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.
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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)