Abstract
BackgroundAn anti-p21Ras scFv can specifically bind with mutant and wild-type p21Ras but cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate scFv penetration into tumor cells and produce an inhibitory effect.MethodsRGD4C-linker-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed, and the fusion proteins were expressed in E. coli and purified with HisPur Ni-NTA. RGD4C-linker-EFGP was used to test the factors affecting RGD4C penetration of the tumor cell membrane. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. Moreover, immunocytochemistry was used to detect the ability of RGD4C-p21Ras-scFv to penetrate SW480 cells. Cell migration, colony formation, cell killing, and apoptosis assays were used to assess the inhibitory effect of RGD4C-p21Ras-scFv on SW480 cells in vitro.ResultsThe RGD4C peptide could target tumor cells, but endocytosis inhibitors and a low temperature inhibited RGD4C peptide endocytosis into cells, and tumor cell entry was time and concentration dependent. Additionally, a change in the cell membrane potential did not affect penetrability. We found that RGD4C-p21Ras-scFv could penetrate SW480 cells; effectively inhibit the growth, proliferation and migration of SW480 cells; and promote apoptosis in SW480 cells. ConclusionThe RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of RAS-mutant colorectal cancer.
Septin remodeling is essential for the formation of cell membrane protrusions (microtentacles) in detached tumor cells
