Autophagy induced by temozolomide combined with radiotherapy in human glioma U251 cells

Abstract

Objective To investigate the autophagy and related mechanisms of human glioma U251 cells induced by temozolomide (TMZ) combined with radiotherapy. Methods MTT assay was used to detect the inhibitory effect of TMZ treatments with different concentrations (0~64 μmol/L) on U251 cells for 24 h and 48 h respectively. The clone formation assay was used to detect the survival fraction of U251 cells under different concentrations of TMZ combined with radiotherapy, and the radiosensitization effect of TMZ was evaluated. The effect of the combined treatment on the apoptosis of U251 cells was detected by flow cytometry. The expression of microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin-1 and phosphated protein kinase B (p-Akt) was detected by Western Blot. Results The inhibitory effect of TMZ on the proliferation of U251 cells was concentration- and time-dependent, and the IC50 values were 42.25 μmol/L and 25.13 μmol/L, respectively for the 24 h and 48 h treatment. TMZ has a radiosensitizing effect on U251 cells. TMZ combined with radiotherapy can induced apoptosis of U251 cells, and significantly up-regulate the expression levels of LC3-I, LC3-I and Beclin-1, and the differences were statistically significant (all P<0.01). This combined treatment can down-regulate the expression of p-Akt protein, and the difference was statistically significant (P<0.05). Conclusions TMZ combined with radiotherapy can activate autophagy on U251 cells. The mechanism may involve the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by inhibiting Akt phosphorylation, the promotion of the expression of autophagy marker proteins (LC3-II, LC3-I and Beclin-1), and subsequent autophagy activation playing an anti-tumor effects. Key words: Malignant glioma; U251 cell; Temozolomide; Apoptosis; Autophagy