title: Repair of radiation damage of U2OS osteosarcoma cells is related to DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity.
abstract: The present study was to investigate the effects of DNA-PKcs deficiencies on radiation sensitivity of human osteosarcoma U2OS cells to γ-ray and to explore the underlying molecular mechanism. In vitro, U2OS cells were transfected with different DNA-PKcs siRNAs or control siRNAs to establish stably siRNA-transfected cell lines U2OS-Si and U2OS-Sc, respectively. Cell viability and apoptosis after irradiation were analyzed using cell counting kit (CCK-8) and flow cytometric assay, respectively. Expressions of apoptosis-related and oxidative stress-responded proteins were assessed using Western blot. The tumorigenesis activity was examined in nude mice xenograft osteosarcoma mode. Results showed that DNA-PKcs siRNA significantly could inhibit U2OS viability and cell proliferation after exposure to irradiation. Compared with the U2OS and U2OS-Sc cells, the U2OS-Si cells induced higher apoptosis rate and loss of mitochondrial membrane potentials, accompanying with more reactive oxygen species (ROS) and malondialdehyde (MDA) production, increased DNA double-strand breaks (DSBs) induced by irradiation. Protein levels of the anti-apoptotic Bcl-2 were downregulated most obviously in U2OS-Si cells after irradiation, while pro-apoptotic factor Bax and caspase-3 upregulated. Moreover, the antioxidants protein expression levels of Nuclear factor-erythroid 2-related factor 2 (Nrf2) and its target heme oxygenase-1 (HO-1) were also significantly reduced in parallel to DNA-PKcs inhibition in U2OS-Si cells. In nude mice xenograft model, DNA-PKcs siRNA remarkably inhibited tumor growth and dissemination. In conclusion, DNA-PKcs siRNA might have a potential for osteosarcoma treatment by sensitizing osteosarcoma cells to γ-ray through modulation on oxidative stress-mediated DNA DSBs repair and mitochondrial pathway apoptosis.
authors.affiliation: Orthopaedic Department of Affiliated Hospital of Xuzhou Medical College, Jiangsu, 223000, China.
authors.forename: Xianye
authors.initials: X
authors.lastname: Tang
authors.forename: Feng
authors.initials: F
authors.lastname: Yuan
authors.forename: Kaijin
authors.initials: K
authors.lastname: Guo
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000528:radiation effects; D020134:Catalytic Domain / Q000235:genetics*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D051379:Mice; D012516:Osteosarcoma / Q000235:genetics* / Q000378:metabolism / Q000532:radiotherapy; D011494:Protein Kinases / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance / Q000528:radiation effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D011494:Protein Kinases; D051747:DNA-Activated Protein Kinase