Changes in the expression of cancer- and metastasis-related genes and proteins after metformin treatment under different metabolic conditions in endometrial cancer cells

Abstract

Research question: Hyperinsulinemia and elevated estrogen levels are known risk factors for endometrial cancer (EC) development and are associated with obesity, type 2 diabetes mellitus (T2DM), insulin resistance, among others. Metformin, an insulin-sensitizing drug, displays anti-tumor effects in cancer patients, including EC, but the mechanism of action is still not completely understood. In the present study, the effects of metformin on gene and protein expression were investigated in pre- and postmenopausal EC in vitro models in order to identify candidates that are potentially involved in the drug's anti-cancer mechanism.

Design: After treating the cells with metformin (0.1 and 1.0 mmol/L), changes in the expression of >160 cancer- and metastasis-related gene transcripts were evaluated with RNA arrays. A total of 19 genes and 7 proteins were selected for a follow-up expression analysis, including further treatment conditions, in order to evaluate the influence of hyperinsulinemia and hyperglycemia on metformin-induced effects.

Results: Changes in the expression of BCL2L11, CDH1, CDKN1A, COL1A1, PTEN, MMP9 and TIMP2 were analyzed on gene and protein level. The consequences resulting from the detected expression changes as well as the influence of varying environmental influences are discussed in detail. With the presented data, we contribute to a better understanding of the direct anti-cancer activity of metformin as well as its underlying mechanism of action in EC cells.

Conclusions: Although further research will be necessary to confirm the data, the influence of different environmental settings on metformin-induced effects could be highlighted with the presented data. Additionally, gene and protein regulation were not similar in the pre- and postmenopausal in vitro models.

Keywords: Endometrial cancer; Hyperglycemia; Hyperinsulinemia; Metformin.

Graphical abstract

Fig. 1

Changes in the expression of >160 genes after treatment of HEC-1A and Ishikawa cells with metformin in a hyperglycemic environment for 7 d. Cells were treated with 1.0 mmol/L metformin under hyperglycemic conditions (17.0 mmol/L glucose) for 7 d and TaqMan RNA arrays were performed afterwards; n = 1. (a) Heat map of the changes in gene expression in the TaqMan “Human Molecular Mechanisms of Cancer” and “Human Tumor Metastasis” RNA arrays for HEC-1A and Ishikawa cells. (b) Networks of 19 genes that were selected for a more detailed follow-up qPCR analysis including further treatment conditions.

Fig. 2

Changes in the expression of selected genes after treatment of HEC-1A and Ishikawa cells with metformin, insulin, or a combination of both substances for 7 d. Cells were treated with 0.1 and 1.0 mmol/L metformin (green), 100 ng/mL insulin (red) or a combination of metformin and insulin (blue) under normo- (5.5 mmol/L glucose) or hyperglycemic conditions (17.0 mmol/L glucose) for 7 d and real-time PCR analysis was performed afterwards. Expression levels were calculated relative to the expression in untreated reference cells under normo- and hyperglycemic conditions (fold-changes were set to 1.0 for these samples as indicated by a dotted line) using the 2^–ΔΔCT method (fold-change). Data presented as dot plots with geometric means of at least three independent experiments; n = 4–7. A mixed effects model analysis was performed with the ΔC_T values followed by Tukey's (analysis of metformin and insulin effects under identical glucose conditions) or Šídák's (analysis of glucose effects between identical treatments; see Figure S1) multiple comparison post-hoc tests; *p ≤ 0.05, **p ≤ 0.01 (metformin effect, black); ^#p ≤ 0.05 (glucose effect, blue); p < 0.15 additionally displayed as values.

Fig. 3

Changes in the expression of selected proteins after treatment of HEC-1A and Ishikawa cells with metformin, insulin, or a combination of both substances for 7 d. (a) Cells were treated with 0.1 and 1.0 mmol/L metformin, 100 ng/mL insulin or a combination of metformin and insulin under normo- (5.5 mmol/L glucose, green) or hyperglycemic conditions (17.0 mmol/L glucose, red) for 7 d and western blotting was performed afterwards. Semi-quantitative, densitometric analysis was carried out in order to determine the relative normalized protein expression (fold-change). Untreated cells cultivated under normoglycemic conditions served as the reference group (fold-change was set to 1.0 for this sample as indicated by a dotted line). Data presented as dot plots with arithmetic means of at least three independent experiments; n = 5–7. Significant differences were determined with a mixed effects model analysis and subsequent Tukey's (analysis of metformin and insulin effects under identical glucose conditions) or Šídák's (analysis of glucose effects between identical treatments) multiple comparison post-hoc tests; *p ≤ 0.05, **p ≤ 0.01 (metformin effect, black); ^#p ≤ 0.05 (glucose effect, blue); p < 0.15 additionally displayed as values. (b) Representative blots are shown with transferred total protein (TP) used for normalization; BCL2L11: 23 kDa, CDH1: 135 kDa, CDKN1A: 21 kDa, COL1A1: 110 kDa, PTEN: 54 kDa, MMP9: 63 kDa, TIMP2: 22 kDa (Figure S3).