The Effect Of PI3K/AKT/MTOR Signaling Pathways On Hypofractionated Radiosensitivity Of NSCLC

Objective:1. To obtain a stable non-small cell lung cancer radioresistant cell lines and identify the genetic pattern of conventional fractioned and hypofractionated radiotherapy2. To investigate the different PTEN/PI3K/AKT/mTOR cell signaling pathways expression patterns of Radioresistant A549R cells, and evaluate the radiosensitivity caused by LY294002on A549R cells.Methods:1. NSCLC cell line A549cells were treated with6MV X-ray irradiation with conventional fractionated (2Gy,17f) and hypofractionated irradiation (4Gy,7f). After extraction of total mRNA of the cells, the whole genome expression microarray was applied to imply differential gene expression. Those genes changed more than2times (P<0.05) were analyzed and the pathway (Q <0.05) methods was used to further analyzed with the chip results.2. The PTEN, PI3K, AKT, mTOR mRNA were detected by RT-PCR method to investigate the different cell signaling pathways expression patterns between Radioresistant A549R cells and normal A549cells.The experiment was divided into control group(A),simple radiation group(B),drug goup(C), and radiation plus drug group(D). A549R cells were adiministered by LY294002(10umol/L). The proliferative capability was measured by Cloning assay in different groups. The data was expressed as mean±SD, comparison among groups was performed by t-test. Significance was accepted when P<0.05.Results:1. After a period of irradiation of the A549cell line, we finally obtained two radiation resist cells which definite as the A549R2Gy-R and the A549R4Gy-R cell lines. A549R2Gy-R cell was radiation resist to conventional fractionated irradiation and A549R4Gy-R cell was radiation resist to hypofractionated irradiation. Microarray analysis shows there are1701(357raised, lowered1344) differentially expressed genes in A549R2Gy-R cells compared to the parental A549cell. In compared with A549cells, the hypofractionated resistance cell A549R4Gy-R has a different mRNA expression pattern of944genes were upregulated and2602genes were downregulated. When we focus the distinction on the A549R2Gy-R cell and A549R4Gy-R cell, we find that318genes were upregulated and699genes were downregulated. Several signaling pathways involved the radiation resistance when compare conventional fractionated radiotherapy with fractioned irradiation with a Pathway significant enrichment analysis, especially the PI3K signaling pathway kinase.2. According to the control group, the expressive level of PTEN、PI3K mRNA of the Radioresistant A549R cells are lower, and AKT1、mTOR mRNA expression are higher. Contrast to the Conventional Fractionated Radioresistant A549R cells, the expressive level of AKT1and mTOR mRNA of hypofractionated radioresistant cells are higher, while PTEN and PI3K expression are not changed significantly (P<0.05)3. Experiment result of radiosensitivity indicated that the abmty of cell line Radioresistant A549R forming colony in the testting group degraded contrasting with A group. With the increasing of radiation dose, forming colony ability decreased obviously. With the same radiation dose, colony rate in the D group was much less than that in the B group(P<0.05).Conclusion:1. Multiple genes and signaling pathways involved in the process of NSCLC radiotherapy resistance. The underline radioresistant mechanisms of conventional and hypofractionated radiotherapy need further elucidated for drug development to provide a new target.2. There were significant differences between the PTEN/PI3K/AKT/mTOR cell signaling pathways expression patterns of the two Radioresistant A549cell groups and control groups. Between the two Radioresistant A549cell groups,some genes show significant differences.LY294002can increase the radiosensitization of hypofractionated Radioresistant A549R4Gy-R cells.