Inhibition Of Cathepsin L Reduces Radiation-induced NF-κB Activation Involved In Radiosensitization Of Human Tumor Cell Lines

Objective: To investigate whether inhibition of Cathepsin L could sensitize humanglioma U251and breast adenocarcinoma MCF-7cells to ionizing radiation (IR) throughregulating NF-κB activation.Methods: Besides cells treated with a selective inhibitor of Cathepsin L,Z-FY-CHOor not, we established Cathepsin L knockdown U251and MCF-7cell lines by transfectingcells with Cathepsin L shRNA plasmids. Western blotting and immunofluorescence assaywere used to detect Cathepsin L protein expression and NF-κB translocation in tumor cellsfollowing IR or not. The DNA binding and transcription activition of NF-κB in tumor cellswas further determined by Luciferase reporter assay. DNA damage repair in p65knockdown cells treated with Z-FY-CHO or not and cellls transfected with control shRNAplasmids after IR was showed by Comet assay to confirm the regulation of Cathepsin L onIR-induced NF-κB activation.Results: The increase of Cathepsin L protein expression and nuclear translocation ofNF-κB p65and p50were observed successively in both tumor cell lines following IR.Western blotting and immunofluorescence assay showed that IR-induced nucleartranslocation of NF-κB was reduced by using a selective inhibitor of Cathepsin L,Z-FY-CHO or Cathepsin L shRNA. Z-FY-CHO or Cathepsin L shRNA could also inhibitIR-induced NF-κB dependent transcription of a luciferase reporter gene and reduced therecruitment of NF-κB to cyclin D1and ATM promoter in U251and MCF-7cells,whichwas tested by ChIP assay. To confirm this regulation of Cathepsin L on IR-induced NF-κBactivation, we established NF-κB knockdown U251and MCF-7cell lines by using p65shRNA. It was observed that Z-FY-CHO significantly decreased IR-induced DNA damagerepair in both U251and MCF-7cell lines. However, Z-FY-CHO did not sensitize eitherNF-κB knockdown cell line to IR. Conclusion: Cathepsin L may play an important role in mediating NF-κB activationto contribute to radio-resistance of U251and MCF-7cell lines. Inhibition of Cathepsin Lcan partially reduce NF-κB activation to enhance IR-induced cytotoxicity, which providesa potential target for tumor radiotherapy.