Numb Gene Enhances Radiation Sensitivity of Nonsmall Cell Lung Cancer Stem Cells.
OBJECTIVE
To study the effects of Numb gene expression on radiation sensitivity of nonsmall cell lung cancer (NSCLC) stem cells.


METHODS
The side population (SP) cells A549-SP were transfected with pcDNA3.1 (pcDNA3.1 group), pcDNA-Numb (pcDNA-Numb group) and shRNA-Numb (shRNA-Numb group). Real-time quantitative polymerase chain reaction and Western blot were performed to determine Numb expression; MTT method was used to measure the proliferation activity change of the NSCLC stem cells both before and after irradiation with different doses of 60Coγ ray; Hoechst staining and Annexin V-FITC/PI were used to detect the apoptosis of the NSCLC stem cells; and colony-forming assay was used to determine the effect of Numb expression on radiation sensitivity of the NSCLC stem cells.


RESULTS
Increased mRNA and protein expressions of the A549-SP cells were found in the pcDNA-Numb group, and decreased mRNA and protein expressions were found in the shRNA-Numb group. The optical density value of the cells decreased in the pcDNA-Numb group but increased in the shRNA-Numb group. The cells with over-expressed Numb showed obvious nuclear condensation and fragmentation; the apoptosis rate increased significantly. The cells with knockdown Numb showed less nuclear damage; the apoptosis rate significantly decreased. After irradiation, the cells in the pcDNA-Numb group showed decreased survival rate, clonality, and the values of D0, Dq, N, and SF2; whereas the cells in the shRNA-Numb group showed the opposite trend.


CONCLUSIONS
Radiation sensitivity of NSCLC stem cells was enhanced with the increase of Numb expression. Determination of Numb expression helped to evaluate the response of lung cancer to radiotherapy, which was important for guiding tumor treatment clinically.
Department of Radiation Oncology, Zhejiang Cancer Hospital , Zhejiang Key Laboratory of Radiation Oncology, Hangzhou, P.R. China .
Guo-Ping
GP
Shan
Department of Radiation Oncology, Zhejiang Cancer Hospital , Zhejiang Key Laboratory of Radiation Oncology, Hangzhou, P.R. China .
Peng
P
Zhang
Department of Radiation Oncology, Zhejiang Cancer Hospital , Zhejiang Key Laboratory of Radiation Oncology, Hangzhou, P.R. China .
Pu
P
Li
Department of Radiation Oncology, Zhejiang Cancer Hospital , Zhejiang Key Laboratory of Radiation Oncology, Hangzhou, P.R. China .
Feng-Lei
FL
Du
Department of Radiation Oncology, Zhejiang Cancer Hospital , Zhejiang Key Laboratory of Radiation Oncology, Hangzhou, P.R. China .
Yi-Wei
YW
Yang
D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000532:radiotherapy*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics / Q000528:radiation effects; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000532:radiotherapy*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000528:radiation effects; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011836:Radiation Tolerance; D014162:Transfection
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C113026:NUMB protein, human; D012333:RNA, Messenger
Numb; gene silencing; nonsmall cell lung cancer stem cells; over-expression; radiation sensitivity; stem cell apoptosis; stem cell proliferation; tumor biology