JA, Scandling JD, Millan MT. Approaches to transplantation tolerance in humans. Transplantation2004;77:932-936.
8.
8. Ishwad CS, Ferrell RE, Davare J, et al. Molecular and cytogenetic analysis of chromosome 7 in uterine leiomyomas. Genes Chromosomes Cancer1995;14:51-55.
9.
9. Bodner K, Bodner-Adler B, Kimberger O, Czerwenka K, Mayerhofer K. Bcl-2 receptor expression in patients with uterine smooth muscle tumors: An immunohistochemical analysis comparing leiomyoma, uterine smooth muscle tumor of uncertain malignant potential, and leiomyosarcoma. J Soc Gynecol Investig2004;11:187-191.
10.
10. Matsuo H. Molecular bases for the actions of ovarian sex steroids in the regulation of proliferation and apoptosis of human uterine leiomyoma. Oncology1999;57:49-58.
11.
11. Zhao J, Dorland M, Taverne MA, et al. In vitro culture of rat pre-antral follicles with emphasis on follicular interactions. Mol Reprod Dev2000;55:65-74.
12.
12. Kawaguchi K, Fujii S, konishi I, Okamura H, Mori T. Ultrastructural study of cultured smooth muscle cells from uterine leiomyoma and myometrium under the influence of sex steroids. Gynecol Oncol1985;21:32-41.
13.
13. Stewart EA, Friedman AJ, Peck K, Nowak RA. Relative overexpression of collagen type I and collagen type III messenger ribonucleic acids by uterine leiomyomas during the proliferative phase of the menstrual cycle. J Clin Endocrinol Metab1994;79:900-906.
14.
14. Zhu BT, Conney AH. Is 2-methoxyestradiol an endogenous estrogen metabolite that inhibits mammary carcinogenesis?Cancer Res1998;58:2269-2277.
15.
15. Lis A, Ciesielski MJ, Barone TA, et al. 2-Methoxyestradiol inhibits proliferation of normal and neoplastic glial cells, and induces cell death, in vitro. Cancer Lett2004;213:57-65.
16.
16. Joubert A, Maritz C, Joubert F. Bax/Bcl-2 exression levels of 2-methoxyestradiol-exposed esophageal cancer cells. Biomed Res2005;26:131-134.
17.
17. Chamon K, Stojek J, Kanakis D, et al. Micromolar concentrations of 2-methoxyestradiol kill glioma cells by an apoptotic mechanism, without destroying their microtubule cytoskeleton. J Neurooncol2005;72:11-16.
18.
18. Bu SZ, Huang Q, Jiang YM, et al. p38 Mitogen-activated protein kinases is required for counteraction of 2-methoxyestradiol to estradiol-stimulated cell proliferation and induction of apoptosis in ovarian carcinoma cells via phosphorylation Bcl-2. Apoptosis2006;11:413-425.
19.
19. Zacharia LC, Gogos JA, Karayiorgou M, et al. Methoxyestradiols mediate the antimitogenic effects of 17beta-estradiol: Direct evidence from catechol-O-methyltransferase-knockout mice. Circulation2003;108:2974-2978.
20.
20. Banerjee SN, Sengupta K, Banerjee S, Saxena NK, Banerjee SK. 2-Methoxyestradiol exhibits a biphasic effect on VEGF-A in tumor cells and upregulation is mediated through ER-alpha: A possible signaling pathway associated with the impact of 2-ME2 on proliferative cells. Neoplasia2003;5:417-426.
21.
21. Dubey RK, Jackson EK, Gillespie DG, et al. Clinically used estrogens differentially inhibit human aortic smooth muscle cell growth and mitogen-activated protein kinase activity. Arterioscler Thromb Vasc Biol2000;20:964-972.
22.
22. Mooberry SL. New insights into 2-methoxyestradiol, a promising antiangiogenic and antitumor agent. Curr Opin Oncol2003;15:425-430.
24. Sweeney C, Liu G, Yiannoutsos C, et al. A phase II multicenter, randomized, double-blind, safety trial assessing the pharmacokinetics, pharmacodynamics, and efficacy of oral 2-methoxyestradiol capsules in hormone-refractory prostate cancer. Clin Cancer Res2005;11:6625-6633.
25.
25. Howe SR, Gottardis MM, Everitt JI, et al. Rodent model of reproductive tract leiomyomata. Establishment and characterization of tumor-derived cell lines. Am J Pathol1995;146:1568-1579.
26.
26. Rauk PN, Surti U, Roberts JM, Michalopoulos, G. Mitogenic effect of basic fibroblast growth factor and estradiol on cultured human myometrial and leiomyoma cells. Am J Obstet Gynecol1995;173:571-577.
27.
27. Swartz CD, Afshari CA, Yu L, Hall KE, Dixon D. Estrogen-induced changes in IGF-I, Myb family and MAP kinase pathway genes in human uterine leiomyoma and normal uterine smooth muscle cell lines. Mol Hum Reprod2005;11:441-450.
28.
28. Teare JM, Islam R, Flanagan R, et al. Measurement of nucleic acid concentrations using the DyNA Quant and the GeneQuant. Biotechniques1997;22:1170-1174.
29.
29. Telford WG, King LE, Fraker PJ. Rapid quantitation of apoptosis in pure and heterogeneous cell population using flow cytometry. J Immunol Methods1994;172:1-16.
30.
30. Vijayanathan V, Venkiteswaran S, Nair SK, et al. Physiologic levels of 2-methoxyestradiol interfere with nongenomic signaling of 17beta-estradiol in human breast cancer cells. Clin Cancer Res2006;12:2038-2048.
31.
31. Schumacher G, Neuhaus P. [2-Methoxyestradiol—A new compound for cancer treatment]. Dtsch Med Wochenschr2006;131:825-830.
32.
32. Figg WD, Kruger EA, Price DK, Kim S, Dahut WD. Inhibition of angiogenesis: Treatment options for patients with metastatic prostate cancer. Invest New Drugs2002;20:183-194.
33.
33. Mabjeesh NJ, Escuin D, LaVallee TM, et al. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell2003;3:363-375.
34.
34. Gokmen-Polar Y, Escuin D, Walls CD, et al. beta-Tubulin mutations are associated with resistance to 2-methoxyestradiol in MDA-MB-435 cancer cells. Cancer Res2005;65:9406-9414.
35.
35. Qadan LR, Perez-Stable CM, Anderson C, et al. 2-Methoxyestradiol induces G2/M arrest and apoptosis in prostate cance. Biochem Biophys Res Commun2001;285:1259-1266.
36.
36. Barchiesi F, Jackson EK, Gillespie DG, et al. Methoxyestradiols mediate estradiol-induced antimitogenesis in human aortic SMCs. Hypertension2002;39:874-879.
37.
37. Lewis JS, Thomas YJ, Pestell RG, et al. Differential effects of 16alpha-hydroxyesterone and 2-methoxyestradiol on cyclin D1 involving the transcription factor ATF-2 in MCF-7 breast cancer cells. J Mod Endocrinol2005;34:91-105.
38.
38. Ghosh R, Ott AM, Seetharam D, Slaga TJ, Kumar AP. Cell cycle block and apoptosis induction in a human melanoma cell line following treatment with 2-methoxyoestradiol: Therapeutic implications?Melanoma Res2003;13:119-127.
39.
39. Wyllie AH, Kerr JF, Currie AR. Cell death: The significance of apoptosis. Int Rev Cytol1980;68:251-306.
40.
40. Reed JC, Talwar HS, Cuddy M, et al. Mitochondrial protein p26 BCL2 reduces growth factor requirements of NIH3T3 fibroblasts. Exp Cell Res1991;195:277-283.
41.
41. Nunez G, London L, Hockenbery D, et al. Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines. J Immunol1990;144:3602-3610.
42.
42. Zhai YL, Nikaido T, Toki T, et al. Prognostic sigificance of bcl-2 expression in leiomyosarcoma of the uterus. Br J Cancer1999;80:1658-1664.
43.
43. 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 Metab1997;82:293-299.
44.
44. Khurana KK, Singh SBTatum AH, Schulz V, Badawy SZ. Maintenance of increased Bcl-2 expression in uterine leiomyomas after GmRH agonist therapy. J Reprod Med1999;44:487-492.
45.
45. Di Lieto A, De Falco M, Pollio F, et al. Clinical response, vascular change, and angiogenesis in gonadotropin-releasing hormone analogue-treated women with uterine myomas. J Soc Gynecol Investig2005;12:123-128.
46.
46. Folkman J, Shing Y. Angiogenesis. J Biol Chem1992;267:10931-10934.
47.
47. Gentry CC, Okolo SO, Fong LF, et al. Quantification of vascular endothelial growth factor-A in leiomyomas and adjacent myometrium. Clin Sci (Lond)2001;101:691-695.
48.
48. Chauhan D, Catley L, Hideshima T, et al. 2-Methoxyestradiol overcomes drug resistance in multiple myeloma cells. Blood2002;100:2187-2194.
49.
49. Ferenczy A, Richart RM, Okagaki T. A comparative ultrastuctural study of leiomyosarcoma, cellular leiomyoma, and leiomyoma of the uterus. Cancer1971;28:1004-1018.
50.
50. Purohit A, Tutill HJ, Day JM, et al. The regulation and inhibition of 17beta-hydroxysteroid dehydrogenase in breast cancer. Molecular Cell Endocrinology2006;248:199-203.
