AG-1031 induced autophagic cell death and apoptosis in C6 glioma cells associated with Notch-1 signaling pathway.

Glioma is the most common primary brain tumor with high morbidity and poor prognosis. The effect of AG-1031, which is developed as an antineoplastic drug, on C6 glioma cells is still not clear. The aim of this research was to explore the effect of AG-1031 on C6 cells, and tried to find out its potential mechanism on cytotoxicity of C6 cells. The 3-(4,5-dimethylthiazol -2-yl) -2,5- diphenyltetrazolium bromide (MTT) assay showed that AG-1031 inhibited cell viability in a concentration-dependent manner, whereas 3-methyadenine (3-MA) reduced this effect. Results from hoechst 33342 staining and Western blot assay indicated that AG-1031 induced C6 cell apoptosis. Western blot assay presented that AG-1031 notably increased the LC3-II/LC3-I ratio and decreased the expression of P62. Besides, our results showed that bafilomycin A1 increased the expression of LC3-II in cells treated with AG-1031, which indicated that AG-1031 can increase autophagy in C6 cells. Meanwhile, Western blot assay showed that AG-1031 can inhibit Notch-1 signaling by testing relative protein expressions. The expression of the intracellular domain of Notch (NICD) also decreased according to immunofluorescence assay. Additionally, the activation of Notch-1 signaling alleviated AG-1031-induced autophagic cell death and apoptosis. Furthermore, phosphorylated Akt and its downstream effector mechanistic target of rapamycin (mTOR) were reduced with AG-1031. These results suggest that AG-1031 may induce autophagic cell death through the inhibition of Akt-mTOR signaling pathway by down-regulating Notch-1 signaling pathway and activating apoptosis in C6 cells via Notch-1 signaling, which develops a new target spot to treat glioma in the future.