Metformin is a potent inhibitor of endometrial cancer cell proliferation--implications for a novel treatment strategy

Abstract

Objectives: Obesity and diabetes are strong risk factors that drive the development of type I endometrial cancers. Recent epidemiological evidence suggests that metformin may lower cancer risk and reduce rates of cancer deaths among diabetic patients. In order to better understand metformin's anti-tumorigenic potential, our goal was to assess the effect of metformin on proliferation and expression of key targets of metformin cell signaling in endometrial cancer cell lines.

Methods: The endometrial cancer cell lines, ECC-1 and Ishikawa, were used. Cell proliferation was assessed after exposure to metformin. Cell cycle progression was evaluated by flow cytometry. Apoptosis was assessed by ELISA for caspase-3 activity. hTERT expression was determined by real-time RT-PCR. Western immunoblotting was performed to determine the expression of the downstream targets of metformin.

Results: Metformin potently inhibited growth in a dose-dependent manner in both cell lines (IC(50) of 1 mM). Treatment with metformin resulted in G1 arrest, induction of apoptosis and decreased hTERT expression. Western immunoblot analysis demonstrated that metformin induced phosphorylation of AMPK, its immediate downstream mediator, within 24 h of exposure. In parallel, treatment with metformin decreased phosphorylation of S6 protein, a key target of the mTOR pathway.

Conclusions: We find that metformin is a potent inhibitor of cell proliferation in endometrial cancer cell lines. This effect is partially mediated through AMPK activation and subsequent inhibition of the mTOR pathway. This work should provide the scientific foundation for future investigation of metformin as a strategy for endometrial cancer prevention and treatment.

Figure 1

Effect of metformin on proliferation of endometrial carcinoma cells. Ishikawa and ECC-1 cells were cultured in the presence of varying concentrations of metformin for 72 hours. Relative growth of cells were determined by MTT. The results are shown as the mean ± SE of triplicate samples and are representative of three independent experiments. (* indicates statistically significant difference)

Figure 2

Metformin inhibited cell cycle progression by arrest in G1 phase. The endometrial cancer cell lines, ECC-1 (A) and Ishikawa (B), were starved overnight and then stimulated with 15% serum and metformin at the noted concentrations for 36 hours. Cell cycle analysis was performed by flow cytometry. Results shown are representative of two independent experiments. (* indicates statistically significant difference)

Figure 3

Metformin induced apoptosis but only at high concentrations. The endometrial cancer cell lines (A) ECC-1 and (B) Ishikawa were grown for 24 hours and then treated with metformin at the indicated concentrations for an additional 24 hours. Apoptosis was assessed using an antibody to caspase-3. The results are shown as the mean +/- SD and are representative of two independent experiments. (* indicates statistically significant difference)

Figure 4

Metformin decreased hTERT mRNA expression in a dose-dependent manner in the (A) ECC-1 and (B) Ishikawa cell lines. Both cell lines were cultured for 24 hours and then treated with the indicated concentrations of metformin for an additional 24 hours. hTERT expression was determined by real-time RT-PCR. The results are shown as the mean ± SE of two independent experiments. (* indicates statistically significant difference)

Figure 5

In both endometrial cancer cell Lines ECC-1 (A & C) and Ishikawa (B & D), metformin increased phosphorylation of AMPK and decreased phosphorylation of S6 in a dose-dependent manner within 16 hours of exposure as determined by Western immunoblotting. Little effect was seen on total AMPK (Pan-AMPK) or total S6 (Pan-S6). Both cell lines expressed wild-type PTEN (E).

Figure 6

Interaction between the metformin and mTOR pathways.