Association Of Protein Kinase CK2 Inhibition With Cellular Radiosensitivity Of Non-small Cell Lung Cancer

PartⅠ:Association of protein kinase CK2 with prognosis of lung cancer patientsPurpose: To investigate the protein kinase CK2 expression in lung cancer cells and tissues,and the accociation of CK2 with the prognosis of lung cancer patients.Method: 1.The CK2 expression in lung cancer cells were analyzed by Genecard.2.The expression of CK2 α,α ’and β in different lung cancer cells and endothelial cells was detected by Western blot assay.3.The CK2 gene level in lung cancer tissues and para-neoplastic normal tissues were analyzed by TCGA database.4.Immunohistochemical staining was used to detect the expression of CK2 subunits in lung cancer tissues of various pathological subtypes as well as adjacent normal lung tissues.5.The association of CK2 gene level with prognosis of lung cancer patients were analyzed by Kaplan-Meier Plotter.6.CK2α subunit expression was measured by immunochemistry,the association of CK2 with the prognosis of lung adenocarcinoma patients was analyzed by SPSS.Result: 1.CK2 was expressed in A549 and H460 cells.2.CK2 protein was ubiquitously expressed in lung cancer cells,and a higher expression was observed in lung cancer cells than that in endothelial cells.3.CK2 gene level was higher in lung cancer tissues than para-neoplastic tissues.4.The protein expression of CK2 subunits in lung cancer tissues was higher than that in adjacent normal tissues.5.High CK2 gene level correlated with poor outcome of lung cancer patients.6.High protein expression of CK2α correlated with poor overall survival in lung adenocarcinoma patients.Conclusion: Protein kinase CK2 was expressed in lung cancer cells and tissues,and higher expression was found in lung cancer tissues than that in para-neoplastic tissues.The expression of CK2 were correlated with poor prognosis of lung cancer patients.PartⅡ: The Effect of ionizing radiation on the subcellular localization and kinase activity of protein kinase CK2 in human non-small cell lung cancer cellsPurpose: In this study,we investigated the effect of ionizing radiation(IR)on the subcellular localization and kinase activity in human non-small cell lung cancer(NSCLC)cells.Methods: 1.Immunofluorescent staining was conducted to measure the subcellular localization of CK2 subunits after IR.2.Western blot assay was used to measure the protein expression of CK2 subunits in cell nucleus and cytoplasm,respectively.3.In vitro kinase assay was used to detect the CK2 kinase activity after IR.Results: 1.Immunofluorescent results showed CK2 subunits shuttle into the nucleus mostly from 1h and last more than 6h after IR.2.Western Blot assay showed the cell nuclear protein expression of CK2 subunits were increased.3.In vitro kinase assay showed an increase of CK2 kinase activity at 6h after IR.Conclusion: The cell nuclear expression and kinase activity of CK2 was increased after IR,indicated that CK2 may be implicated in the regulation of IR induced biological process.Part Ⅲ: The role of protein kinase CK2 inhibition in the radiation response of non-small cell lung cancer cellsPurpose: This study investigated the possibility of using CK2 inhibition as a new approach for increasing the efficacy of ionizing radiation(IR)in non-small cell lung cancer(NSCLC)and its underlying mechanisms.Methods: 1.Kinase assay was conducted to measure the activity of CK2 in Quinalizarin treated A549 and H460 cells.2.CCK8 assay was used to detect the cell viability after treatment with 12.5μM,25μM,50μM Quinalizarin for 24 h,48h,or 72 h.3.Colony formation assay was used to measure the radiosensitivity of cells treated with indicated concentration of Quinalizarin or transfected with si-CK2α.4.Flow cytometric analysis was conducted to measure cell apoptosis and cell cycle distribution.5.Ed U staining was preformed to detect cell DNA replication.6.γ-H2 AX and 53BP1 staining was preformed to reflect DNA double-strand breaks and repair.7.In vivo assay was conducted to detect the effect of Quinalizarin and IR on tumor growth in the H460 cell xenograft model.Results: 1.Treatment with Quinalizarin significantly reduced the activity of CK2 in A549 and H460 cells.2.Quinalizarin suppressed the cell viability of A549 and H460 cells.3.CK2α knockdown or Quinalizarin significantly enhanced the radiosensitivity of A549 and H460 cells.4.The combination of Quinalizarin and IR obviously increase the proportion of cells in G2/M phase but failed to further increase apoptosis.5.The combination of Quinalizarin and IR reduced cell DNA replication.6.Quinalizarin increased IR induced the number of γ-H2 AX foci and reduced the number of 53BP1 foci.7.CK2 inhibition combined with IR reduced tumor growth in the H460 cell xenograft model.Conclusion: CK2 is a promising target for the enhancement of radiosensitivity in NSCLC cells.Part Ⅳ: Protien kinase CK2 inhibition enhances the radiosensitivity of non-small cell lung cancer cells through IL-6/JAK/STAT3 signalingPurpose: To explore the signal pathway of protein kinase CK2 inhibition to enhance the radiosensitivity of non-small cell lung cancer(NSCLC)cells.Methods: 1.The effect of ionizing radiation(IR)on the m RNA level of IL-6 in A549 and H460 cells was detected by PCR.2.IL-6 secretion of cells treated with 25μM Quinalizarin and IR was measured by Elisa assay.3.Western blot assay was conducted to detect the protein expression of p-JAK/JAK and p-STAT3/STAT3 of cells treated with 25μM Quinalizarin and IR.4.Colony formation assay was used to measure the radiosensitivity of cells treated with 100ng/ml IL-6 and 25μM Quinalizarin.5.The protein expression of p-JAK/JAK and p-STAT3/STAT3 of cells treated with 100ng/ml IL-6 and 25μM Quinalizarin were detected by Western blot.Results: 1.IR enhanced the m RNA level of IL-6.2.Quinalizarin obviously suppressed the IR induced secretion of IL-6.3.Quinalizarin significantly suppressed the IR induced p-JAK and p-STAT3 protein expression.4.Quinalizarin suppressed the IL-6 enhanced radiation resistance.5.Quinalizarin reduced the IL-6 induced p-JAK and p-STAT3 protein expression.Conclusion: Protein kinase CK2 inhibition enhances the radiosensitivity of NSCLC cells through inhibit the IL-6/JAK/STAT3 pathway.