Metformin inhibits leptin-induced growth and migration of glioblastoma cells

Abstract

Metformin, a derivative of biguanide, is a first-line therapy for type 2 diabetes mellitus. Since the drug has been shown to significantly reduce the risk of various cancers and cancer mortality in diabetic patients, it is being considered as a potential anticancer therapeutic or preventive agent. In cellular models, metformin inhibits the growth of many types of cancer cells; however, its effects on glioblastoma multi-forme (GBM) are not well characterized. Here, we analyzed the effects of metformin on the growth and migration of LN18 and LN229 GBM cells cultured under basal conditions or exposed to leptin, a cytokine that has recently been implicated in GBM development. We found that 2-16 mM metformin reduced basal and leptin-stimulated growth of GBM cells in a dose-dependent manner. Furthermore, the drug significantly inhibited the migration of GBM cells. The action of metformin was mediated through the upregulation of its main signaling molecule, the adenosine monophosphate-activated protein kinase (AMPK), as well as through the downregulation of the signal transducer and activator of transcription 3 (STAT3) and the Akt/PKB serine/threonine protein kinase. In leptin-treated cells, the drug reversed the effects of the cytokine on the AMPK and STAT3 pathways, but modulated Akt activity in a cell-dependent manner. Our results suggest that metformin or similar AMPK-targeting agents with optimized blood-brain-barrier penetrability could be developed as potential treatments of GBM and could be used in conjunction with other target drugs such as leptin receptor antagonists.

Figures

Figure 1

Effects of metformin on the normal growth of glioblastoma multiforme (GBM) cells. Cell growth in the presence or absence of 0–16 mM concentrations of metformin was assessed as described in Materials and methods. The number of cells in untreated cultures is taken as 100%. Differences between untreated and treated cells (± SE) were calculated as described in Materials and methods; *p≤0.05; **p≤0.01.

Figure 2

Effects of metformin on the leptin-dependent growth of glioblastoma multiforme (GBM) cells. Cell growth in the presence or absence of 200 ng/ml leptin and/or 8 or 16 mM metformin was assessed as described in Materials and methods. The number of cells in untreated cultures is taken as 100%. Differences between untreated and leptin-treated cells, and leptin-treated vs. leptin plus metformin (± SE) were calculated as described in Materials and methods and are indicated by connecting lines; *p≤0.05; **p≤0.01.

Figure 3

Effects of leptin and metformin on glioblastoma multiforme (GBM) cell migration. Cell migration in the presence or absence of 200 ng/ml leptin and/or 8 or 16 mM metformin was assessed as described in Materials and methods. The number of cells in untreated cultures is taken as 100%. Differences between untreated and leptin-treated or metformin-treated cells, and leptin-treated vs. leptin plus metformin (± SE) were calculated as described in Materials and methods and are indicated by connecting lines; *p≤0.05; **p≤0.01.

Figure 4

Effects of leptin and metformin on intracellular signaling in glioblastoma multiforme cells. The cells were pretreated or not with 16 mM metformin and stimulated or not with 200 ng/ml leptin for 0–60 min, as described in Materials and methods. The levels of phosphorylated (p) and total AMPK, STAT3, and Akt proteins, and control protein GAPDH were assessed by western blot analysis using specific antibodies, as detailed in Materials and methods.