Effect Of Silencing Of PKM2 Expression On Radiosensitivity Of Lung Adenocarcinoma A549 Cell And Xenograft

Objectives: To investigate the impact of pyruvate kinase M2 isoform(PKM2) silencing on the radiosensitivity of lung adenocarcinoma line A549 in vitro and in vivo and the potential mechanisms.Materials and Methods: 1. The expression levels of PKM2 in normal lung bronchial epithelial cell BEAS-2B and five NSCLC cell lines were tested by Western blot. A plasmid of psh RNA-PKM2 for expressing a short hairpin RNA targeting PKM2 and psh RNA-Control were simulitantly constructed and transfered into A549 cells. The protein expression of PKM2 was measured in vitro and in vivo, respectively. 2. Trypan blue staining assay and colony formation assay evaluate the frequency of viable cells and colony-forming ability in psh RNA-PKM2 cells, with or without 3-MA and/or z-VAD-fmk. Immunofluorescence assay analyses the g-H2 AX positive cells with nuclear foci. 3. TUNEL assay and flow cytometry were used to detect apoptosis of PKM2 silencing A549 cells with or without inhibitors in different groups after radiotherapy. 4. Transmission electron microscopy and Western Blot were detected autophagic vesicle and autophagosome and expression levels of autophagy-related proteins with or without inhibitors, respectively. 5. Immunohistochemistry was used to detect the efficiency of xenograft’s psh RNA-PKM2. The volume of tumor was measured with a caliper over time. TUNEL detects the transplanted tumor cell apoptosis and TEM observes autophagosome and autophagic vesicle formation.Results:1. Compared with normal lung epithelial cells, A549, H460, H1299, H292 and H520 increased the expression of PKM2. And A549 cell with PKM2 silencing was successfully constructed. 2. The percentage of viable cells in the PKM2-silencing and irradiated cells were reduced and the numbers of colonies formed by psh RNA-PKM2 cells were significantly decreased compared with that of mock-treated and control cells, in contrast with the addition of inhibitors. The frequency of g-H2AX+ cells significantly increased when combined IR with PKM2 silencing. 3. The apoptosis rate was significantly increased when combined IR with PKM2 silencing, while decreased after adding z-VAD-fmk and 3-MA. 4. Numerous autophagosomes were detected in the IR+psh RNA-PKM2-transfected cells. The relative ratios of LC3-II/I greatly increased in the IR+psh RNA-PKM2-transfected cells, decreased with 3-MA and z-VAD-fmk. Combination of PKM2 silencing with IR dramatically reduced the levels of AKT and PDK1 phosphorylation, but obviously elevated the levels of ERK1/2 and GSK3b phosphorylation. 5. Treated with psh RNA-PKM2 and IR significantly suppressed the growth of implanted tumors in mice and increased the frequency of apoptotic cells. Simultaneously, more autophagosomes were observed in the tumors treated with psh RNA-PKM2 and IR than in single treatment.Conclusion: Silencing of PKM2 expression may enhance the radiosensitivity of human lung adenocarcinoma cell line A549 cells and xenografts by inducing autophagy and apotosis which is expected to become effective radiation sensitizers, but this needs to be confirmed by further studies. In conclusion, PKM2 may be used as a novel strategy for IR of NSCLC.