Long-term metformin effect on endometrial cancer 2 development depending on glucose environment in 3 vitro 4

Background: The incidence of endometrial cancer has increased worldwide over the past years. 15 Common risk factors include obesity and metabolic disturbances, like hyperinsulinemia and insulin 16 resistance, as well as prolonged and elevated estrogen exposure. Metformin, an anti-hyperglycemic 17 and insulin-sensitizing biguanide, displayed anti-proliferative effects in recent studies. Therefore, 18 metformin may act as a therapeutic and prophylactic anti-cancer agent in several tissues, including 19 endometrium. Methods: Two different endometrial cancer cell lines, reflecting type I (Ishikawa) 20 and type II endometrial cancer (HEC-1A) were cultured under normoglycemic (5.5mM) or 21 hyperglycemic (17.0mM) conditions and treated with different concentrations of metformin (0.01 – 22 5.0mM). Results: Effects of metformin on proliferation, cell viability, clonogenicity and migration 23 were investigated after treatment for 7d. Long-term treatment with metformin showed effects on 24 cellular viability, proliferation and migration of endometrial cancer cells in a concentration- 25 dependent manner in vitro . Additionally, glucose levels affected the outcome of the experiments. 26 Conclusion: Our in vitro findings support the hypothesis that metformin has a direct effect on 27 endometrial tissues and reflects the importance of the local glucose environment, suggesting that 28 metformin may be considered as a potential adjuvant agent in endometrial cancer therapy due to 29 its direct and indirect effects on endometrial development. 30

metformin could be effective as an adjuvant in cancer therapy along with its traditional role in the 48 treatment of type II diabetes [12][13][14][15][16][17]. However, most experimental studies analyzed metformin effects 49 at unphysiologically high concentrations (up to 100mM) during short-term treatment of 24-72h 50 [18,19]. We therefore believe that those effects described in the literature are related to cytotoxicity 51 rather than the desired anti-cancer effects of metformin [11]. Considering that the beneficial impact 52 of metformin in EC remains to be determined, this study investigated the direct effects of low 53 metformin concentrations (0.01-5.0mM) during long-term treatment (7d) on EC cell growth, viability, 54 clonogenicity and motility. Furthermore, cells were exposed to metformin in an environment with 55 normal (5.5mM, equivalent to 100mg/dL) or high (17.0mM, equivalent to 306mg/dL) glucose levels 56 to mimic a diabetic condition, in order to investigate the metformin effect within different metabolic 57 conditions. Furthermore, as increased estrogen levels are considered as an additional risk factor, ß-58 estradiol was added to the cell culture, a factor that was often omitted in prior studies [18,19].

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The underlying aim of this study was to investigate the potential direct effects of long-term

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During experiments, cells were maintained in a normoglycemic (NG) environment (5.5mM 73 glucose), representing physiological blood glucose levels of 100mg/dL. For the experiments under 74 hyperglycemic (HG) conditions, the medium was supplemented with glucose (Sigma-Aldrich) to 75 achieve a final concentration of 17.0mM glucose, equivalent to 306mg/dL as seen in diabetic patients.

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A 100mM stock solution of metformin (Sigma-Aldrich) was freshly prepared on the day of 77 administration in normo-or hyperglycemic culture medium, respectively, and cells were treated with 78 different concentrations of the drug (0.01-5.0mM) during experiments. Furthermore, cell culture 79 media were supplemented with 10nM ß-estradiol (E2; Sigma-Aldrich) during treatments to mimic 80 high estrogen levels, a common risk factor for EC development, in the experimental setting.

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The cells were seeded into 6-well plates at a density of 50,000 cells/well in a normal or high glucose, 121 drug-free medium for 24h. Afterwards, cells were treated with selected concentrations of metformin 122 (0.5 or 5.0mM) for 7d with medium changes every 2-3d. On day 7, confluent monolayers were 123 wounded using a sterile pipette tip and the medium was replaced by fresh medium to remove cellular 124 debris. Representative images were taken with an inverse light microscope (Leica, Munich, Germany) 125 at 40× magnification directly and 24h after wounding of the monolayer and the migration area A was 126 measured using the ImageJ software [21]. The % wound closure was calculated as follows: (At=0h -127 At=24h) / At=0h x 100%, where At=0h is the area of the wound measured immediately after scratching and

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At=24h is the area of the wound measured 24h after the scratch was performed. Measurements were 129 taken after 24h in order to limit the observations to migration rather than cellular proliferation [23].

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Moreover, hyperglycemia is associated with obesity and insulin resistance, leading to 292 hyperinsulinemia that stimulated cellular growth and hyperplasia in different tissues [28][29][30].

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Tumorigenesis of obesity-associated EC was linked to enhanced cellular glucose uptake and 294 increased metabolism [31], but can also be related to increased proliferation in vitro, as seen in the 295 present study between untreated control cells. Consequently, agents like metformin, that decrease

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Although studies evaluating the relationship between metformin and EC incidence revealed

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In conclusion, the present study highlighted the importance of the metabolic environment in EC 319 development and progression, and potential actions of metformin as a therapeutic agent in the 320 treatment for EC subtypes. Furthermore, it was shown that metformin acts on endometrial tissue via 321 direct effects, in addition to its well-known indirect effects, i.e. lowering serum glucose levels. These 322 findings suggest that the drug might be a potential adjuvant agent in EC therapy. However, further 323 studies are required to elucidate the role of metformin in EC prevention and treatment in more detail.