Effect Of Targeting PDK1 On Radiosensitivity Of Nasopharyngeal Carcinoma And Its Molecular Mechanism

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The expression of PDK1 in nasopharyngeal carcinoma and association between its expression and clinicopathological characteristicsObjective:To investigate the expression of PDK1 in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells,and to analyze the relationship between PDK1 expression and clinicopathological characteristics and prognosis of patients with nasopharyngeal carcinoma(NPC).Methods:Western blot was used to detect the expression of PDK1 in normal nasopharyngeal epithelial cell line NP-69 and 5 kinds of nasopharyngeal carcinoma cell line(CNE1,CNE2,HNE1,HONE1,and HK1).Immunohistochemistry(IHC)was used to detect the expression of PDK1 in nasopharyngeal tumor tissues,the correlation between PDK1 expression and clinicopathologic parameters of patients was analyzed.Kaplan-Meier analysis was used to analyze the relationship between PDK1 expression and prognosis.Results:PDK1 expression was increased in the nasopharyngeal carcinoma cell lines than that of NP-69,and it was detected in cytoplasm/membrane in tumor tissues.PDK1 expression was not significantly associated with clinicopathologic parameters,but the progression free survival(PFS)rates were poorer in patients with higher expression of PDK1.Conclusions:PDK1 expression level was higher in NPC cell lines than NP-69.Higher expression of PDK1 was significantly associated with poorer PFS in NPC.Part ? The effect of PDK1 knockdown on radiosensitivity of NPC cellsObjective:To investigate the effect of PDK1 knockdown on radiosensitivity of nasopharyngeal carcinoma cells in vitro and in vivo.Methods:HK1-ShPDKl and CNE2-ShPDK1 cells were constructed by RNA interference and verified by Western blot.Colony formation assay was used to evaluate the effect of PDK1 knockdown on radiosensitivity of nasopharyngeal carcinoma cells,the multi-target single-hit model was used to calculate survival fraction curve.Immunofluorescence was performed to detect the level of y-H2AX after irradiation.Then,cell cycle distribution and apoptosis were detected by flow cytometry.Finally,a mouse xenograft model was constructed to evaluate the effect of PDK1 knockdown on proliferation and radiosensitivity of NPC cells.Results:HK1-ShPDKl and CNE2-ShPDK1 cells were successfully constructed and verified.The clone formation rates were significantly reduced in PDK1 depleted cells after different doses of irradiation than that of the control group.Compared to irradiation alone,PDK1 knockdown significantly increased the radiation-induced apoptosis in NPC cells.In addition,irradiation alone induced a typical G2/M arrest in HK1 and CNE2 cells,while G1 arrest was further promoted in PDK1 depleted cells after irradiation.Moreover,PDK1 depleted cells contained a significantly higher level of y-H2AX positive foci after irradiation in comparison with control cells.In vivo experiment showed that the growth of tumor was inhibited after irradiation,the inhibition was more evident and tumor weight was also significantly reduced in the PDK1 depleted cells,while this situation was not observed in the absence of irradiation.Conclusions:Downregulation of PDK1 enhanced radiosensitivity of NPC cells in vitro and in vivo.Part ? The molecular mechanism of PDK1 knockdown in regulating radiosensitivity of NPC cellsObjective:To clarify the molecular mechanism of PDK1 knockdown in regulating radiosensitivity of nasopharyngeal carcinoma cells.Methods:Western blot was adopted to detect the phosphorylation of Akt and its downstream molecules in HK1-ShPDKl and CNE2-ShPDK1 cells.Moreover,PDK1 overexpression cell lines were constructed and Coimmunoprecipitation was performed to measure the interaction between PDK1 and its downstream molecules.Results:After exposed to irradiation,the phosphorylation of Akt-Thr308 was significantly inhibited by PDK1 depletion in NPC cells,while the levels of Akt remained stable.Consistent with the reduction of Akt activation,phosphorylation of GSK3?,mTOR and p70S6k were diminished.The levels of ?-catenin were also decreased.In addition,we observed that the epithelial marker(E-cadherin)was obviously upregulated while the mesenchymal markers(N-cadherin and Vimentin)were downregulated in PDK1 depleted cells which indicated that depletion of PDK1 could suppress epithelial-mesenchymal transition(EMT)in NPC cells.However,there was no detectable interaction between PDK1 and its downstream targets.Conclusions:These data coherently suggests that depletion of PDK1 sensitized NPC cells to irradiation by suppressing various targets of Akt signaling pathway and reversing EMT phenotype.Part ? The radiosensitizing effect of PDK1 inhibitor in radioresistant NPC cellsObjective:To investigate the effect of PDK1 inhibitor on radiosensitivity of radioresistant nasopharyngeal carcinoma cells.Methods:To establish radioresistant subclone cell lines,HK1 and CNE2 cells were exposed to 6 Gy irradiation once every week for 10 times at a total dose of 60 Gy.CCK8 assay were performed to measure the effect of PDK1 inhibitor on cell viability.Colony formation assay was used to evaluate the colony formation rate of radioresistant cells under specific doses of PDK1 inhibitor.Multi-target single-hit model was used to calculate survival fraction curve.Western blot was used to analyze the effect of PDK1 inhibitor in combination with irradiation on DNA damage and apoptosis.Results:The acquired radioresistant HK1-R and CNE2-R were successfully generated and its radioresistance was confirmed by colony formation assay.Western blot showed that a higher level of N-cadherin and Vimentin were in HK1-R and CNE2-R cells,while E-cadherin was decreased in these cells,which suggests the mesenchymal traits of radioresistant NPC cells.CCK8 results showed that cell viability was significantly inhibited by GSK2334470 in HK1-R and CNE2-R cells,while these effects were more obvious in combination with irradiation.Moreover,we found that GSK2334470 resulted in significant increase of DNA damage and induction of apoptosis after irradiation.Conclusions:Inhibition of PDK1 with GSK2334470 sensitized irradiation in radioresistant NPC cells.