ShRNA-mediated Ku80 Gene Silencing Inhibits Cell Proliferation and Sensitizes to γ-radiation and Mitomycin C-induced Apoptosis in Esophageal Squamous Cell Carcinoma Lines

Journal of Radiation Research, Jul 2008

To investigate the effects of Ku80 depletion on cell growth and sensitization to γ-radiation and MMC-induced apoptosis in esophageal squamous cell carcinoma lines. Six human carcinoma cell lines (LNcaP, K562, MDA-MB-231, MCF-7, EC9706, and K150) and normal HEK293 cell line were examined for basal levels of Ku80 protein by western blotting analysis. The suppression of Ku80 expression was performed using vector-based shRNA in EC9706 cells. Cell proliferation was determined with MTT assay and colony formation assay and tumorigenicity in a xenograft model in vitro and in vivo. Sensitivity of EC9706 cells treated with shRNA vector to γ-radiation and MMC was determined with colony formation assay and MTT assay. The cell cycle distribution was determined by Flow cytometry. Apoptosis induced by γ-radiation and MMC was analyzed using GENMED-TUNEL FACS kit. Ku80 showed higher basal levels in six carcinoma cell lines than in HEK293. The suppression of Ku80 expression decreased cellular proliferation, colony formation and inhibited tumorigenicity in a xenograft model. Furthermore, it sensitized apoptosis of the cancer cells induced by γ-radiation and MMC. Ku80 plays an important role not only in tumorigenesis but also in radiation resistance and chemotherapy resistance in esophageal cancer cells. Hence Ku80 may serve as a promising therapeutic target, particularly for recurrent esophageal tumors.

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ShRNA-mediated Ku80 Gene Silencing Inhibits Cell Proliferation and Sensitizes to γ-radiation and Mitomycin C-induced Apoptosis in Esophageal Squamous Cell Carcinoma Lines

Qing-Shan YANG Jin-Long GU Li-Qing DU Li-Li JIA Li-Li QIN Yong WANG Fei-Yue FAN Ku80/shRNA/Proliferation/Apoptosis/Esophageal cancer. To investigate the effects of Ku80 depletion on cell growth and sensitization to -radiation and MMC-induced apoptosis in esophageal squamous cell carcinoma lines. Six human carcinoma cell lines (LNcaP, K562, MDA-MB-231, MCF-7, EC9706, and K150) and normal HEK293 cell line were examined for basal levels of Ku80 protein by western blotting analysis. The suppression of Ku80 expression was performed using vector-based shRNA in EC9706 cells. Cell proliferation was determined with MTT assay and colony formation assay and tumorigenicity in a xenograft model in vitro and in vivo. Sensitivity of EC9706 cells treated with shRNA vector to -radiation and MMC was determined with colony formation assay and MTT assay. The cell cycle distribution was determined by Flow cytometry. Apoptosis induced by -radiation and MMC was analyzed using GENMED-TUNEL FACS kit. Ku80 showed higher basal levels in six carcinoma cell lines than in HEK293. The suppression of Ku80 expression decreased cellular proliferation, colony formation and inhibited tumorigenicity in a xenograft model. Furthermore, it sensitized apoptosis of the cancer cells induced by -radiation and MMC. Ku80 plays an important role not only in tumorigenesis but also in radiation resistance and chemotherapy resistance in esophageal cancer cells. Hence Ku80 may serve as a promising therapeutic target, particularly for recurrent esophageal tumors. - Esophageal cancer is one of the most common tumors and its incidence is increasing steadily worldwide. Despite improved treatments with surgery and radiotherapy, esophageal cancer is still the leading cause of cancer death in many counties, especially in China.1) Although our understanding of the molecular biology of esophageal cancer has increased in recent years, a detailed knowledge of the molecular mechanisms underlying its formation and progression remains elusive. Furthermore, no specific molecular targets for clinical practice have yet been identified. It will be desirable in the future to develop such potent molecular targets for novel clinical treatments based on the etiology of esophageal cancer formation. Ku80 is one component of a protein complex, the Ku80/ Ku70 heterodimer, which was originally identified as a major target of autoantibody from Japanese patients with scleroderma-polymyositis overlap syndromes two decades ago.2) Ku80 is well known for its crucial role in DNA repair. The double-strand break (DSB) is the major DNA lesion leading to chromosomal aberrations. Unrepaired DSB is likely to result in cell death. It will be likely to cause mutations or carcinogenesis under misrepaired DSB. DSB are repaired by homologous recombination (HR) and by nonhomologous end-joining (NHEJ). NHEJ is the predominant mechanism in higher eukaryotes, whereas single cell organisms (such as yeast) rely more heavily on HR. Here, we are particularly concerned with NHEJ pathway. It has been shown that Ku80 can maintain the genome stability by repairing DSB through NHEJ. This process requires several factors: Firstly, Ku heterodimer is capable of both sequence independent and sequence specific DNA binding and bridging. Then, Ku complex recruits DNA-PKcs to the DSB and activates its kinase function. Lastly, this complex stimulates DNA repair and transduces the damage/stress signal response, which may affect apoptosis and cell proliferation.3,4) These results were identified through studies of ionizing radiation-sensitive Chinese hamster cell lines and cells from severe combined immune-deficient (SCID) mice.5,6) Besides its important role in DNA repair, many reports have implicated that Ku80 is also involved in other cellular processes, such as telomere maintenance, antigen receptor gene arrangement, regulation of specific gene transcription, apoptosis, tumor suppression, as well as regulation of the G2 and M phases of the cell cycle,7,8) suggesting that Ku80 is multifunctional housekeepers in cells. In recent years, studies on Ku80 protein in tumor occurrence, development, treatment and prognosis are increasing extensively. Several investigations have shown abnormal expression of Ku80 protein in various cancers,913) including esophageal cancers,14) Over-expression of Ku80 made cancer acquire resistance to radiation and chemical drug.1517) These previous findings suggested that Ku80 plays an important role in occurrence, progression and prognosis of tumor, so Ku80 protein is very likely to become a new target for radiation sensitizer. However, exact role of the Ku80 is not clear in esophageal cancer cells. A novel molecular approach to specifically target Ku80 mRNA was used in this experiment in order to validate our obtained outcomes and clarify the relationship between Ku80 and oncogenesis of esophageal cancers. Results showed that this approach inhibited the expression of Ku80 protein. Then, t (...truncated)


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Qing-Shan Yang, Jin-Long Gu, Li-Qing Du, Li-Li Jia, Li-Li Qin, Yong Wang, Fei-Yue Fan. ShRNA-mediated Ku80 Gene Silencing Inhibits Cell Proliferation and Sensitizes to γ-radiation and Mitomycin C-induced Apoptosis in Esophageal Squamous Cell Carcinoma Lines, Journal of Radiation Research, 2008, pp. 399-407, 49/4, DOI: 10.1269/jrr.07096