Inhibition of glioblastoma tumorsphere by combination of 2-deoxyglucose and metformin

Abstract

Objective: The treatment failure of glioblastoma is thought to be the presence of refractory cancer cells. It has been suggested that deprivation of tumor bioenergetics by inhibition of multiple energy pathways could be an effective new therapeutic approach for various human tumors. However, the effectiveness of this idea has not been evaluated in glioblastoma tumorsphere (GBM TS) model. We hypothesized that the dual inhibition of glycolysis and oxidative phosphorylation could suppress GBM TS effectively.

Methods: We evaluated the effect of 2-deoxyglucose (2DG) or metformin each alone, and their combination in GBM TS model. The viability of GBM TS was tested in different conditions, and protein expression related to AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway were examined. The influence of combination treatment on cytotoxicity, stemness and invasion properties of GBM TSs was evaluated with sphere formation assay and 3-demensional culture system. Glucose metabolism was assessed with 18fluorodeoxyglucose positron emission tomography (PET) scan. Transcriptome assay was performed with gene set enrichment analysis. Lastly, in vivo efficacy tests were performed with mouse orthotopic xenograft model in different conditions.

Results: Viability of GBM TS was not decreased by any single or combination treatments of 2DG and metformin. Of note, mTOR-signaling proteins were down-regulated in AMPK-independent manner. Sphere formation was significantly suppressed with 2DG and metformin combination treatment and the proteins related to stemness were down-regulated. Invasion capacity of GBM TS was inhibited by combination treatment in the 3D invasion model assay. PET scan showed 18fluorodeoxyglucose uptake was decreased in the GBM TS treated with the combination of 2DG and metformin. Transcriptome gene set enrichment analysis showed subset of genes related to extracellular matrix and adhesion were negatively correlated when compared before and after combination treatment. Combination of 2DG and metformin prolonged survival of tumor bearing mice, and the tissue examination revealed decreased invasion of GBM TS.

Conclusion: The combination of 2DG and metformin did not show significant cytotoxicity for GBM TS. However, 2DG and metformin effectively decreased the stemness and invasion capacity of GBM TS, and showed potential survival benefit in mouse orthotopic xenograft models. We believe this dual inhibition of cellular bioenergetics would be worth expedited clinical evaluation in the treatment of glioblastoma patients by targeting GBM TS.