High-Dose Metformin Plus Temozolomide Shows Increased Anti-tumor Effects in Glioblastoma In Vitro and In Vivo Compared with Monotherapy

Abstract

Purpose: The purpose of the study is to investigate the efficacy of combined treatment with temozolomide (TMZ) and metformin for glioblastoma (GBM) in vitro and in vivo.

Materials and methods: We investigated the efficacy of combined treatment with TMZ and metformin using cell viability and apoptosis assays. A GBM orthotopic mice model was established by inoculation of 5×105 U87 cells and treatedwith metformin, TMZ, and the combination for 4weeks. Western blotting and immunofluorescence of tumor specimens were analyzed to investigate AMP-activated protein kinase (AMPK) and AKT pathway.

Results: The combination of TMZ and metformin showed higher cytotoxicity than single agents in U87, U251, and A172 cell lines. A combination of high-dose metformin and TMZ showed the highest apoptotic activity. The combination of TMZ and metformin enhanced AMPK phosphorylation and inhibited mammalian target of rapamycin phosphorylation, AKT phosphorylation, and p53 expression. The median survival of each group was 43.6, 55.2, 53.2, 65.2, and 71.3 days for control, metformin treatment (2 mg/25 g/day or 10 mg/25 g/day), TMZ treatment (15 mg/kg/day), combination treatment with low-dose metformin and TMZ, and combination treatment with high-dose metformin and TMZ, respectively. Expression of fatty acid synthase (FASN) was significantly decreased in tumor specimens treated with metformin and TMZ.

Conclusion: The combination of metformin and TMZ was superior to monotherapy using metformin or TMZ in terms of cell viability in vitro and survival in vivo. The combination of high-dose metformin and TMZ inhibited FASN expression in an orthotopic model. Inhibition of FASN might be a potential therapeutic target of GBM.

Keywords: Fatty acid synthase; Glioblastoma; Metformin; Temozolomide.

Fig. 1.

The cytotoxic effects of metformin (Met) and temozolomide (TMZ) on U87 (A), U251 (B), and A172 (C) cell lines. Cell viability was measured by MTT assay and cell viability (percentage) was determined by normalizing each group to the control. Values are presented as mean±standard error of mean (n=3). *p < 0.05.

Fig. 2.

Flow cytometry analysis of apoptosis and necrosis. The high-dose combination of metformin (Met; 20 mM) and temozolomide (TMZ; 500 μM) showed the highest apoptotic activity.

Fig. 3.

Combined treatment with metformin (Met) and temozolomide (TMZ) enhanced AMP-activated protein kinase (AMPK) phosphorylation and inhibited mammalian target of rapamycin (mTOR) phosphorylation, AKT phosphorylation, and p53 expression. PBS, phosphate buffered saline.

Fig. 4.

Survival of orthotopic mice is represented in a Kaplan-Meier plot. Met, metformin; TMZ, temozolomide.

Fig. 5.

(A) AMP-activated protein kinase (AMPK) immunohistochemistry showed strong positivity in tumor specimens treated with a combination of metformin (Met) and temozolomide (TMZ) (×200). Similarly, high immunofluorescence showed that the combined treatment increased nuclear accumulation of phosphorylated AMPK (p-AMPK). (B) 4',6- Diamidino-2-phenylindole (DAPI) staining was used to determine nuclear localization (×200).

Fig. 6.

Western blot analysis showed inhibition of fatty acid synthase (FASN) expression after combined treatment with high-dose metformin (Met) and temozolomide (TMZ).