Cathepsin L Involved In X-rays-induced Cell Death In Human U251 Glioma Cells

AIM To investigate the possible mechanism of Cathepsin L in the X-rays-induced cell death of human U251 glioma cells.METHODS The proliferative inhibition of U251 cells induced by X-rays was assessed with Clonogenic assay and WST-1. The activation of proapoptotic effect,cell cycle arrest and the change of the mitochondrial membrane potential (MMP) were characterized by Hoechst stain and flow cytometry. Immunofluorescence, Western blot analysis and RT-PCR were used to study the Cathepsin L mechanism in X-rays-induced death of U251 cells.RESULTS Clonogenic assay and WST-1 indicated that X-rays decreased the viability of U251 cells in a dose- and time-dependent manner. Apoptosis was induced in U251 cells by X-rays as detected by Hoechst staining; flow cytometry analysis indicated that X-rays increased cell population at sub-G1 phase, an index of apoptosis, and G2/M cell arrest. The pan-caspase inhibitor Z-VAD-fmk suppressed radiation-induced U251 cell death. Furthermore, radiation-induced cell death was associated with a collapse of the mitochondrial membrane potential (MMP). Immunofluorescence and western blot indicated that X-rays induced Cathepsin L immunoreactivity and expression in U251 cells. Inhibition of Cathepsin L by Z-FY-DMK sensitized the cytotoxicity of radiation. As for cell cycle, pretreatment of U251 cells with Z-FY-DMK induced an accumulation of cells in G2/M than in radiation-induced alone cells. Z-FY-DMK decreased cyclin B1 and Bcl-2 expression, increased cyclin D1 and Bax expression.CONCLUSION Together, these findings indicated that X-rays induced the proliferation inhibition, cell cycle arrest and apoptosis in human U251 glioma cells. In addition, we have identified a new mechanism where the lysosomal enzyme Cathepsin L acts as a death signal integrator and a critical role for Cathepsin L inhibition in X-rays-induced G2/M arrest and apoptosis in U251 cells.