RGD4C peptide mediates anti-p21Ras scFv entry into tumor cells and produces an inhibitory effect on the human colon cancer cell line SW480

Abstract

Background We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. Methods RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. Results RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. Conclusion The 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-driven cancers.

BR2 cell penetrating peptide effectively delivers anti-p21Ras scFv to tumor cells with ganglioside expression for therapy of ras-driven tumor

“…Tumor-specific CPPs penetrate tumor cells by binding with their receptors on the cell surface [ 23 – 25 ]. The integrin is the receptor of RGD cell membrane penetrating peptide, Huang et al in our laboratory used RGD to delivery anti-p21Ras scFv into integrin expressing tumors [ 26 , 27 ]. The matrix metalloproteinase 2(MMP2) is the receptor of activatable cell-penetrating peptide ACPP, Du et al in our laboratory used ACPP cell membrane penetrating peptide to delivery anti-p21Ras scFv into MMP2 expressing tumors [ 28 , 29 ].…”

“…Compared with the anti-p21Ras scFv carried by RGD or ACPP, the anti-p21Ras scFv carried by BR2 into tumor cells had the same antitumor effect [ 26 , 29 ]. The receptor of RGD is integrin, the receptor of ACPP is MMP2, and the receptor of BR2 is GS.…”

RGD peptide in cancer targeting: Benefits, challenges, solutions, and possible integrin–RGD interactions

“…Colorectal cancer (CRC) is a complex disease with several molecular subgroups that can interact with RGD peptides. 10 , 233 , 234 The interaction of integrins with RGD peptides is important in the development, metastasis, and angiogenesis of colorectal cancer and colon cancer. Several integrins, including αvβ3, αvβ5, αvβ6, α5β1, and α2β1, can interact with RGD peptides in CRC.…”

Mechanisms of Action and Limitations of Monoclonal Antibodies and Single Chain Fragment Variable (scFv) in the Treatment of Cancer

“…Other alternative vector strategies for delivering scFvs to the tumor cells included “cell-penetrating peptides” (CPPs), which are natural or synthetic peptides with the ability to interact with cellular membranes for the internalization of cells and effective intracellular delivery. CPPs had low cytotoxicity and immunogenicity [ 160 ] and have been widely used as vehicles for the administration of scFv with a Ras-blocking effect in colon cancer [ 161 ] and lung cancer [ 162 ]; these peptides improved the intracellular administration and orientation, thus favoring the antitumoral activity of the scFv.…”