The Effects And Mechanisms Of Autophagy On Radioresistance Of Human Lung Adenocarcinoma Cells

Background and ObjectiveIncidence and mortality rate of lung cancer have ranked the first among all cancers in China for recent years. As indicated by the latest statistics of the world and USA, the number of new cancer cases was significantly increased, of which the incidence and mortality rate of lung cancer rise, ranking first in men. Lung cancer is divided into small cell lung cancer(SCLC) and non-small cell lung cancer(NSCLC) based upon the pathological pattern. NSCLC accounts for 85% of all lung cancers, while lung adenocarcinoma is one of the common histological types of NSCLC. Primary means for lung adenocarcinoma treatment include surgery, chemotherapy, radiotherapy, molecular targeted therapy, etc. Over the past decade, with the continuous research and development of large-scale precision radiotherapy equipment, along with the advancement in computer technology and medical radiological technology, radiotherapy technology has improved significantly, and side effects and complications have remarkably reduced, making the radiotherapy to be one of major and effective means for tumor treatment. Lung adenocarcinoma is occult in onset, and most patients are found to be locally advanced or with distant metastasis at time of diagnosis, thus tending to lose the opportunities of radical surgery. Postoperative auxiliary radiotherapy and concurrent chemoradiotherapy have been commonly used as the comprehensive treatment strategies clinically. It’s quite common that patients with local recurrence or failure of control after radiotherapy, therefore, radiotherapy resistance is one of primary deficits that may reduce clinical effects of radiotherapy and affect prognosis of patients. Mechanisms of radiotherapy resistance that are commonly seen are tumor stem cells, repair of DNA damage, hypoxic status of tumors, P53 mutation, enhancement of autophagy, Micro RNAs dysregulation, overexpression of various growth factor, etc. Autophagy is a process of programmed death that the aging organelles or macromolecular substances are degraded as amino acid, fatty acid and nucleotide in the lysosome for repeated utilization, which is of great importance in maintaining dynamic balance of cells and genomic integrity. In the early experiment, human lung adenocarcinoma cells SPC-A1 were induced in vitro by gradually increasing the docetaxel concentration, and thus the SPC-A1 anti-docetaxel cell line SPC-A1/DTX was built. Besides, preliminary experimental study has demonstrated that the high-level autophagy may promote the drug resistance of human lung adenocarcinoma cells to the docetaxel. However, the present studies on the way the autophagy participating in the forming process and mechanism of radiotherapy resistance of lung adenocarcinoma cells at home and abroad are still inadequate. The purpose of this study was to investigatet the role and mechsnisms of radioresisitance of human lung adenocarcinoma cells which were drug resistance to the docetaxel, and discussed that inhibition of autophagy could enhance the radiosensitivity of SPC-A1/DTX cells.Materials and Methods1、Transmission electron microscopy were applied to assess autophagy activity of parent lung adenocarcinoma cells(SPC-A1) and drug resistant lung adenocarcinoma cells(SPC-A1/DTX). The detail groups were as follows:group control and group treated with IR(6Gy).2、Fluorescence microscope were applied to detect the density of GFP-LC3 of parent lung adenocarcinoma cells(SPC-A1) and drug resistant lung adenocarcinoma cells(SPC-A1/DTX). The detail groups were as follows:(1)group control and group treated with IR(6Gy).(2)control; treated with 3-MA/si RNA-Survivin alone; treated with 6Gy/6Gy+ si RNA control; treated with 3-MA/si RNA-Survivin followed 6Gy.3、Pretreatment of autophagy inhibitor 3-MA or transfection with Survivin si RNA with SPC-A1/DTX, western blot analysis was used to detect autophagy related protein expression of LC3, p62, beclin-1 and Survivin. The detail groups were as follows:(1)group control and group treated with IR(6Gy).(2)control; treated with 3-MA/si RNA-Survivin alone; treated with 6Gy/6Gy+ si RNA control; treated with 3-MA/si RNA-Survivin followed 6Gy.4、Apoptosis and cell cycle distribution were analyzed by Flow cytometry.5、Cell viability and proliferation were examined by MTT and colony formation assay respectively.Results1、The chemoresistant SPC-A1/DTX cells with higher autophagy activity were also radioresistant compared with the parental SPC-A1 cells, and the ED50 value was higher than that of the parental SPC-A1 cells(the mean was approximately 7.5Gy in SPC-A1/DTX cells, while 4Gy in SPC-A1 cells)(P<0.05).2、SPC-A1/DTX cells showed an increase in autophagosome, punctate localization of green fluorescent protein-LC3 which was characteristic of autophagy, and autophagy related protein expression of LC3Ⅱ/Ⅰ、p62、beclin-1 compared to SPC-A1 cells. Elevated autophagy levels of SPC-A1/DTX were seen obviously.3 、 Autophagy inhibitor 3-MA transfection or transfection with Survivin si RNA combination with irradiation suppressed the expression of autophagy markers such as LC3, p62 and beclin-1(P<0.05), which represented decreased autophagy activity in SPC-A1/DTX cells.4、Irradiation-induced G2/M phase delay was increased and S phase arrest was decreased by combination of 3-MA or transfection with Survivin si RNA combination with irradiation leading to radiosensitization of SPC-A1/DTX cells. which then resulted in enhanced cell deaths in SPC-A1/DTX cells(P<0.05).ConclusionsOur results demonstrated that irradiation-induced autophagy provided a cytoprotective mechanism against radiotherapy in SPC-A1/DTX cells. Autophagy contributed to radioresistance of docetaxel-resistant human lung adenocarcinoma(LAD) cells, and blocking autophagy would be a potential strategy for reversing chemoradiotherapy cross resistance of LAD patients.