PARP-1 Promotes Autophagy Via The Ampkmtor Pathway In CNE-2 Cells Following Ionizing Radiation,And Down-regulation Of Autophagy Contributes To The Radiation Sensitization Of CNE-2 Cells

[Background]Nasopharyngeal carcinoma(NPC) is a type of epithelial tumor, which is prevalent in Southern China including Guangdong, Guangxi, Hunan, Fujian, Zhejiang, Hong Kong and Southeast Asia. Because of the special anatomical structure of NPC, radiotherapy is the manly treatment method of early and locally advanced NPC. However, the local recurrence and distant metastasis rate of NPC following the radiotherapy, chemotherapy and targeted therapy is about 20 percent, and it is mainly due to the radioresistance to NPC. Therefore, to find out the mechanism underling the radioresistance related to NPC is important. It may afford the new ideas to treat NPC.Recent research showed that the radioresistance related to NPC is contributed to autophagy. Our previous study showed that ionizing radiation(IR) induced autophagy in CNE-2 cells, and it played a pro-survival function in IR-induced cell death. In addition, PARP-1 activation contributed to IR-induced cell autophagy, and it was an upstream mediator of autophagy. In the xenotransplanted tumor model of NPC in nude mice assay, the tumor growth of Autophagy protein 5(ATG-5) gene silencing group was slower, and the tumor weight of ATG-5 gene silencing group as significantly less than the control group. It demonstrated that down-regulation of autophagy contributed to the radiation sensitization of NPC cells.This study is aimed to find out the relation of PARP-1, liver kinase B1(LKB1)-adenosine monophosphate-activated protein kinase(AMPK)-mammalian target of rapamycin(m TOR)(LKB1-AMPK-m TOR), and autophagy. Furthermore, interfering the PARP-1 and LKB1-AMPK-m TOR pathway to down-regulation of autophagy, observe the dead rate of NPC cells to radiation sensitization.ResearchPart 1 PARP-1 promotes autophagy via the AMPK-m TOR pathway inCNE-2 cells following ionizing radiation[Objective]This study aims to verify whether PARP-1 is the upstream mediator of LKB1-AMPK-m TOR pathway related to IR-induced autophagy of CNE-2 cells. Firstly, prove that PARP-1 induces LKB1 activation and consequently AMPK activation, and finally induces autophagy. Secondly, prove that AMPK inhibition does not influence PARP-1 activation.[Method]1. Western blot was apply to detect the LC3-II expression, the maker of autophagy.2. PARP-1, p-LKB1, p-AMPK and p-P70S6 K were detected by Western blot while CNE-2 cells deal with irradiation, 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside(AICAR),PARP-1 gene silencing and Compound C, individually.[Results]1. LC3-II expression was increasing in CNE-2 cells after irradiation.2. While CNE-2 cells deal with irradiation, PARP-1, p-AMPK and LC3-II expression were increasing compared with the control, and p-P70S6 K was decreasing compared with the control.3. While CNE-2 cells deal with irradiation and AICAR, PARP-1 expression was not changed, p-AMPK expression increasingly, p-P70S6 K expression decreasingly, and LC3-II expression increasingly.4. While CNE-2 cells deal with irradiation and PARP-1 gene silencing, PARP-1 expression was decreasing, p-AMPK expression decreasingly, pP70S6 K increasingly,and LC3-II expression decreasingly.5. While CNE-2 cells deal with Compound C, PARP-1 expression was not changed, p-AMPK expression decreasingly, p-P70S6 K expression increasingly, and LC3-II expression decreasingly.6. While CNE-2 cells deal with irradiation, AICAR, and PARP-1 gene silencing, p-LKB1 expression was not changed. While CNE-2 cells deal with irradiation and Compound C, p-LKB1 was disappeared.[Conclusion]Irradiation induced autophagy in CNE-2 cells. And PARP-1 promoted autophagy via the AMPK-m TOR pathway in CNE-2 cells following irradiation.Part II Down-regulation of the autophagy contributes to the radiationsensitization of CNE-2 cells[Objective]Down-regulation of PARP-1-AMPK-m TOR pathway to inhibit the autophagy activity, observe the radiation sensitization of CNE-2 cells.[Method]1. CNE-2 cells deal with irradiation and without irradiation, then treated with PARP-1 gene silencing and Compound C, individually, and cells proliferation was assayed at the indicated time points by MTT.2. CNE-2 cells deal with irradiation and without irradiation, then treated with PARP-1 gene silencing and Compound C, individually, and cells proliferation was analyzed by colony formation assay.[Results]The MTT assay and the colony formation assay showed that there was no significant difference between PARP-1 gene silencing and the control group(p>0.05). In addition, PARP-1 gene silencing following irradiation significantly attenuated the proliferation of CNE-2 cells(P<0.05). Furthermore, the AMPK inhibitor Compound C sensitized CNE-2 cells to irradiation, as indicated by the inhibition of proliferation(P<0.05). Finally in Compound C-treated cells without irradiation, CNE-2 cell proliferation was also significantly decreased compared with the control group(P<0.05).[Conclusion]Down-regulation of PARP-1-AMPK-m TOR pathway to inhibit the autophagy activity contributes to the radiation sensitization of CNE-2 cells.