European Journal of Obstetrics & Gynecology and Reproductive Biology
Effects of metformin on mammalian target of rapamycin in a mouse model of endometrial hyperplasia
Introduction
Rapamycin has been in use as an anti-rejection drug for over 10 years and research into its mode of action has identified a kinase, well-conserved in eukaryotic organisms, named “mammalian target of rapamycin” (mTOR). mTOR-S6K1 signaling has a key role in cellular growth and proliferation and it is at the crossroads of nutrient hormonal signaling [1], [2], [3]. mTOR is activated by two stimuli: nutrient/energy availability and mitogens that activate PI3K/Akt pathway [2]. Active PI3-kinase localizes to the cell membrane and catalyzes the conversion of phosphatidylinositol (4,5)-biphosphate [PtdIns(4,5)P2] into PtdIns(3,4,5)P3, whose concentrations are regulated negatively by the tumor suppressor PTEN. PtdIns(3,4,5)P3 recruits and activates Akt, which, in turn, phosphorylates and inactivates the tuberous sclerosis protein complex (TSC), which is a heterodimer composed of TSC1 and TSC2 [1], [3].
The best characterized downstream effectors of mTOR are the ribosomal protein S6 kinases (S6K) and 4E-BP1 [1]. S6K1 is phosphorylated directly by mTOR during stimulation with either nutrients or growth factors. mTOR complex phosphorylates S6K1 and regulates ribosomal protein synthesis. S6K1 activation causes increased protein synthesis for growth. Inhibition of mTOR by depletion of nutrients, absence of growth factors, activation of AMP-activated protein kinase (AMPK) or by specific mTOR inhibitors downregulates ribosomal synthesis of protein for growth and stimulates autophagy [4], [5], [6].
Metformin is an oral biguanide used in diabetes, insulin resistance and polycystic ovarian syndrome [7]. Metformin may decrease neoplastic proliferation by attenuating the increased insulin receptor activation associated with a high-energy diet as well as increased phosphorylation of AMP kinase [8]. Effects of metformin on mTOR may be explained by the activation of AMPK, which regulates cellular energy metabolism [7], [9]. There are a few studies evaluating the effect of metformin on endometrium and these reports are mainly based on the effect of metformin on endometrial receptivity [10], but not on endometrial hyperplasia. To the authors’ knowledge (Pubmed search 1950–2008; search items “mTOR”, “metformin”, “endometrium”, and/or “endometrial hyperplasia”) there has been no study evaluating the preventive effect of metformin on endometrial hyperplasia by inhibiting mTOR signaling. On the basis of these findings, we hypothesized that metformin might block the mTOR cascade and might lead to prevention of endometrial hyperplasia. To test this hypothesis, the effects of metformin on mTOR mediated S6K1 activation and relation to endometrial hyperplasia were examined in a mouse model of endometrial hyperplasia induced by unopposed estradiol or tamoxifen.
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Materials and methods
Before the study, approval of the experimental protocols was obtained from the local ethical committee at Süleyman Demirel University. Forty-eight 10-week-old Balb/c mice of Harlan (Harlan Europe Company, The Netherlands) were allowed to acclimatize for 7 days. Mice were housed in a controlled environment at 23 ± 2 °C on an illumination schedule of 12 h of light and 12 h of darkness each day. Mice were fed standard food and tap water ad libitum. After acclimatization, all mice underwent midline
Results
The densities of glands in the control group and metformin treated group were 29.8 ± 5.5/unit area and 30.2 ± 7.9/unit area, respectively (p > 0.05). Mice treated either with tamoxifen (54.4 ± 5.1/unit area) or estradiol (90 ± 5.1/unit area) had significantly increased density of glands compared to vehicle-only or metformin-only groups (p < 0.001). Addition of metformin either to tamoxifen or estradiol treated mice significantly decreased the density of glands. The density of glands in mice treated with
Discussion
Despite advances in identifying cellular pathways of proliferation, drugs targeting these molecular pathways have not been studied well. Endometrial hyperplasia is a common disorder which may progress to endometrial intraepithelial neoplasia and endometrial cancer [14]. PI3/Akt pathway leading to mTOR activation might be the main physiological key to this proliferative disorder [14], [15]. PI/AKT-mTOR cascade is also the subject of PTEN which is frequently inactivated in human cancers [15], [16]
Condensation
Metformin treated mice had important changes in S6K1 activation and in endometrial hyperplasia model induced by estradiol or Tamoxifen.
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