Effect of metformin on the human T98G glioblastoma multiforme cell line

Abstract

Metformin is a guanidine derivative found in Galega officinalis that is commonly used to treat diabetes mellitus. The mechanism of action of metformin involves regulation of the adenosine monophosphate-activated protein kinase/mammalian target of rapamycin signaling pathway, which is implicated in the control of protein synthesis and cell proliferation. This led to the hypothesis that metformin reduces the risk of cancer and slows tumor growth. Thus, in the present study, the effectiveness of metformin as an antiglioma agent was evaluated using the human T98G glioblastoma multiforme cell line. The viability of the T98G cells was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was monitored by measuring caspase-3 levels, as well as by terminal deoxynucleotidyl transferase dUTP nick end labeling and staining with acridine orange and ethidium bromide. The results demonstrate that metformin reduced cell viability and caused apoptotic morphological changes in the T98G cells. Furthermore, the caspase-3 levels in the metformin-treated T98G cells were higher than those in the control cells. Metformin induced apoptosis in the T98G cell line in a concentration-dependent manner. Metformin may provide an important contribution to the treatment of glioblastoma multiforme.

Keywords: T98G; apoptosis; glioblastoma cell line; metformin.

Figure 1

MTT assay viability results in T98G cells. Viability of the T98G cells in the 24 h presence of (A) metformin (1, 5, 10, 50 and 100 mM) and (B) metformin (1, 5, 10, 50 and 100 mM) + 2.5 mM H₂O₂ combination. All data are expressed as the mean and SE from three independent experiments. ^*P<0.001 vs. control, ^#P<0.001 vs. 2.5 mM H₂O₂. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 2

Fold increase in caspase 3 activity in T98G cells. (A) Metformin (1, 5, 10, 50 and 100 mM) and (B) metformin (1, 5, 10, 50 and 100 mM) + 2.5 mM H₂O₂ combination. All data are expressed as the mean and SE from three independent experiments. ^*P<0.001 vs. control, ^#P<0.001 vs. 2.5 mM H₂O₂.

Figure 3

Ethidium bromide and acridine orange fluorescein dye in the T98G cell line (magnification ×400). Apoptotic cells are indicated with an arrowhead. (A) Control, (B) 2.5 mM H₂O₂, (C) 1 mM metformin, (D) 1 mM metformin + 2.5 mM H₂O₂, (E) 5 mM metformin, (F) 5 mM metformin + 2.5 mM H₂O₂, (G) 10 mM metformin, (H) 10 mM metformin + 2.5 mM H₂O₂, (I) 50 mM metformin, (J) 50 mM metformin + 2.5 mM H₂O₂, (K) 100 mM metformin, (L) 100 mM metformin + 2.5 mM H₂O₂.

Figure 4

Apoptosis by TUNEL assay of the T98G cell line (magnification ×400). Apoptotic cells are indicated with an arrowhead. (A) Control, (B) 2.5 mM H₂O₂, (C) 1 mM metformin, (D) 1 mM metformin + 2.5 mM H₂O₂, (E) 5 mM metformin, (F) 5 mM metformin + 2.5 mM H₂O₂, (G) 10 mM metformin, (H) 10 mM metformin + 2.5 mM H₂O₂, (I) 50 mM metformin, (J) 50 mM metformin + 2.5 mM H₂O₂, (K) 100 mM metformin, (L) 100 mM metformin + 2.5 mM H₂O₂. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.