Aspirin Induces Both Autophagy And Apoptotic Death In Human Glioma Cell Line U251

Aim: To study the mechanism and inhibitory effect of aspirin on U251 cells in vitro, and study the effects of autophagy in Aspirin-induced death of U251 cells.Methods: The effects of aspirin on proliferation of U251 cells were assessed using the MTT assay. Cell Cycle Analysis was by flow cytometry. Annexinâ…¤-FITC Apoptosis Detection Kit was used to detect apoptosis of cells. MDC staining was used to examine autophagy induced by aspirin in U251 cells. Western blot was employed to detect expression of Bcl-2,Caspase-3,LC3,Beclin1. Total RNA was extracted from U251 cells and Real Time PCR was used to detect the mRNA levels of LC3 and Beclin1; To examine the involvement of autophagy in aspirin-induced death of U251 cells, the autophagy specific inhibitor 3-methyadenine (3-MA) was added with aspirin and the cytotoxicity of aspirin was measured by trypan blue exclusion assay and DNA synthesis assay.Results: The growth inhibiton of U251 cells by aspirin was in a time and dose-dependent stytle. After treatment with 8 mM, cell cycle was arrested at G2/M phase. Aspirin also significantly enhanced apoptosis of U251 cells with down-regulated anti-apoptotic protein Bcl-2, and activation of Caspase-3. Autophagy was induced in U251 cells as detected by MDC staining; Western blot and Real Time PCR analysis revealed that expression of LC3 and Beclin1 increased after aspirin treatment. The blockade of autophagy with specific inhibitor 3-methyladenine(3-MA) amplified the cytotoxicity of aspirin in U251 cells.Conclusions: Aspirin could significantly inhibit the growth of U251 cells through inducing cell apoptosis in vitro, which may depend on mitochondrial apoptotic pathway. 3-methyladenine(3-MA) can amplify the cytotoxicity of aspirin in U251 cells, this demonstrates that autophagy may paly a protective role in the early time of aspirin treatment.