The results were examined with a confocal microscope (FV300/FV500 Olympus). LNCaP vs PC-3RR, DU145RR and LNCaPRR) recognized by LC-MS/MS. Open in a separate window Physique 6 Disease and function analysis of the common important differentially expressed proteins associated with CaP radioresistance in paired CaP cell lines.The model for understanding the mechanism of radioresistance and identifying new therapeutic targets. In the current study, comparison of three established CaP-RR cell lines to CaP parental cell lines recognized 19 common protein differences related to 3 significant signaling pathways involved in CaP radioresistance using a label-free LC-MS/MS proteomic technique. In addition, the recognized main pathway proteins were further validated in CaP-RR cell lines and PC-3RR-luc tumor xenografts by western blot and IHC, respectively. Furthermore, one selected potential glycolysis marker, ALDOA, was functionally verified in CaP-RR cells for increasing radiosensitivity. In this study, we established three novel CaP-RR (PC-3RR, DU145RR and LNCaPRR) cell lines derived from clones that experienced survived after irradiation which represent androgen-responsive (LNCaP) and androgen-nonresponsive (PC-3 and DU145) stages during CaP progression and examined the newly established cell lines with respect to proliferation, invasion and migration, and colony formation after a range of ionizing radiation exposure. We exhibited that reduced cell proliferation (Fig. 1S), increased invasion and migration and increased colony formation ability in three CaP-RR cell lines compared to untreated CaP-control cell lines13, indicating the reduced cell growth and increased progression and radiation resistance in the newly established sublines. In addition, the two cell populations (CaP-RR Batimastat sodium salt vs CaP-control cells) were significantly separated by PCA (Fig. 2S). These data confirmed that this CaP-RR cells are radioresistant and obviously different from CaP-control cells, which is suitable for proteomics analysis. After comparing three paired CaP and CaP-RR cell lines, we recognized protein difference varying from 299 to 391. To investigate the association of recognized protein profiles with signaling pathways, we found 151/299, 180/391, 163/360 proteins were mapped with pathway proteins in paired PC3/PC-3RR, DU145/DU145RR and LNCaP/LNCaPRR cell lines, respectively, indicating the link of the recognized proteins with signaling pathways in CaP radioresistance. These mapped proteins were found to be up-regulated or down-regulated, with different locations in CaP cells including cytoplasm, nucleus, plasma membrane, extracellular space. Our results indicate that this proteins differentially expressed in CaP and CaP-RR cells are associated with signaling pathways which demonstrate multiple functions in CaP radioresistance, suggesting that it is important to investigate these functions in the future studies. In this study, 19 proteins overlapped among three paired CaP cell lines, which were involved in different functions including glycolysis, EMT, Batimastat sodium salt signal transduction and redox. ALDOA was reported to affect the glycolysis pathway in PC-3 cells14 and functions as an oncogene in the highly metastatic pancreatic malignancy15. AHSG is usually a tumor antigen Batimastat sodium salt found in glioblastoma, breast malignancy and pancreatic malignancy16. As glycolytic proteins, ALDOA and AHSG were both up-regulated in CaP-RR cell lines analyzed by LC-MS/MS, indicating glycolysis is usually involved in CaP GDF1 radioresistance. Recent studies exhibited that EMT affects therapeutic resistance17. Vimentin is usually a symbol of the acquisition of mesenchymal characteristics. In this study, 2-, 6- and 7-fold changes of Vimentin were found to be increased in CaP-RR (PC-3RR, DU145RR, LNCaPRR) cells compared with CaP (PC-3, DU145 and LNCaP) cells, respectively, indicating that EMT is usually correlated with CaP radioresistance. This result is also in line with our previous statement13. YWHAE.