Research ArticleExperimental Studies
Open Access

Metformin Inhibits the Estrogen-mediated Epithelial-Mesenchymal Transition of Ectopic Endometrial Stromal Cells in Endometriosis

YUNKAI XIE, WEIMIN KONG, XIAOLING ZHAO, DAN LUO, SHUNING CHEN, HE ZHANG and YULIANG RAN
In Vivo November 2023, 37 (6) 2490-2497; DOI: https://doi.org/10.21873/invivo.13356

Abstract

Background/Aim: Endometriosis is an estrogen-dependent disease characterized by the ectopic implantation and growth of endometrial tissue outside the uterus. Endometrial stromal cells (ESCs) play a crucial role in the pathogenesis of endometriosis. Epithelial-mesenchymal transition (EMT) has recently been described in endometriosis and was induced by estrogen. Metformin has been shown to inhibit EMT in various diseases, but its role in endometriosis remains unclear. Materials and Methods: We collected endometrial tissue samples from patients with endometriosis and healthy controls and isolated primary ESCs. We performed gene expression analysis using the Gene Expression Omnibus (GEO) dataset and validated the results by immunohistochemistry in tissue samples. We also assessed the effects of metformin on the proliferation, migration and invasion of ectopic ESCs (EESCs) by Cell Counting Kit-8 and Transwell migration and invasion assays, respectively. We analyzed the protein expression of EMT-related markers (N-cadherin, vimentin, twist, and snail) and β-catenin by Western blotting and immunohistochemistry. Results: We found that vimentin was highly expressed in ectopic endometrial tissues compared to normal endometrial tissues. Metformin treatment inhibited the proliferation, migration and invasion of EESCs in a dose-dependent manner. Metformin treatment also downregulated the expression of EMT-related markers and reduced the expression and nuclear translocation of β-catenin in EESCs. Conclusion: Our results suggest that metformin inhibits estrogen-induced EMT and regulates the expression of β-catenin in EESCs. This study provides new insights into the potential therapeutic role of metformin in endometriosis.

Key Words:

Endometriosis (EMs) is a chronic estrogen-dependent disease characterized by the ectopic implantation and growth of endometrial tissues outside the uterus, resulting in pelvic pain and infertility in women of reproductive age (1). Approximately 10% of women of reproductive age suffer from EMs, and it is estimated that there are at least 190 million women with EMs worldwide, with an increasing trend (2, 3). Approximately 25-50% of infertile patients have endometriosis, while 30-50% of patients with endometriosis experience infertility or subfertility (4). Retrograde menstruation is considered the most likely cause of endometriosis. It refers to the backward flow of menstrual blood containing endometrial cells through the fallopian tubes into the peritoneal cavity, where they implant and grow on pelvic structures (5). However, the implantation processes of ectopic endometrial tissue are complex and may involve hormonal and immunological factors, the peritoneal environment, genetic and epigenetic processes, inflammation, and angiogenesis (6-9).

Ectopic endometrial stromal cells (EESCs) play a key role in the invasion of endometriosis. Studies have shown that the migration and invasion of EESCs may be the first step in the invasion process of the retrograde implantation theory (10, 11). Endometriosis is also considered to be a hormone-dependent disease closely related to steroid metabolism and related pathways (12, 13). 17β-Estradiol (E2) is important in endometriosis; it can promote the growth of endometriosis tissue and the invasion of EESCs by various mechanisms (14-17). EMT plays an important role in the pathogenesis of endometriosis. It is characterized by the loss of epithelial cell markers and the gain of mesenchymal cell markers (such as N-cadherin and vimentin), which confer increased invasiveness and motility to the cells (18).

Metformin is an antidiabetic agent used in type 2 diabetes mellitus (T2DM) and has been shown to inhibit the invasion and migration of a variety of tumor cells through the AMPK and EMT signaling pathways (19). In endometrial cancer, metformin has been reported to suppress EMT in in vitro and in vivo studies (20, 21). In addition, metformin has been found to inhibit the growth of estrogen-dependent endometrial cancer cells (22). It has also been considered as a potential treatment option for endometriosis and has been shown to have an inhibitory effect on endometriosis both in vitro and in rat models (23, 24). In a clinical trial, women with endometriosis showed a reduction in dysmenorrhea, pelvic pain and dyspareunia, and an increase in the percentage of pregnancies after treatment with metformin (25). The Wnt/β-catenin signaling pathway has been shown to regulate the migration and invasion of various types of tumor cells (26-29). However, the effect of metformin on Wnt/β-catenin pathway in endometriosis is still unclear. Therefore, the current study investigated the effects of high-concentration metformin on the proliferation and invasion of ESCs and whether these effects were related to the Wnt/β-catenin pathway.

Materials and Methods

Tissue collection. Ectopic endometrial tissues were obtained from the cystic walls of ovarian endometriomas of 12 women with endometriosis (mean age=35.17±2.205 years) and normal endometrial tissues were obtained from 11 women of reproductive age (mean age=36.82±2.26 years) without endometriosis undergoing hysteroscopy. All enrolled women underwent the procedure during the early proliferative phase of their menstrual cycle. The diagnosis was confirmed by histologic examination and malignant cases were excluded. Each participant provided informed consent before undergoing biopsy, and human tissues were used with the approval of the institutional review board of Beijing Obstetrics and Gynecology Hospital affiliated to Capital Medical University (2019-KY-002-01).

Primary cell culture. Fresh ectopic endometrial tissues (n=12) were washed 3 times with PBS and minced with micro scissors. The tissues were then digested with type II collagenase (1 mg/ml, Solarbio, Beijing, PR China) for 60 min and then filtered through 100 μm and 40 μm pore size nylon meshes to isolate EESCs. Isolated EESCs were cultured in DMEM/F12 (1:1) (Biosharp, Anhui, Hefei, PR China) containing 10% fetal bovine serum (FBS; Corning, Corning, NY, USA), 100 U/ml penicillin (Biosharp), and 100 U/ml streptomycin (Biosharp) in a cell incubator at 37°C with 5% CO2. Metformin (MCE, Monmouth Junction, NJ, USA) and 17β-Estradiol (MCE) were respectively added to the culture medium in different experiments.

Cell Counting Kit-8 (CCK-8) assay. Cells were resuspended and 100 μl of cell suspension containing 104 cells was added to each well of a 96-well plate, with five replicate wells per plate. Then, 10 μl of Cell Counting Kit-8 solution (MCE) was added to each well. The plates were incubated for 60 min at 37°C in a 5% CO2 incubator. The absorbance of each well was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

Bioinformatics analysis. Two endometriosis datasets (GSE7305, GSE23339) were obtained from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/). We used the R package inSilicoMerging (https://bioconductor.org/packages/3.0/bioc/html/inSilicoMerging.html) to merge and normalize the datasets. We performed a t-test to analyze the expression of genes associated with EMT. We used Gene Set Enrichment Analysis (GSEA) software (GSEA, version 3.0) to compare the biological pathways between the endometriosis and control groups. We predefined a set of pathway genes most closely related to EMT as gene sets to assess pathway gene enrichment.

Immunohistochemistry (IHC). Ectopic endometrial tissues (n=12) and normal endometrial tissues (n=11) were collected and fixed in 4% paraformaldehyde, then embedded in paraffin. IHC analysis was performed following the manufacturer’s instructions (ZSGB-BIO, Beijing, PR China). The slides were deparaffinized in xylene and rehydrated in graded ethanol. Antigen retrieval was performed with 1 mM EDTA buffer (pH=9.0), and endogenous peroxidase activity was blocked with 3% H2O2. Tissues were incubated overnight at 4°C with Vimentin antibody (1:500, CST, #5741). Hematoxylin was used for counterstaining. Five random fields of view were observed under a light microscope, and two experienced gynecologic pathologists evaluated the samples using IHC scores (30).

Transwell migration and invasion assay. For the migration assay, 5×104 cells were seeded each into a 24-well transwell chamber (8 μm pore size) (Thermo Scientific, Waltham, MA, USA, #140629). For the invasion assay, the chambers were coated with Matrigel (Corning) and seeded with 5×104 cells in each chamber. The EESCs were cultured for 72 h for migration and invasion. Cells that did not penetrate the filter were wiped off, and those on the lower surface of the filter membrane were stained with 0.4% crystal violet. The number of migrating or invading cells was counted in a single chamber of three samples under five 10x microscopic fields of view (mean±standard deviation).

qRT-PCR analysis. Total RNA was extracted from EESCs using Trizol reagent (Thermo Scientific). The first strand of cDNA was synthesized from 1 μg of total RNA using the Reverse Transcription System Kit (Thermo Scientific). mRNA levels were quantified using KAPA SYBR FAST UNI qPCR Kits (Kapa Biosystems, Boston, MA, USA, # KK4601) and detected using the QuantStudio™5 Real-Time PCR System (Thermo Scientific). Actin expression was used as internal control and each assay was conducted in triplicate. The primer sequences were as following: Actin, forward, 5′-CATGTAC GTTGCTATCCAGGC-3′, reverse, 5′-CTCCTTAATGTCACGCA CGAT-3′; CTNNB1, forward, 5′-AAAGCGGCTGTTAGTCACT GG-3′, reverse, 5′-CGAGTCATTGCATACTGTCCAT-3′. ESR2, forward, 5′-AGCACGGCTCCATATACATACC-3′, reverse, 5′-TGGACC ACTAAAGGAGAAAGGT-3′.

Western blot analysis. Whole-cell protein was extracted from EESCs using RIPA lysis buffer (Beyotime, Shanghai, PR China) containing protein phosphatase inhibitors (Thermo Scientific) and protease inhibitors (Thermo Scientific). The cytosolic and nuclear fractions of cells were lysed using an Extract Kit for Nuclear and Cytoplasmic Protein Extraction (Beyotime) to obtain nuclear proteins. Protein concentrations were measured using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Proteins were separated by electrophoresis on 10% polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore). The PVDF membranes were blocked with 5% fat-free milk and then incubated with primary antibodies against Actin (1:1,000, CST, Boston, MA, USA, #3700), Lamin-B1 (1:1,000, CST, #9087), Vimentin (1:1,000, CST, #5741), N-Cadherin (1:1,000, CST, #13116), Twist (1:1,000, CST, #90445), Snail (1:1,000, #3879) and β-catenin (1:1,000, CST, #8480). After washing with TBST buffer, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:10,000, CST). Protein bands were detected using an ECL detection kit (Thermo Scientific).

Immunofluorescence assay. EESCs were seeded onto cover glasses in a 6-well plate and incubated for 8 h at 37°C, 5% CO2. After treatment with 17β-estradiol and/or metformin for 72 h, cells were fixed with 4% paraformaldehyde for 15 mins and then permeabilized with 0.5% Triton X-100 for 20 mins at room temperature. Cells were washed with PBS and blocked with 2% bovine serum albumin for 30 mins before being incubated with primary antibodies against β-catenin and Vimentin overnight at 4°C. ells were then incubated with fluorochrome-conjugated anti-rabbit IgG (1:1,000; CST, #4412) for 60 min at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and fluorescent images were captured using a fluorescence microscope.

Statistical analysis. Continuous variables are expressed as mean±SEM. Statistical charts were generated, and analyses were conducted using GraphPad Prism 7.0 (GraphPad Software, Boston, MA, USA) and SPSS 22.0 (IBM, Armonk, NY, USA) software. Unpaired t-tests, one-way ANOVA, and two-way ANOVA were used to determine statistical significance, and Tukey’s post hoc test was used to determine significance. All experiments were independently repeated three times and p-values <0.05 were considered significant.

Results

Ectopic endometrial tissue expresses high levels of EMT-related genes. To investigate the expression levels of EMT-related genes in endometriosis tissues, we firstly analyzed two data sets (GSE7305, GSE23339) of the GEO database. In two datasets containing a total of 18 endometriosis tissue samples and 17 normal endometrial tissue samples, the mesenchymal markers Vimentin (VIM, p<0.001), MMP9 (MMP9, p<0.01), and Slug (SNAI2, p<0.05) were highly expressed, and the epithelial marker, E-Cadherin (CDH1, p<0.0001) was lowly expressed in endometriosis tissues (Figure 1A). We further performed IHC staining for the Vimentin protein in clinical tissue samples. IHC staining showed a higher Vimentin expression in the endometriosis group (n=12) than the control group (n=11) (7.73±1.81 vs. 5.55±2.39, p=0.03) (Figure 1B). These results indicate that ectopic endometrial cells in endometriosis exhibit a more pronounced mesenchymal phenotype than normal endometrial cells.

Figure 1.
Figure 1.

Ectopic endometrial tissue expresses high levels of EMT-related genes. (A) The analysis of EMT-related gene expression in endometriosis and normal endometrium from GEO database. (B): IHC analysis of vimentin expression in ectopic endometrial tissue and normal endometrial tissue. Scale bars represent 50 μm. *p<0.05.

Metformin inhibits the proliferation, migration, and invasion of EESCs induced by estrogen. To explore the effects of estrogen and metformin on EESCs, 17 β-estradiol (10−10 M, 10−8 M) and metformin (1 mM, 5 mM) were used to treat EESCs for 96 h. Results showed there were effects of estrogen and metformin on the proliferation of EESCs in a dose-dependent manner (Figure 2A-C). High concentrations (10−8 M) of estradiol could promote the proliferation of EESCs after 72 h of treatment (Figure 2A). High dose of metformin (5 mM) significantly inhibited cell proliferation of EESCs after treatment of 72 h (Figure 2B). When two drugs act simultaneously on cells, metformin (5 mM) can inhibit the estrogen-induced proliferative activity of EESCs cells (Figure 2C). Transwell assays (Figure 2D) showed that 72 h of estrogen (10-8 M) treatment enhanced the migration and invasion of EESCs, whereas metformin (5 mM) treatment for 72 h could antagonize the effects of estrogen and inhibit the migration and invasion of EESCs.

Figure 2.
Figure 2.

Metformin inhibits estrogen-induced proliferation, migration, and invasion of EESCs and decreases the expression of EMT-related proteins. (A-C) The cell proliferation ability was detected by the CCK8 assay. (D) Transwell assay results after cells were treated with metformin (5 mM) and/or estrogen (10−8 M) for 72 h. (E) Eutopic and ectopic EESCs were treated with 17β-Estradiol and/or metformin for 72 h and subjected to immunofluorescence staining for Vimentin. (F) Expression levels of EMT markers (N-Cad, Vimentin, Twist, and Snail) were detected in the EESCs in the different treatment groups. Data are presented as the mean±standard deviation and analyzed using one-way and two-way ANOVA. Tukey’s post hoc test was used to determine significance. *p<0.05, **p<0.01 and ****p<0.0001.

The expressions of EMT-related proteins in EESCs decreased after metformin treatment. To investigate the effects of estrogen and metformin on EMT-related proteins in EESCs, the EESCs were then treated with estrogen and metformin for 72 h. Immunofluorescence experiments revealed that estrogen treatment enhanced Vimentin expression in EESCs, while metformin treatment reduced Vimentin expression (Figure 2E). In further studies, the expression levels of EMT-related proteins in EESCs were evaluated using western blot. As shown in Figure 2F, metformin treatment induced a significant decrease in N-Cadherin and Vimentin in EESCs compared with control and estrogen-treated groups. The expression of transcription factor proteins Twist and Snail were also decreased in the metformin treated group compared to the control and estrogen treated group, according to immunoblotting assays.

Metformin decreases β-catenin expression and nuclear translocation. Estrogen acts mainly through estrogen receptor beta (ER-β), which is abnormally highly expressed in EESCs, to promote the growth of ectopic endometrial tissue in endometriosis (31). To further investigate the mechanism of the effect of metformin on EESC, we first examined the mRNA levels of ESR2 in EESCs with qRT-PCR analysis. The results showed that metformin treatment did not affect the expression of estrogen receptor β (Figure 3A). To investigate the mechanism by which metformin affects EMT in EESCs, we performed a gene set enrichment analysis (GSEA) of the dataset from the GEO database by customizing the gene set of EMT-related pathways (including WNT, SMAD, PI3K-AKT, TGFB1 and NOTCH signaling pathways). We found that among the pathways closely related to EMT, the WNT signaling pathway was the most enriched in genes (NES=−1.37, FDR=0.0877) (Figure 3B). To investigate the effect of metformin and estrogen on β-catenin expression and action in EESCs, we performed a qRT-PCR analysis to investigate the mRNA levels of CTNNB1 in EESCs of different treatment groups. The result showed that the mRNA levels of EESCs were significantly decreased in Met-treated groups compared with control and estrogen-treated groups (Figure 3C). Western-blot showed that the levels of β-catenin in the nucleus of EESCs were significantly decreased in Met-treated groups and Met+E2-treated groups (Figure 3E). Immunofluorescence assays proved that metformin treatment significantly decreased the nuclear localization of β-catenin in ESCs (Figure 3D). These results showed that metformin may influence EMT in EESCs by regulating the expression and nuclear translocation of β-catenin.

Figure 3.
Figure 3.

Metformin decreases β-catenin expression and nuclear translocation. (A) mRNA levels of ESR2 were determined in the cells in the different treatment groups. (B) Gene set enrichment analysis in endometriosis of pathway genes most closely related to EMT. (C) mRNA levels of CTNNB1 were determined in the cells in the different treatment groups. (D) Immunofluorescence staining for β-catenin of EESCs in the different treatment groups. (E) Cells were fractionated into the cytosol and nucleus, and the expression levels of total and nuclear β-catenin were detected in the ESCs in the different treatment groups. Scale bars represent 100 μm. Data are presented as the mean±standard deviation and analyzed using one-way ANOVA. Tukey’s post hoc test was used to determine significance. **p<0.01 and ****p<0.0001.

Discussion

Endometrial fragments flow through the fallopian tubes into the pelvic cavity with the menstrual flow during menstruation, where they are able to implant and develop (32). Cells that dispersed into the pelvis during menstruation include epithelial, stromal, vascular, and immune, where the abundant stromal cells play a key role in the initial phases of tissue adhesion, invasion, and proliferation (33). Besides, it has been shown that under estrogen treatment conditions, EESCs had different epigenetic changes compared to normal stromal cells (34). In our study, we observed increased expression of mesenchymal markers in ectopic endometrial tissue compared with normal endometrium, suggesting that ectopic endometrial cells have increased invasive potential.

Estrogens play a crucial role in the pathogenesis of endometriosis (35). Several genes involved in the EMT pathway are differentially expressed in EESCs and estrogen can induce EMT in endometriosis through various mechanisms (36-38). A previous study reported that estrogen upregulated β-catenin expression in EESCs and facilitated their EMT (39). Similarly, our study found that estrogen promoted the migration and invasion of EESCs and induce the expression of β-catenin without affecting estrogen receptor expression. Those results were consistent with the antagonistic effect of metformin on estrogen action in many estrogen-dependent tumors (40, 41).

β-catenin is a key nuclear effector of canonical Wnt/β-catenin signaling. Nuclear accumulation of β-catenin promotes transcription of EMT-promoting genes (42, 43). In endometriosis, silencing of β-catenin was shown to abolish estrogen-induced EMT in both endometrial epithelial cells and EESCs (36). Our study found a decrease in both total and nuclear β-catenin at the protein expression level. Those results suggest that the inhibitory effect of metformin on EESCs was linked to the decrease of β-catenin in the nucleus.

Conclusion

In conclusion, our study revealed, for the first time, the effect of metformin on EMT of EESCs and found that metformin inhibited the proliferation, invasion, and metastasis of EESCs, which was antagonistic to estrogen. Furthermore, we showed that metformin can reduce the nuclear translocation of β-catenin, a key mediator of EMT, suggesting that metformin exerts its effect through modulating β-catenin signaling. Our findings indicate that metformin may have therapeutic potential for endometriosis patients.

Acknowledgements

This work was supported by The National Natural Science Foundation of China (No.81871142).

Footnotes

  • Authors’ Contributions

    WMK and YLR designed the study. YKX and XLZ performed experiments and collected the data. DL, SNC and HZ assisted in drafting and revising the manuscript. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare that they have no competing interests in relation to this study.

  • Received July 17, 2023.
  • Revision received August 22, 2023.
  • Accepted August 30, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. Giudice LC
    : Clinical practice. Endometriosis. N Engl J Med 362(25): 2389-2398, 2010. DOI: 10.1056/NEJMcp1000274
    OpenUrlCrossRefPubMed
  2. ↵
    1. Shafrir AL,
    2. Farland LV,
    3. Shah DK,
    4. Harris HR,
    5. Kvaskoff M,
    6. Zondervan K,
    7. Missmer SA
    : Risk for and consequences of endometriosis: A critical epidemiologic review. Best Pract Res Clin Obstet Gynaecol 51: 1-15, 2018. DOI: 10.1016/j.bpobgyn.2018.06.001
    OpenUrlCrossRefPubMed
  3. ↵
    1. Signorile PG,
    2. Cassano M,
    3. Viceconte R,
    4. Marcattilj V,
    5. Baldi A
    : Endometriosis: a retrospective analysis of clinical data from a cohort of 4,083 patients, with focus on symptoms. In Vivo 36(2): 874-883, 2022. DOI: 10.21873/invivo.12776
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Gruber TM,
    2. Mechsner S
    : Pathogenesis of endometriosis: The origin of pain and subfertility. Cells 10(6): 1381, 2021. DOI: 10.3390/cells10061381
    OpenUrlCrossRef
  5. ↵
    1. Peiris AN,
    2. Chaljub E,
    3. Medlock D
    : Endometriosis. JAMA 320(24): 2608, 2018. DOI: 10.1001/jama.2018.17953
    OpenUrlCrossRef
  6. ↵
    1. Porpora MG,
    2. Scaramuzzino S,
    3. Sangiuliano C,
    4. Piacenti I,
    5. Bonanni V,
    6. Piccioni MG,
    7. Ostuni R,
    8. Masciullo L,
    9. Benedetti Panici PL
    : High prevalence of autoimmune diseases in women with endometriosis: a case-control study. Gynecol Endocrinol 36(4): 356-359, 2020. DOI: 10.1080/09513590.2019.1655727
    OpenUrlCrossRef
    1. Young VJ,
    2. Brown JK,
    3. Saunders PT,
    4. Horne AW
    : The role of the peritoneum in the pathogenesis of endometriosis. Hum Reprod Update 19(5): 558-569, 2013. DOI: 10.1093/humupd/dmt024
    OpenUrlCrossRefPubMed
    1. Lin YH,
    2. Chen YH,
    3. Chang HY,
    4. Au HK,
    5. Tzeng CR,
    6. Huang YH
    : Chronic niche inflammation in endometriosis-associated infertility: Current understanding and future therapeutic strategies. Int J Mol Sci 19(8): 2385, 2018. DOI: 10.3390/ijms19082385
    OpenUrlCrossRef
  9. ↵
    1. Greenbaum H,
    2. Weil C,
    3. Chodick G,
    4. Shalev V,
    5. Eisenberg VH
    : Evidence for an association between endometriosis, fibromyalgia, and autoimmune diseases. Am J Reprod Immunol 81(4): e13095, 2019. DOI: 10.1111/aji.13095
    OpenUrlCrossRef
  10. ↵
    1. Witz CA,
    2. Dechaud H,
    3. Montoya-Rodriguez IA,
    4. Thomas MR,
    5. Nair AS,
    6. Centonze VE,
    7. Schenken RS
    : An in vitro model to study the pathogenesis of the early endometriosis lesion. Ann N Y Acad Sci 955(1): 296-307, 2002. DOI: 10.1111/j.1749-6632.2002.tb02790.x
    OpenUrlCrossRefPubMed
  11. ↵
    1. Ferreira MC,
    2. Witz CA,
    3. Hammes LS,
    4. Kirma N,
    5. Petraglia F,
    6. Schenken RS,
    7. Reis FM
    : Activin A increases invasiveness of endometrial cells in an in vitro model of human peritoneum. Mol Hum Reprod 14(5): 301-307, 2008. DOI: 10.1093/molehr/gan016
    OpenUrlCrossRefPubMed
  12. ↵
    1. Greene AD,
    2. Lang SA,
    3. Kendziorski JA,
    4. Sroga-Rios JM,
    5. Herzog TJ,
    6. Burns KA
    : Endometriosis: where are we and where are we going? Reproduction 152(3): R63-R78, 2016. DOI: 10.1530/REP-16-0052
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Zhao Y,
    2. Gong P,
    3. Chen Y,
    4. Nwachukwu JC,
    5. Srinivasan S,
    6. Ko C,
    7. Bagchi MK,
    8. Taylor RN,
    9. Korach KS,
    10. Nettles KW,
    11. Katzenellenbogen JA,
    12. Katzenellenbogen BS
    : Dual suppression of estrogenic and inflammatory activities for targeting of endometriosis. Sci Transl Med 7(271): 271ra9, 2015. DOI: 10.1126/scitranslmed.3010626
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Han SJ,
    2. Lee JE,
    3. Cho YJ,
    4. Park MJ,
    5. O’Malley BW
    : Genomic function of estrogen receptor β in endometriosis. Endocrinology 160(11): 2495-2516, 2019. DOI: 10.1210/en.2019-00442
    OpenUrlCrossRef
    1. Monsivais D,
    2. Dyson MT,
    3. Yin P,
    4. Navarro A,
    5. Coon JS Th.,
    6. Pavone ME,
    7. Bulun SE
    : Estrogen receptor β regulates endometriotic cell survival through serum and glucocorticoid-regulated kinase activation. Fertil Steril 105(5): 1266-1273, 2016. DOI: 10.1016/j.fertnstert.2016.01.012
    OpenUrlCrossRef
    1. Tamura M,
    2. Deb S,
    3. Sebastian S,
    4. Okamura K,
    5. Bulun SE
    : Estrogen up-regulates cyclooxygenase-2 via estrogen receptor in human uterine microvascular endothelial cells. Fertil Steril 81(5): 1351-1356, 2004. DOI: 10.1016/j.fertnstert.2003.09.076
    OpenUrlCrossRefPubMed
  17. ↵
    1. Chantalat E,
    2. Valera MC,
    3. Vaysse C,
    4. Noirrit E,
    5. Rusidze M,
    6. Weyl A,
    7. Vergriete K,
    8. Buscail E,
    9. Lluel P,
    10. Fontaine C,
    11. Arnal JF,
    12. Lenfant F
    : Estrogen receptors and endometriosis. Int J Mol Sci 21(8): 2815, 2020. DOI: 10.3390/ijms21082815
    OpenUrlCrossRef
  18. ↵
    1. Konrad L,
    2. Dietze R,
    3. Riaz MA,
    4. Scheiner-Bobis G,
    5. Behnke J,
    6. Horné F,
    7. Hoerscher A,
    8. Reising C,
    9. Meinhold-Heerlein I
    : Epithelial-mesenchymal transition in endometriosis-when does it happen? J Clin Med 9(6): 1915, 2020. DOI: 10.3390/jcm9061915
    OpenUrlCrossRef
  19. ↵
    1. Chen YC,
    2. Li H,
    3. Wang J
    : Mechanisms of metformin inhibiting cancer invasion and migration. Am J Transl Res 12(9): 4885-4901, 2020.
    OpenUrl
  20. ↵
    1. Laskov I,
    2. Abou-Nader P,
    3. Amin O,
    4. Philip CA,
    5. Beauchamp MC,
    6. Yasmeen A,
    7. Gotlieb WH
    : Metformin increases E-cadherin in tumors of diabetic patients with endometrial cancer and suppresses epithelial-mesenchymal transition in endometrial cancer cell lines. Int J Gynecol Cancer 26(7): 1213-1221, 2016. DOI: 10.1097/IGC.0000000000000761
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Qiang P,
    2. Shao Y,
    3. Sun YP,
    4. Zhang J,
    5. Chen LJ
    : Metformin inhibits proliferation and migration of endometrial cancer cells through regulating pi3k/akt/mdm2 pathway. Eur Rev Med Pharmacol Sci 23(4): 1778-1785, 2019. DOI: 10.26355/eurrev_201902_17140
    OpenUrlCrossRef
  22. ↵
    1. Zou J,
    2. Hong L,
    3. Luo C,
    4. Li Z,
    5. Zhu Y,
    6. Huang T,
    7. Zhang Y,
    8. Yuan H,
    9. Hu Y,
    10. Wen T,
    11. Zhuang W,
    12. Cai B,
    13. Zhang X,
    14. Huang J,
    15. Cheng J
    : Metformin inhibits estrogen-dependent endometrial cancer cell growth by activating the AMPK-FOXO1 signal pathway. Cancer Sci 107(12): 1806-1817, 2016. DOI: 10.1111/cas.13083
    OpenUrlCrossRefPubMed
  23. ↵
    1. Yilmaz B,
    2. Sucak A,
    3. Kilic S,
    4. Aksakal O,
    5. Aksoy Y,
    6. Lortlar N,
    7. Sut N,
    8. Gungor T
    : Metformin regresses endometriotic implants in rats by improving implant levels of superoxide dismutase, vascular endothelial growth factor, tissue inhibitor of metalloproteinase-2, and matrix metalloproteinase-9. Am J Obstet Gynecol 202(4): 368.e1-368.e8, 2010. DOI: 10.1016/j.ajog.2009.10.873
    OpenUrlCrossRefPubMed
  24. ↵
    1. Zhou Y,
    2. Xu JN,
    3. Zeng C,
    4. Li X,
    5. Zhou YF,
    6. Qi Y,
    7. Xue Q
    : Metformin suppresses prostaglandin E2-induced cytochrome P450 aromatase gene expression and activity via stimulation of AMP-activated protein kinase in human endometriotic stromal cells. Reprod Sci 22(9): 1162-1170, 2015. DOI: 10.1177/1933719115590664
    OpenUrlCrossRefPubMed
  25. ↵
    1. Foda AA,
    2. Aal IAA
    : Metformin as a new therapy for endometriosis, its effects on both clinical picture and cytokines profile. Middle East Fertil Soc J 17(4): 262-267, 2012. DOI: 10.1016/j.mefs.2012.09.001
    OpenUrlCrossRef
  26. ↵
    1. Guo Q,
    2. Xu J,
    3. Huang Z,
    4. Yao Q,
    5. Chen F,
    6. Liu H,
    7. Zhang Z,
    8. Lin J
    : ADMA mediates gastric cancer cell migration and invasion via Wnt/β-catenin signaling pathway. Clin Transl Oncol 23(2): 325-334, 2021. DOI: 10.1007/s12094-020-02422-7
    OpenUrlCrossRef
    1. Wang J,
    2. Cai H,
    3. Liu Q,
    4. Xia Y,
    5. Xing L,
    6. Zuo Q,
    7. Zhang Y,
    8. Chen C,
    9. Xu K,
    10. Yin P,
    11. Chen T
    : Cinobufacini inhibits colon cancer invasion and metastasis via suppressing Wnt/β-catenin signaling pathway and EMT. Am J Chin Med 48(03): 703-718, 2020. DOI: 10.1142/S0192415X20500354
    OpenUrlCrossRef
    1. Tian S,
    2. Peng P,
    3. Li J,
    4. Deng H,
    5. Zhan N,
    6. Zeng Z,
    7. Dong W
    : SERPINH1 regulates EMT and gastric cancer metastasis via the Wnt/β-catenin signaling pathway. Aging (Albany NY) 12(4): 3574-3593, 2020. DOI: 10.18632/aging.102831
    OpenUrlCrossRef
  29. ↵
    1. Moon JH,
    2. Lee SH,
    3. Lim YC
    : Wnt/β-catenin/Slug pathway contributes to tumor invasion and lymph node metastasis in head and neck squamous cell carcinoma. Clin Exp Metastasis 38(2): 163-174, 2021. DOI: 10.1007/s10585-021-10081-3
    OpenUrlCrossRef
  30. ↵
    1. Russell-Goldman E,
    2. Hornick JL,
    3. Qian X,
    4. Jo VY
    : NKX2.2 immunohistochemistry in the distinction of Ewing sarcoma from cytomorphologic mimics: Diagnostic utility and pitfalls. Cancer Cytopathol 126(11): 942-949, 2018. DOI: 10.1002/cncy.22056
    OpenUrlCrossRef
  31. ↵
    1. Yilmaz BD,
    2. Bulun SE
    : Endometriosis and nuclear receptors. Hum Reprod Update 25(4): 473-485, 2019. DOI: 10.1093/humupd/dmz005
    OpenUrlCrossRef
  32. ↵
    1. Chapron C,
    2. Marcellin L,
    3. Borghese B,
    4. Santulli P
    : Rethinking mechanisms, diagnosis and management of endometriosis. Nat Rev Endocrinol 15(11): 666-682, 2019. DOI: 10.1038/s41574-019-0245-z
    OpenUrlCrossRefPubMed
  33. ↵
    1. Armstrong GM,
    2. Maybin JA,
    3. Murray AA,
    4. Nicol M,
    5. Walker C,
    6. Saunders PTK,
    7. Rossi AG,
    8. Critchley HOD
    : Endometrial apoptosis and neutrophil infiltration during menstruation exhibits spatial and temporal dynamics that are recapitulated in a mouse model. Sci Rep 7(1): 17416, 2017. DOI: 10.1038/s41598-017-17565-x
    OpenUrlCrossRef
  34. ↵
    1. Houshdaran S,
    2. Oke AB,
    3. Fung JC,
    4. Vo KC,
    5. Nezhat C,
    6. Giudice LC
    : Steroid hormones regulate genome-wide epigenetic programming and gene transcription in human endometrial cells with marked aberrancies in endometriosis. PLoS Genet 16(6): e1008601, 2020. DOI: 10.1371/journal.pgen.1008601
    OpenUrlCrossRef
  35. ↵
    1. Clemenza S,
    2. Vannuccini S,
    3. Ruotolo A,
    4. Capezzuoli T,
    5. Petraglia F
    : Advances in targeting estrogen synthesis and receptors in patients with endometriosis. Expert Opin Investig Drugs 31(11): 1227-1238, 2022. DOI: 10.1080/13543784.2022.2152325
    OpenUrlCrossRef
  36. ↵
    1. Xiong W,
    2. Zhang L,
    3. Liu H,
    4. Li N,
    5. Du Y,
    6. He H,
    7. Zhang Z,
    8. Liu Y
    : E(2) -mediated EMT by activation of β-catenin/Snail signalling during the development of ovarian endometriosis. J Cell Mol Med 23(12): 8035-8045, 2019. DOI: 10.1111/jcmm.14668
    OpenUrlCrossRef
    1. Liu HY,
    2. Zhu ZY,
    3. Chen XM,
    4. Lu JQ,
    5. Song Y,
    6. Xia W
    : A review of the effects of estrogen and epithelial-mesenchymal transformation on intrauterine adhesion and endometriosis. Transpl Immunol 79: 101679, 2023. DOI: 10.1016/j.trim.2022.101679
    OpenUrlCrossRef
  38. ↵
    1. Chen YJ,
    2. Li HY,
    3. Huang CH,
    4. Twu NF,
    5. Yen MS,
    6. Wang PH,
    7. Chou TY,
    8. Liu YN,
    9. Chao KC,
    10. Yang MH
    : Oestrogen-induced epithelial-mesenchymal transition of endometrial epithelial cells contributes to the development of adenomyosis. J Pathol 222(3): 261-270, 2010. DOI: 10.1002/path.2761
    OpenUrlCrossRefPubMed
  39. ↵
    1. Xiong W,
    2. Zhang L,
    3. Yu L,
    4. Xie W,
    5. Man Y,
    6. Xiong Y,
    7. Liu H,
    8. Liu Y
    : Estradiol promotes cells invasion by activating β-catenin signaling pathway in endometriosis. Reproduction 150(6): 507-516, 2015. DOI: 10.1530/REP-15-0371
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Zhang J,
    2. Xu H,
    3. Zhou X,
    4. Li Y,
    5. Liu T,
    6. Yin X,
    7. Zhang B
    : Role of metformin in inhibiting estrogen-induced proliferation and regulating ERα and ERβ expression in human endometrial cancer cells. Oncol Lett 14(4): 4949-4956, 2017. DOI: 10.3892/ol.2017.6877
    OpenUrlCrossRef
  41. ↵
    1. Campagnoli C,
    2. Berrino F,
    3. Venturelli E,
    4. Abbà C,
    5. Biglia N,
    6. Brucato T,
    7. Cogliati P,
    8. Danese S,
    9. Donadio M,
    10. Zito G,
    11. Pasanisi P
    : Metformin decreases circulating androgen and estrogen levels in nondiabetic women with breast cancer. Clin Breast Cancer 13(6): 433-438, 2013. DOI: 10.1016/j.clbc.2013.08.012
    OpenUrlCrossRefPubMed
  42. ↵
    1. Valenta T,
    2. Hausmann G,
    3. Basler K
    : The many faces and functions of β-catenin. EMBO J 31(12): 2714-2736, 2012. DOI: 10.1038/emboj.2012.150
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Ghahhari NM,
    2. Babashah S
    : Interplay between microRNAs and WNT/β-catenin signalling pathway regulates epithelial–mesenchymal transition in cancer. Eur J Cancer 51(12): 1638-1649, 2015. DOI: 10.1016/j.ejca.2015.04.021
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Metformin Inhibits the Estrogen-mediated Epithelial-Mesenchymal Transition of Ectopic Endometrial Stromal Cells in Endometriosis
YUNKAI XIE, WEIMIN KONG, XIAOLING ZHAO, DAN LUO, SHUNING CHEN, HE ZHANG, YULIANG RAN
In Vivo Nov 2023, 37 (6) 2490-2497; DOI: 10.21873/invivo.13356
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords