Synthetic Peptide Pep-RBD Suppresses Human MDA-MB-231Breast Cancer Cell Adhesion By Interrupting Rap1Signaling Pathway

Rap1(Ras Associate Protein1) is a member of Ras family of small GTPase, whichare small cytosolic proteins that act like cellular switches and are vital for effective signaltransduction. Rap1is inactive when in its GDP-bound form, and becomes active when itbinds to GTP.Based on the previous studies, the Rap binding domain of RalGDS binds to theGTP-bound form of Rap1with a very high affinity (KD=10nM), the affinity for theGDP-bound form is undetectably low, we have designed several peptides, called PepN,Pep-RBD and Pep-RBD-CPPs-FAM, which may have the ability to interfere withRap1GTP. The aim of this study was found those peptides, which come from the Rap1binding domain of Guanine nucleotide dissociation stimulator (RalGDS-RBD), whether ornot has ability for interfering with Rap1GTP. The result of flow cytometer shows thatPep-RBD-CPPs-FAM can permeate MDA-MB-231cell membrane, when cell incubatedwith Pep-RBD-CPPs-FAM for5min. The result of GST-RBD assay suggests that the levelof Rap1GTP has decreased dramatically when Pep-RBD or Pep-RBD-CPPs-FAM fordifferent concentration and time course. Surprisely, the result of GST-RBD assay showesthat the level of RasGTP or RacGTP in MDA-MB-231cell has not been affected by thosepeptides. Furthermore, we show that Pep-RBD or Pep-RBD-CPPs-FAM can inhibitintegrin-mediated cell adhesion without altering cell viability.In summary, the peptide Pep-RBD can decrease the level of Rap1GTP inMDA-MB-231cell effectively. This study focuses on the research thatPep-RBD-CPPs-FAM and Pep-RBD can interact with Rap1GTP in MDA-MB-231humanbreast carcinoma cells, which will provide a significant preview to the research ofRap1GTP specific inhibitor.