ADκ Fused With Cell-penetrating Peptide Inhibits The Growth And Migration Of Tumor Cells

Cancer has seriously threatened human life and health as its high incidence and mortality.Studies have shown that the overexpression of cyclin D1 is associated with tumor migration and poor prognosis.Cyclin D1 has become an important target for tumor therapy.In our previous studies,an anti-cyclin D1 single-chain variable fragment(sc Fv)with good anti-tumor effect in vitro and in vivo was successfully obtained through the screening of the phage antibody library.However,the anti-cyclin D1 sc Fv(ADκ)cannot directly cross the membrane into the cell to act on the intracellular cyclin D1.The cell-penetrating peptide can be used as a delivery vehicle to transport therapeutic agents across the cell membrane without damaging the cell membrane,which can effectively improve the cellular uptake and therapeutic effect of drugs.In addition,studies have shown that coupling cell-penetrating peptides to sc Fv can enhance the delivery of sc Fv into cells,which provides a new way for the tumor targeted therapy with sc Fv and has a good application prospect.In our previous study,we successfully constructed prokaryotic expression vectors of anti-cyclin D1 sc Fv fused with cell-penetrating peptide,ADκ-PTN and ADκ-TAT,and then purified the two kinds of recombinant proteins.The results showed that the fusion proteins ADκ-PTN and ADκ-TAT have good binding activity with cyclin D1,which indicated that fusion with cell-penetrating peptide does not affect the antigenbinding activity of sc Fv.However,it is still not clear whether the anti-cyclin D1 sc Fv fused with cell-penetrating peptide can cross the cell membrane of tumor cells and interact with intracellular cyclin D1,and whether it can inhibit the growth and migration of tumor cells.In this study,we investigated the inhibitory effects of ADκ fused with cell-penetrating peptide,ADκ-PTN and ADκ-TAT,on the growth and migration of tumor cells.The results are as follows:Firstly,the purified ADκ,ADκ-PTN and ADκ-TAT were used to treat breast cancer cells MCF-7 and liver cancer cells Hep G2.MTT assay was conducted to clarify whether ADκ,ADκ-PTN and ADκ-TAT could kill MCF-7 and Hep G2 cells,respectively,and determine the optimal treatment concentration.The results showed that both ADκ-PTN and ADκ-TAT could significantly reduce the survival rate of MCF-7 cells and Hep G2 cells,and then inhibit the growth and proliferation of tumor cells.Secondly,the fluorescence microscope observation experiments of MCF-7 cells and Hep G2 cells treated with fluorescence-labeled ADκ,ADκ-PTN and ADκ-TAT,and indirect immunofluorescence experiments of MCF-7 and Hep G2 cells treated with ADκ,ADκ-PTN and ADκ-TAT showed that anti-cyclin D1 sc Fv fused with penetrating peptides,ADκ-PTN and ADκ-TAT,could penetrate the cell membrane and enter into MCF-7 cells and Hep G2 cells.Furthermore,immunoprecipitation assay and Western Blot analysis showed that ADκ-PTN and ADκ-TAT could not only penetrate the cell membrane into the cell,but also specifically bind to intracellular cyclin D1.Finally,we analyzed the antitumor effects of ADκ、ADκ-PTN and AD-κ-TAT.Annexin V-FITC and PI double staining combined with flow cytometry analysis showed that both of ADκ-PTN and ADκ-TAT significantly induced apoptosis of MCF-7 cells and Hep G2 cells.The results of cell scratch assay showed that the wound healing rate of ADκ-PTN and ADκ-TAT treated groups was significantly lower than that of ADκ,indicating that both ADκ-PTN and ADκ-TAT could inhibit the migration of MCF-7cells and Hep G2 cells,respectively.Transwell assay showed that both MCF-7 cells and Hep G2 cells,the number of cells passing through the compartment in the ADκ-PTN and ADκ-TAT groups was significantly lower than that in the ADκ and PBS negative control group,but there was no significant difference between the ADκ group and the PBS negative control group,which indicated that ADκ-PTN and ADκ-TAT decreased the ability of invasion and infiltration of MCF-7 cells and Hep G2 cells in vitro,respectively.In conclusion,the anti-cyclin D1 sc Fv fused with cell-penetrating peptides can cross the cell membrane of tumor cells and interact with intracellular cyclin D1,and then effectively inhibit the growth and migration of tumor cells.This study not only lays the theoretical foundation for tumor therapy with antibody targeting cyclin D1,but also provides an experimental basis for tumor therapy targeting intracellular antigens.