Glucose promotes cell proliferation, glucose uptake and invasion in endometrial cancer cells via AMPK/mTOR/S6 and MAPK signaling

Abstract

Objectives: Obesity and diabetes are well-known risk factors for the development of endometrial cancer. A high rate of aerobic glycolysis represents a key mechanism by which endometrial cancer cells consume glucose as its primary energy source. The up-regulated glycolytic pathway is a common therapeutic target whose inhibition has implications for anti-tumor activity in cancer cells. This study aimed to investigate the effect of various concentrations of glucose on cell proliferation in endometrial cancer.

Methods: ECC-1 and Ishikawa cells were treated with low glucose (1mM), normal glucose (5mM) and high glucose (25mM), and cytotoxicity, apoptosis, cell cycle, adhesion/invasion, and changes of AMPK/mTOR/S6 and MAPK pathways were evaluated.

Results: Our results revealed that high glucose increased cell growth and clonogenicity in two endometrial cancer cell lines in a dose dependent manner. Low glucose induced the activity of cleaved caspase 3 and caused cell cycle G1 arrest. High glucose increased the ability of adhesion and invasion by decreasing E-cadherin and increasing Snail expression. In addition, high glucose increased glucose uptake and glycolytic activity through modulating the AMPK/mTOR/S6 and MAPK pathways.

Conclusions: Our findings suggest that glucose stimulated cell proliferation through multiple complex signaling pathways. Targeting glucose metabolism may be a promising therapeutic strategy in the treatment of endometrial cancer.

Keywords: Endometrial cancer; Glucose; Glycolysis; Invasion.

Figure 1. Glucose promotes cell proliferation in ECC-1 and Ishikawa cells

ECC-1 and Ishikawa cells were cultured for 24 hours and treated with no glucose (NO), low glucose (1 mM, LG), normal glucose (5 mM, NG) and high glucose (25 mM, HG) in 96-well plates for 48 hours (A) and 72 hours (B). Cell proliferation was assessed by MTT assay. The effect of glucose on long term growth in endometrial cancer cells was assessed through colony-forming assay (C and D). *p < 0.05 and **p < 0.01.

Figure 2. Low glucose induced apoptosis in endometrial cancer cells

The ECC-1 and Ishikawa cells were cultured in the presence of varying concentration of glucose (LG, NG and HG) in regular medium for 14 hours. Cleaved caspase-3 activity was determined by ELISA assay (A). The protein expression of BCL-2 and MCL-1 was detected by Western blotting in the both cells after exposure to glucose for 18 hours (B).

Figure 3. Low Glucose induced cell cycle G0/G1 arrest in endometrial cancer cells

ECC-1 and Ishikawa cells were cultured for 24 hours and then treated with different concentrations of glucose (LG, NG and HG) for 48 hours. Cell cycle analysis was performed by Cellomte. Low glucose induced cell cycle G0/G1 phase arrest in the both cells (A and B). The expression of CDK4 and CDK6 were examined by Western blotting in ECC-1 and Ishikawa cells after exposure to glucose for 24 hours at the indicated concentrations (C).

Figure 4. High glucose induced the ability of adhesion and invasion in endometrial cancer cells

ECC-1 and Ishikawa cells were treated with glucose as indicated in a laminin coated 96 well plates or BME coated 96 transwell plates for 12 hours. Adhesion and invasion were assessed using a plate reader. High glucose significantly increased the ability of adhesion and invasion in the both cells (A and B). (B). The expression of Snail and E-Cadherin were determined by Western blotting in the ECC-1 and Ishikawa cell lines after exposure to glucose for 24 hours (C). *p < 0.05 and **p < 0.01.

Figure 5. High glucose promoted the activity of glycolysis in endometrial cancer cells

ECC-1 and Ishikawa cells were treated with different concentrations of glucose for 12 hours. The levels of cellular glucose uptake were measured by 2-NBDG assay (A). The expression of GLUT1, phos-LDHA and PDH was analyzed using Western blotting (B). High glucose significantly increased glucose uptake, accompanied by an increase in the expression of GLUT1 and Phos-LDHA and a decrease in PDH.

Figure 6. Glucose affects the AMPK and AKT/mTOR/S6 pathways

ECC-1 and Ishikawa cells were treated with glucose as indicated in their regular media for 3, 6 and 12 hours. The expression of phosphorylated pS6, AKT and p42/44 was analyzed using western blotting (A). The phosphorylation of AMPK was detected by Western blotting after incubation of glucose for 12 hours in both cells (B).