The Artificially Synthesized Gene And Recombinantly Expressed Mutant Small Peptide RLj-RGD4 Inhibited Mouse Lewis Lung Cancer Cells

rLj-RGD3 is a recombinant protein with three RGD(Arg-Gly-Asp)motifs and had a molecular weight of 14.5kDa derived from the buccal gland of Japanese lamprey.A previous study showed that rLj-RGD3 exhibits potent antitumor function.To reduce the potential risk of immunogenicity in the biopharmaceutical application of this antitumor protein,the present study investigated the inhibitory effect of the truncated peptide rLj-RGD4 on mouse lung cancer cells.The small peptide rLj-RGD4 is a deletion mutant of the wild-type rLj-RGD3 peptide,and the gene of rLj-RGD4 was artificially synthesized and ligated into the pET23 b vector.A recombinant plasmid positive for the rLj-RGD4 gene,pET23b-RGD4,was transformed into Escherichia coli(E.coli)BL21 cells,and the recombinant mutant protein was expressed and purified.The recombinant protein rLj-RGD4 contained four RGD sequences and had a molecular weight of only 6.27 kDa.rLj-RGD3 protein as a positive control group during the experiment.The result of cell proliferation assay demonstrated that rLj-RGD4 inhibited the proliferation of mouse Lewis lung cancer cells in a dose-dependent manner.The half maximal inhibitory concentration(IC50)of rLj-RGD4 was 45.83 ?mol/L;In order to determine the effect of rLj-RGD4 protein on Lewis cells migration toward bFGF,we used the transwell containing insert filter(8.0 gm pore size,polycarbonate filter)The results showed that rLj-RGD4 significantly inhibited Lewis lung cancer cell migration in a dose-dependent manner.Cell invasion requires penetration through the basement membrane layer located between cell membrane and ECM.In the present study,an artificial basement membrane,composed of matrigel(concentration: 4 mg/mL),was coated onto the polycarbonate membrane of a Transwell cell culture plate to simulate the basement membrane in vivo.The results showed that rLj-RGD4 significantly inhibited Lewis lung cancer cell invasion in a dose-dependent manner.The results of Hoechst staining and flow cytometry demonstrated that rLj-RGD4 significantly induced apoptosis of Lewis lung cancer cells.In the present study,western blot analysis was conducted to verify the effects of rLj-RGD4 on the expression of the caspase-3 and Bcl-2 proteins in Lewis lung cancer cells.The results showed that rLj-RGD4 induced upregulation of caspase-3 expression and downregulation of Bcl-2 expression in a dose-dependent manner.Compared with rLj-RGD3,rLj-RGD4 exhibited a slightly weaker effect.However,due to its low molecular weight(only 6.27 kDa)and potent antitumor function,rLj-RGD4 shows more promising prospects for utilization as an antitumor drug.