Metformin Increases Exosome Biogenesis and Secretion in U87 MG Human Glioblastoma Cells: A Possible Mechanism of Therapeutic Resistance

Abstract

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor. Metformin, an anti-diabetic drug, can suppress tumor cells. Exosomes from GBM cells contribute to intercellular communication, tumor aggressiveness, and therapeutic resistance. We studied the effect of metformin on the exosomal secretory pathway in U87MGcells. Cell survival against metformin was investigated using MTT assay. Expression of miRNA-21, miRNA-155, and miRNA-182, as well as the genes involved in exosome biogenesis and secretion such as Rab27a, Rab27b, Rab11, CD63, and Alix were calculated by real time-PCR. The expression of CD63 protein was analyzed by western blotting, while the subcellular distribution of CD63 protein was monitored by flow cytometry. Exosomes were characterized by transmission and scanning electron microscopes, and flow cytometry. Amount of exosomes was assayed using acetylcholinesterase activity assay and ELISA. The expression of autophagic markers LC3 and P62 were assessed using ELISA. Data showed that metformin decreased cell survival and expression of miRNA-21, miRNA-155, and miRNA-182 (p <0.05). Expression of Rab27a, Rab27b, Rab11, CD63, and Alix as well as protein level of CD63 up-regulated in treated cells (p <0.05). Concurrently, flow cytometry analysis showed that surface CD63/total CD63 ratio was increased in treated cells (p <0.05). We found that acetylcholinesterase activity and CD63 protein of exosomes from treated cells increased (p <0.05). The expression of LC3 and P62 was not affected by metformin (p >0.05). Data indicates metformin could promote exosome biogenesis and secretion in U87MGcells, proposing the therapeutic response against metformin.