Role of metformin in inhibiting estrogen‑induced proliferation and regulating ERα and ERβ expression in human endometrial cancer cells

Zhang,Jingbo; Xu,Hui; Zhou,Xueyan; Li,Yanyu; Liu,Tong; Yin,Xiaoxing; Zhang,Bei

doi:10.3892/ol.2017.6877

Role of metformin in inhibiting estrogen‑induced proliferation and regulating ERα and ERβ expression in human endometrial cancer cells

Authors:
- Jingbo Zhang
- Hui Xu
- Xueyan Zhou
- Yanyu Li
- Tong Liu
- Xiaoxing Yin
- Bei Zhang
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Affiliations: Department of Obstetrics and Gynecology, Xuzhou Central Hospital, Xuzhou, Jiangsu 221004, P.R. China, School of Pharmacy, Xuzhou Medical College, Xuzhou, Jiangsu 221004, P.R. China
Published online on: September 4, 2017 https://doi.org/10.3892/ol.2017.6877
Pages: 4949-4956

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Abstract

Diabetes mellitus (DM) is an important factor that contributes to the development of type I endometrial cancer (EC). Previous studies have demonstrated that metformin decreases mortality and risk of neoplasms in patients with DM. Since estrogen and estrogen receptor (ER) expression has been associated with the development of EC, the present study aimed to investigate the effects of metformin on cell proliferation and ER expression in EC cell lines that are sensitive to estrogen. The viability and proliferation of Ishikawa and HEC‑1‑A cells were measured following treatment with metformin and/or a 5' AMP‑activated protein kinase (AMPK) inhibitor (compound C) with or without treatment with estradiol (E2). In addition, the levels of ERα, ERβ, AMPK, ribosomal protein S6 kinase β‑1 (p70S6K), myc proto‑oncogene protein (c‑myc) and proto‑oncogene c‑fos (c‑fos) were measured following treatment. Metformin significantly decreased E2‑stimulated cell proliferation; an effect that was rescued in the presence of compound C. Metformin treatment markedly increased the phosphorylation of AMPK while decreasing p70S6K phosphorylation, indicating that metformin exerts its effects through stimulation of AMPK and subsequent inhibition of the mammalian target of rapamycin (mTOR) signaling pathway. In addition, metformin significantly inhibited ERα expression while increasing ERβ expression, whereas treatment with compound C reversed these effects. Reverse transcription‑quantitative polymerase chain reaction analysis demonstrated that c‑fos and c‑myc expression were attenuated by metformin, an effect that was rescued in the presence of compound C. Therefore, metformin regulates the expression of ERs, and inhibits estrogen‑mediated proliferation of human EC cells through the activation of AMPK and subsequent inhibition of the mTOR signaling pathway.

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1	Fader AN, Arriba LN, Frasure HE and von Gruenigen VE: Endometrial cancer and obesity: Epidemiology, biomarkers, prevention and survivorship. Gynecol Oncol. 114:121–127. 2009. View Article : Google Scholar : PubMed/NCBI
2	Rižner T: Estrogen biosynthesis, phase I and phase II metabolism, and action in endometrial cancer. Mol Cell Endocrinol. 381:124–139. 2013. View Article : Google Scholar : PubMed/NCBI
3	Klinge CM: Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res. 29:2905–2919. 2001. View Article : Google Scholar : PubMed/NCBI
4	Sakaguchi H, Fujimoto J, Aoki I, Toyoki H, Khatun S and Tamaya T: Expression of oestrogen receptor alpha and beta in uterine endometrial and ovarian cancers. Eur J Cancer. 38 Suppl 6:S74–S75. 2002. View Article : Google Scholar : PubMed/NCBI
5	Utsunomiya H, Suzuki T, Harada N, Ito K, Matsuzaki S, Konno R, Sato S, Yajima A and Sasano H: Analysis of estrogen receptor alpha and beta in endometrial carcinomas: Correlation with ER beta and clinicopathologic findings in 45 cases. Int J Gynecol Pathol. 19:335–341. 2000. View Article : Google Scholar : PubMed/NCBI
6	Hu K, Zhong G and He F: Expression of estrogen receptors ERalpha and ERbeta in endometrial hyperplasia and adenocarcinoma. Int J Gynecol Cancer. 15:537–541. 2005. View Article : Google Scholar : PubMed/NCBI
7	Saegusa M and Okayasu I: Changes in expression of estrogen receptors alpha and beta in relation to progesterone receptor and pS2 status in normal and malignant endometrium. Jpn J Cancer Res. 91:510–518. 2000. View Article : Google Scholar : PubMed/NCBI
8	Bolton JL and Thatcher GR: Potential mechanisms of estrogen quinone carcinogenesis. Chem Res Toxicol. 21:93–101. 2008. View Article : Google Scholar : PubMed/NCBI
9	Hammes SR and Levin ER: Minireview: Recent advances in extranuclear steroid receptor actions. Endocrinology. 152:4489–4495. 2011. View Article : Google Scholar : PubMed/NCBI
10	Prossnitz ER and Barton M: The G-protein-coupled estrogen receptor GPER in health and disease. Nat Rev Endocrinol. 7:715–726. 2011. View Article : Google Scholar : PubMed/NCBI
11	Yamashita S, Takayanagi A and Shimizu N: Temporal and cell-type specific expression of c-fos and c-jun protooncogenes in the mouse uterus after estrogen stimulation. Endocrinology. 137:5468–5475. 1996. View Article : Google Scholar : PubMed/NCBI
12	Bjørge T, Lukanova A, Jonsson H, Tretli S, Ulmer H, Manjer J, Stocks T, Selmer R, Nagel G, Almquist M, et al: Metabolic syndrome and breast cancer in the me-can (metabolic syndrome and cancer) project. Cancer Epidemiol Biomarkers Prev. 19:1737–1745. 2010. View Article : Google Scholar : PubMed/NCBI
13	Del Barco S, Vazquez-Martin A, Cufi S, Oliveras-Ferraros C, Bosch-Barrera J, Joven J, Martin-Castillo B and Menendez JA: Metformin: Multi-faceted protection against cancer. Oncotarget. 2:896–917. 2011. View Article : Google Scholar : PubMed/NCBI
14	Dowling RJ, Goodwin PJ and Stambolic V: Understanding the benefit of metformin use in cancer treatment. BMC Med. 9:332011. View Article : Google Scholar : PubMed/NCBI
15	Rocha GZ, Dias MM, Ropelle ER, Osório-Costa F, Rossato FA, Vercesi AE, Saad MJ and Carvalheira JB: Metformin amplifies chemotherapy-induced AMPK activation and antitumoral growth. Clin Cancer Res. 17:3993–4005. 2011. View Article : Google Scholar : PubMed/NCBI
16	Cantrell LA, Zhou C, Mendivil A, Malloy KM, Gehrig PA and Bae-Jump VL: Metformin is a potent inhibitor of endometrial cancer cell proliferation-implications for a novel treatment strategy. Gynecol Oncol. 116:92–98. 2010. View Article : Google Scholar : PubMed/NCBI
17	Zhang Q, Celestino J, Schmandt R, McCampbell AS, Urbauer DL, Meyer LA, Burzawa JK, Huang M, Yates MS, Iglesias D, et al: Chemopreventive effects of metformin on obesity-associated endometrial proliferation. Am J Obstet Gynecol. 209:24.e1–24.e12. 2013. View Article : Google Scholar
18	Dowling RJ, Zakikhani M, Fantus IG, Pollak M and Sonenberg N: Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res. 67:10804–10812. 2007. View Article : Google Scholar : PubMed/NCBI
19	Viollet B, Guigas B, Garcia Sanz N, Leclerc J, Foretz M and Andreelli F: Cellular and molecular mechanisms of metformin: An overview. Clin Sci (Lond). 122:253–270. 2012. View Article : Google Scholar : PubMed/NCBI
20	Esteva FJ, Moulder SL, Gonzalez-Angulo AM, Ensor J, Murray JL, Green MC, Koenig KB, Lee MH, Hortobagyi GN and Yeung SC: Phase I trial of exemestane in combination with metformin and rosiglitazone in nondiabetic obese postmenopausal women with hormone receptor-positive metastatic breast cancer. Cancer Chemother Pharmacol. 71:63–72. 2013. View Article : Google Scholar : PubMed/NCBI
21	Campagnoli C, Berrino F, Venturelli E, Abbà C, Biglia N, Brucato T, Cogliati P, Danese S, Donadio M, Zito G and Pasanisi P: Metformin decreases circulating androgen and estrogen levels in nondiabetic women with breast cancer. Clin Breast Cancer. 13:433–438. 2013. View Article : Google Scholar : PubMed/NCBI
22	Markowska A, Pawałowska M, Filas V, Korski K, Gryboś M, Sajdak S, Olejek A, Bednarek W, Spiewankiewicz B, Lubin J and Markowska J: Does Metformin affect ER, PR, IGF-1R, β-catenin and PAX-2 expression in women with diabetes mellitus and endometrial cancer? Diabetol Metab Syndr. 5:762013. View Article : Google Scholar : PubMed/NCBI
23	Xie Y, Wang YL, Yu L, Hu Q, Ji L, Zhang Y and Liao QP: Metformin promotes progesterone receptor expression via inhibition of mammalian target of rapamycin (mTOR) in endometrial cancer cells. J Steroid Biochem Mol Biol. 126:113–120. 2011. View Article : Google Scholar : PubMed/NCBI
24	Zhang J, Zhang B, Yin Z, Chen F, Liu T, Xu H, Liu Y and Zhou X: Effects of metformin on the estrogen-induced proliferation and the expression of ER in human endometrial cancer cells. Zhonghua Fu Chan Ke Za Zhi. 49:932–937. 2014.(In Chinese). PubMed/NCBI
25	Emerling BM, Viollet B, Tormos KV and Chandel NS: Compound C inhibits hypoxic activation of HIF-1 independent of AMPK. FEBS Lett. 581:5727–5731. 2007. View Article : Google Scholar : PubMed/NCBI
26	Jefferies HB, Fumagalli S, Dennis PB, Reinhard C, Pearson RB and Thomas G: Rapamycin suppresses 5′TOP mRNA translation through inhibition of p70s6k. EMBO J. 16:3693–3704. 1997. View Article : Google Scholar : PubMed/NCBI
27	Ellenson LH and Wu TC: Focus on endometrial and cervical cancer. Cancer Cell. 5:533–538. 2004. View Article : Google Scholar : PubMed/NCBI
28	Farnell YZ and Ing NH: The effects of estradiol and selective estrogen receptor modulators on gene expression and messenger RNA stability in immortalized sheep endometrial stromal cells and human endometrial adenocarcinoma cells. J Steroid Biochem Mol Biol. 84:453–461. 2003. View Article : Google Scholar : PubMed/NCBI
29	Soliman PT, Wu D, Tortolero-Luna G, Schmeler KM, Slomovitz BM, Bray MS, Gershenson DM and Lu KH: Association between adiponectin, insulin resistance, and endometrial cancer. Cancer. 106:2376–2381. 2006. View Article : Google Scholar : PubMed/NCBI
30	Zakikhani M, Dowling RJ, Sonenberg N and Pollak MN: The effects of adiponectin and metformin on prostate and colon neoplasia involve activation of AMP-activated protein kinase. Cancer Prev Res (Phila). 1:369–375. 2008. View Article : Google Scholar : PubMed/NCBI
31	Gotlieb WH, Saumet J, Beauchamp MC, Gu J, Lau S, Pollak MN and Bruchim I: In vitro metformin anti-neoplastic activity in epithelial ovarian cancer. Gynecol Oncol. 110:246–250. 2008. View Article : Google Scholar : PubMed/NCBI
32	Terry KL and Missmer SA: Epidemiology of ovarian and endometrial cancers. Pathol Epidemiol Cancer. 2017. View Article : Google Scholar
33	Zhao H, Jiang Y, Liu Y, Yun C and Li L: Endogenous estrogen metabolites as biomarkers for endometrial cancer via a novel method of liquid chromatography-mass spectrometry with hollow fiber liquid-phase microextraction. Horm Metab Res. 47:158–164. 2015.PubMed/NCBI
34	Patel SM, Iqbal N, Kaul S, Ratcliffe SJ, Rickels MR, Reilly MP, Scattergood T, Basu A, Fuller C and Cappola AR: The effects of metformin and leuprolide acetate on insulin resistance and testosterone levels in non-diabetic postmenopausal women: A randomized, placebo-controlled trial. Fertil Steril. 94:2161–2166. 2010. View Article : Google Scholar : PubMed/NCBI
35	Brown KA, Hunger NI, Docanto M and Simpson ER: Metformin inhibits aromatase expression in human breast adipose stromal cells via stimulation of AMP-activated protein kinase. Breast Cancer Res Treat. 123:591–596. 2010. View Article : Google Scholar : PubMed/NCBI
36	Erdemoglu E, Güney M, Giray SG, Take G and Mungan T: Effects of metformin on mammalian target of rapamycin in a mouse model of endometrial hyperplasia. Eur J Obstet Gynecol Reprod Biol. 145:195–199. 2009. View Article : Google Scholar : PubMed/NCBI
37	Burns KA and Korach KS: Estrogen receptors and human disease: An update. Arch Toxicol. 86:1491–1504. 2012. View Article : Google Scholar : PubMed/NCBI
38	Ström A, Hartman J, Foster JS, Kietz S, Wimalasena J and Gustafsson JA: Estrogen receptor beta inhibits 17beta-estradiol-stimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad Sci USA. 101:1566–1571. 2004. View Article : Google Scholar : PubMed/NCBI
39	Helguero LA, Faulds MH, Gustafsson JA and Haldosén LA: Estrogen receptors alfa (ERalpha) and beta (ERbeta) differentially regulate proliferation and apoptosis of the normal murine mammary epithelial cell line HC11. Oncogene. 24:6605–6616. 2005. View Article : Google Scholar : PubMed/NCBI
40	Paruthiyil S, Parmar H, Kerekatte V, Cunha GR, Firestone GL and Leitman DC: Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest. Cancer Res. 64:423–428. 2004. View Article : Google Scholar : PubMed/NCBI
41	Grober OM, Mutarelli M, Giurato G, Ravo M, Cicatiello L, De Filippo MR, Ferraro L, Nassa G, Papa MF, Paris O, et al: Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation. BMC Genomics. 12:362011. View Article : Google Scholar : PubMed/NCBI
42	Matthews J and Gustafsson JA: Estrogen signaling: A subtle balance between ER alpha and ER beta. Mol Interv. 3:281–292. 2003. View Article : Google Scholar : PubMed/NCBI
43	Ali SH, O'Donnell AL, Balu D, Pohl MB, Seyler MJ, Mohamed S, Mousa S and Dandona P: Estrogen receptor-alpha in the inhibition of cancer growth and angiogenesis. Cancer Res. 60:7094–7098. 2000.PubMed/NCBI
44	Ali SH, O'Donnell AL, Balu D, Pohl MB, Seyler MJ, Mohamed S, Mousa S and Dandona P: High levels of oestrogen receptor-alpha in tumorigenesis: Inhibition of cell growth and angiogenic factors. Cell Prolif. 34:223–231. 2001. View Article : Google Scholar : PubMed/NCBI
45	Loose-Mitchell DS, Chiappetta C and Stancel GM: Estrogen regulation of c-fos messenger ribonucleic acid. Mol Endocrinol. 2:946–951. 1988. View Article : Google Scholar : PubMed/NCBI
46	Murphy LJ, Murphy LC and Friesen HG: Estrogen induction of N-myc and c-myc proto-oncogene expression in the rat uterus. Endocrinology. 120:1882–1888. 1987. View Article : Google Scholar : PubMed/NCBI
47	Weisz A, Cicatiello L, Persico E, Scalona M and Bresciani F: Estrogen stimulates transcription of c-jun protooncogene. Mol Endocrinol. 4:1041–1050. 1990. View Article : Google Scholar : PubMed/NCBI
48	Akinyeke T, Matsumura S, Wang X, Wu Y, Schalfer ED, Saxena A, Yan W, Logan SK and Li X: Metformin targets c-MYC oncogene to prevent prostate cancer. Carcinogenesis. 34:2823–2832. 2013. View Article : Google Scholar : PubMed/NCBI
49	Blandino G, Valerio M, Cioce M, Mori F, Casadei L, Pulito C, Sacconi A, Biagioni F, Cortese G, Galanti S, et al: Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC. Nat Commun. 3:8652012. View Article : Google Scholar : PubMed/NCBI
50	Dang CV: MYC on the path to cancer. Cell. 149:22–35. 2012. View Article : Google Scholar : PubMed/NCBI