Abstract
BackgroundWe 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.MethodsRGD4C-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.ResultsRGD4C-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.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-driven cancers.
Highlights
We prepared an anti-p21Ras single-chain variable fragment antibody (scFv) which could bind with mutant and wild-type p21Ras
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 rasdriven cancers
BCA assay showed that the concentration of purified anti-p21Ras scFv, which was not codon optimized, was 0.96 mg/ml, and that of RGD4C-linker-scFv was 1.06 mg/ml
Summary
We prepared an anti-p21Ras scFv which could bind with mutant and wild-type p21Ras. Surgery remains the only effective curative option for colorectal cancer [6], 50– 60% of tumors have metastasized when diagnosed, resulting in metastatic colorectal cancer (mCRC), which is incurable in most cases [7, 8]. The application of targeted molecular drugs, such as bevacizumab, cetuximab and panitumumab [9], has led to a significant improvement in the survival rate of patients with metastatic colorectal cancer [10]. Ras (K-ras/Nras) mutation testing is highly recommended in the National Comprehensive Cancer Network (NCCN) guidelines and other guidelines [13,14,15]
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