Effects of de novo designed protein binders targeting EGFR and FGFR2 on pancreatic cancer cells
Objective:To investigate the effect of de novo designed epidermal growth factor receptor(EGFR)binding protein EGn and fibroblast growth factor receptor(FGFR)Ⅲc binding protein FG2 on pancreatic cancer cells.Methods:EGn and FG2 sequences were obtained from an existing study,and the proteins were amplified and expressed in Escherichia coli BL21(DE3)followed by purification through Ni-NTA affinity chromatography and size exclusion chromatography.The purified protein was tested with SDS-PAGE followed by Coomassie Brilliant Blue staining for purity evaluation.The expression level of EGFR and FGFR2 Ⅲc in PANC-1,MIA PaCa-2 and BxPC-3 cells was compared using Western blotting and real-time fluorescence quantitative PCR.The binding of EGn and FG2 in different pancreatic cancer cell lines was assessed by flow cytometry.The effect of EGn and FG2 on the phosphorylation of the target receptors or the down-stream extracellular signal-regulated kinase(ERK)molecules was analyzed by Western blotting.The effect of EGn and FG2 on the proliferation of PANC-1 and MIA PaCa-2 cells was evaluated by CCK-8 assay and colony formation assay.The effect of EGn and FG2 on the migration of PANC-1 cells was examined by Transwell assay.Results:EGn and FG2 proteins were successfully purified and can specifically bind to pancreatic cancer cell lines expressing target receptors,with the binding efficiency positively correlated with target receptor expression levels.EGn and FG2 can competitively inhibit the down-stream signaling of target receptors in PANC-1 cells,and significantly suppress the proliferation and migration of PANC-1 cells(P<0.05).However,their inhibitory effects on the down-stream signaling or proliferation in MIA PaCa-1 and BxPC-3 cells were relatively limited.Conclusion:EGn and FG2 demonstrate inhibitory effects on the proliferation and migration of PANC-1 cells with high expression of EGFR and ectopic high-expression of FGFR2 Ⅲc,indicating their potential therapeutic efficacy in pancreatic cancer and supporting the potential application of de novo designed binding proteins in targeted therapy for pancreatic cancer.
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