Radiosensitizing Effect Of Autophagy/cathepsin L On Glioma Stem Cells

Aim: In this study, we investigate the effect of cathepsin L and autophagy on gliomastem cells sensitivity to radiation, and clarify the critical linkages of Cathepsin L/autophagy activity and radiation resistance.Methods: GSCs have been established in vitro culture system and xenografts in nudemice model. GSCs are pretreated with appropriate dose of Cathepsin L inhibitorZ-FY-DMK or Resveratrol and siRNA Cathespin L before treating by different doses ofX-ray. The colony formation assay is used to detect the radiosensitivity difference of eachtreated GSCs group. The fluorescent staining by Hoechst33258staining, flow cytometryand Annexin V-FITC/PI Staining served to study treatment effects on apoptosis andapoptosis rate. JC-1kits test the mitochondrial membrane potential of GSCs. The changeof the CD133protein could be detected by immunofluorescence andimmunohistochemistry methods. The DNA of GSCs in various groups is damaged afterradiation. Alkaline comet assay reflected the recovery of the DNA. Immunofluorescenceand Western blot are used to study the expression level of the proteins (γH2AX、DNA-PKcs、p-ATM) related to the DNA damage recovery. The HE staining could analyzethe histology Morphological differentiation of the transplantable tumor in various groups.The MDC staining and the Transmission electron microscope detect the changeof autophagy, which is in the GSCs and xenograft tumor in various groups.Results: Compared to other treated groups, the survival fraction reduced in thecombined treatment group (inhibition of Cathepsin L combined with X-ray) and theapoptosis rate increased remarkable. Furthermore, the combined treatment group wasassociated with a collapse of the mitochondrial membrane potential (MMP).Immunofluorescence show that expression level of CD133was significantly decreased inthe combined treatment group; Alkaline comet assay indicated that less efficient repair inresponse to DNA damage in the combined treatment group. Immunofluorescence and Western blotting analysis indicated that the expression level of DNA damage repair relatedproteins (DNA-PKcs、p-ATM) was significantly decreased in the combined treatmentgroup. The relative tumor volume of xenografts tumors in the combined treatment groupwere significantly lower than that of alone treatment group xenograft tumors in nude mice.Cells previously treated with Resveratrol and IR generated a decreased number ofcolonies as compared to untreated cells. Immunofluorescence show that expression level ofCD133was significantly decreased in the combined treatment group; Alkaline comet assayindicated that less efficient repair in response to DNA damage in the combined treatmentgroup. Apoptosis level in GSCs was significantly increased by the combined treatmentwith Resveratrol and IR. Western blotting analysis indicated that the expression level ofCathepsin L was significantly decreased in the combined treatment group.TEM results also show that a large number of autophagic vacuole in the combinedtreatment group. The relative tumor volume of xenografts tumors in the combinedtreatment group were significantly lower than that of alone treatment group xenografttumors in nude mice(p<0.05). Western blotting analysis indicated that the expression levelof Cathepsin L, Bcl-2was significantly decreased in the combined treatment group. Theexpression level of LC3Ⅱ was significantly increased in the combined treatment group.TEM results show that a large number of autophagic vacuole in the combined treatmentgroup.Conclusion: In the research, we discovered that: when the GSCs was treated by X-raycombined with the Cathepsin L in vivo and in vitro experiment, whose activity wasinhibited, the GSCs stopped growing and the growth of hepatocarcinoma was inhibited.The expression of CD133protein reduced, apoptosis appeared and MMP decreased. Therate of the DNA damage repair slowed down. The expression of DNA-PKcs and p-ATMreduced. When the GSCs was treated by X-ray and the Resveratrol, the GSCs stoppedgrowing and the growth of hepatocarcinoma was inhibited. The rate of the DNA damagerepair slowed down. The level of apoptosis and autophagy improved. The combination(Resveratrol+IR) inhibited cell viability more than IR treatment did alone. The rate of theDNA damage repair slowed down. The level of apoptosis and autophagy improved.Theregulation of autophagy/Cathepsin L may abrogate the resistance of GSCs to radiation andcan lead to the development of novel therapeutic approaches for the treatment of GBMs.