Guozi Yang¹, Jiuwei Cui², Jiuwei Cui², Lihua Dong¹, Zhenyu Pan¹

¹Department of RadiationOncology, the First Hospital of Jilin University,

²Cancer Center, the FirstHospital of Jilin University

Objective:Low-dose radiation (LDR) has been proposedto induce an adaptive response in normal cells but not in tumor cells, whichmay protect normal tissue against the damage of conventional radiationtherapy.However, the cell signaling pathways implicated in the different adaptiveresponse to LDR are not well established. The aim of this study istoinvestigate the molecular mechanism underlying the different adaptiveresponse of normal lung cells and lung cancer cells to LDR. Method: Normallung epithelial cell line HBE135-E6E7 (HBE) and human lung adenocarcinoma cellline A549 were used in this study. Cell viability assay and cell cycle weremeasured for the two cell lines at different time points (3h, 6h, 12h, 24h and48h) following 75mGy X-rays using MTT and flow cytometry respectively. A redoxsensitive transcription factor Nrf2 and its downstream event GSH were alsodetermined by western blot and ELIS Arespectively. For the adaptive responsestudy, HBE cells were divided into fourgroups: control (sham irradiated), D1(75mGy), D2 (5 Gy) and D1+D2 (75mGy+5Gy). The interval between D1 and D2 was24h by which time the effect of LDR on HBE cells was most significantdemonstrated in the above studies. The expression of nuclear Nrf2 and theactivation of its upstream regulators Akt and GSK3β were determined by westernblot analysis. Furthermore, the Nrf2 siRNA and LY 294002 were used toinhibitNrf2 expression and PI3K activity respectively. Result: We found that75mGy X-rays significantly promoted the proliferation of HBE cells at 24hpost-irradiation, but did not affect the growth of A549 cells. At this timepoint, pretreatment of LDR protected HBE cells against the damage of ROSinduced by subsequent high dose radiation through up-regulating nuclear Nrf2and its downstream event GSH. And a 25-nM concentration of siRNA targeting Nrf2restored this protective effect. However, these phenomena were not detected inA549 cells, which suggested that the difference of adaptive response in twocells lines is associated with Nrf2-mediated antioxidative function. In thestudy of Nrf2 upstream pathway, we found that the treatment of HBE cells with75mGy X-rays increased the Akt (Ser473) phosphorylation in a time-dependentmannerfrom 6h to 24h after radiation, there by adding the regulation imposed byit on GSK3β, which promotes nuclear accumulation of Nrf2 and eventuallyresulted in the increase of intracellular antioxidant GSH. On inhibitingPI3K/Akt pathway, we observed decreased levels of phosphorylated GSK3β(Ser9),which was accompanied by reduced nuclear retention of Nrf2 and intracellarlevel of GSH. Conclusion: The study indicates that the activation ofPI3K/Akt/GSK3β/Nrf2 pathway is a criticalmechanism of LDR-induced adaptiveresponse in normal lung cells, which does notexist in lung cancer cells.

Key Words: Low dose radiation  Adaptive response  Nrf2 signaling