Cathepsin L Involved In Resveratrol-induced Glioma Cell Cycle Arrest And Apoptosis

Objective: Cathepsin L, a lysosomal cysteine proteinase, is involved in tumor progression, including cell apoptosis and cell cycl in a variety of malignancies, including gliomas. The aim of this study is to preliminarily investigate the role of cathepsin L in resveratrol induced glioma cell apoptosis and cell cycle arrest and to try to explore its possible mechanism using glioma cell lines U251 and U87 cells as models.Methods: Cell viability was tested by using the method of CCK-8,colony formation and annexin V/PI flow cytometry werer carried out to investigate the toxic effects of resveretrol on U251 and U87 glioma cells. Western blot was used to detect the expression level of cathepsin L, cyclin A, cyclin B1, cyclin D1 and apoptosis related proteins Bcl- 2 / Bax. Cathepsin L was also analyzed by immunofluorescence staining. Cell cycle distribution was measured by PI staining flow cytometry. Using si RNA to downregulate the expression of cathepsin L. U87 cells was converted to p53 mutated cells using p53mut-flag plasmid to observe chages on the sensitivity of the resveratrol.Results: Western blot showed that cathepsin L was significantly activated. Immunofluorescence experiment further verified the expression of cathepsin L in glioma cells increased after the resveratrol treatment. CCK-8, colony-forming assay and Annexin V/PI double staining flow cytometry results suggest resveratrol could induce glioma cells apoptosis; besides, U87 cells were more sensitive to resveratrol than U251 cells after the same doses were administrated. PI single staining results showed resveratrol could cause U251 cells in G1 phase arrest, while U87 cell in S phase. Cyclins expression level is continuously reduced in glioma cells, which also indicated cell cycle arrest. Down regulation of cathepsin L can enhance cytotoxicity caused by resveratrol and cell cycle arrest. Clonogenic formation rate is decreased obviously. The expression level of apoptosis related proteins Bcl- 2 / Bax is different from that in U251 and U87 cells. This is likely related to cell’s genetic background on p53. After U87 was transformed to p53-mut cells, both cell viability and clonogenic rate increase to some extent.Conclusion: Cathepsin L plays a role in growth inhibitory and cell cycle arrest on glioma cells induced by resveretrol. Inhibition of cathepsin L can sensitize glioma cells to resveretrol and enhance cell cycle arrest. Resveretrol-induced glioma cell death was in a p53 dependent manner.