Design For Tumor-Targeting Antimicrobial Peptide Chimera And Its Anticancer Mechanism Study

Nowadays, the primary treatment of cancer includes surgery, radiotherapy,chemotherapy, immune therapy, monoclonal antibody therapy or other methods.While many chemotherapeutic agents have been developed, most have deleteriousside effects，because they are cytotoxic to both cancer cells and healthy cells andtissues. How to target cancer cells with high specificity and kill cancer cells with highefficiency remains an urgent demand for anticancer drugs. Antimicrobial peptides(AMPs), especially cationic antimicrobial peptides (CAPs), derived from variousspecies (including insects, amphibians, and humans) are bioactive peptides withpotential applications as anticancer agents. As new candidates for anticancer drugs,AMPs possess the advantages of high positive charges, amphipathic α-helices, shortamino acid sequences, low molecular weights, and weak antigenicity.We first used a whole bioinformatic analysis method as a platform to identifynew anticancer antimicrobial peptides (AMPs). Factor analysis was used to determinehow six parameters (net positive charges,%hydrophobic residues, meanhydrophobicity <H>, Grand average of hydropathicity, amphiphilicity <μH>, andsecondary structure) contributed to structure and activity relationship. We found thatthe net charges was the relative independent factor, which could be used for peptidesdesign. We derived a conserved sequence template (F/L/I+L/F+P+L/I/F+L/I/V+G/A+N/S/K+L/F+L+S/N/G+K/G/S+L+L) from temporins peptides. Thehydrophobic residues were at positions1,2,4,5,8,9,12, and13. Polar residues withpositive charges were most common at position3,7, and11, and position10was themost common site for polar residues with no charges or negative charges.Correspondingly, Glu-10was replaced by a Ser residue (temporin-Las) and thisanalog as well as the parent temporin-La were extended N-terminally with RGDwithout spacing to incorporate into the temporin sequence the integrin αvβ3homingdomain (RGD-La and RGD-Las). Based on these results, we designed a temporin-La analogue (temporin-Las) and related constructs containg the RGD tripeptide, theintegrin αvβ3homing domain (RGD-La and RGD-Las).MTT assay showed that temporin-La and its synthetic analogs inhibited cancercell proliferation in a dose-dependent manner. We detected a link between the netcharges and integrin αvβ3expression of cancer cell lines and the antitumor activitiesof these peptides. Peptide-cell pull-down experiments revealed that the bindingefficiency of temporin-Las (+4) was higher than that of temporin-La (+3), whileRGD-Las had the strongest affinity. Furthermore, the effect of cell surface charges onpeptide affinity was examined. The zeta potential of the cancer cell lines showed thatall the cell lines carried negative charges. Evidence was provided that the affinitybetween RGD-Las and tumor cell membranes was stronger than other tested peptidesusing a pull-down assay. Morphological changes on the cell membrane induced bytemporin-La and RDG-Las, respectively, were examined by scanning electronmicroscopy (SEM). Additionally, time-dependent morphological changes weredetected by confocal microscopy, where the binding process of RGD-Las to the cellmembrane could be monitored. Since no apoptotic mechanism could be detected ontumor cells treated with RGD-Las, a membrane-disturbing action seems to be themajor mechanism for cell death. At the same time, we observed a lot of pores on thedamaged cell membranes, consistent with the carpet model mechanism. The resultsindicate that the electrostatic interaction between these cationic peptides and theanionic cell membrane is a major determinant of selective cell killing. Thus, the RGDtripeptide is a valuable ligand motif for tumor targeting, which leads to an increasedanticancer efficiency by RGD-Las. These AMPs-derived peptides have clinicalpotential as specifically targeting agents for the treatment of αvβ3positive tumors.Poor penetration of anti-cancer drugs into tumors can be an important factor limitingtheir efficacy.Studying mouse SMMC-7721tumor models, we show that chimera RGD-Lasincreased tissue permeability in a tumor-specific manner, allowing co-administereddrugs (DOX) to penetrate into extravascular tumor tissue. Importantly, this effect didnot require the drugs to be chemically conjugated to the peptide. HE results showedthat the RGD-Las may induce necrosis of SMMC-7721xenografts in nude mice. Thus, co-administration of chimera RGD-Las may be a valuable way to enhance theefficacy of anti-cancer drugs while reducing their side effects, a primary goal ofcancer therapy research.In this study, we successfully set a whole bioinformatic analysis method as aplatform to identify new anticancer antimicrobial peptides (AMPs). We found that thenet charges was the relative independent factor, which could be used for peptidesdesign. We detected a link between the net charges and integrin αvβ3expression ofcancer cell lines and the antitumor activities of these peptides.The designed peptidesin tumor imaging showed that chimera RGD-Las could target tumor tissue in vivo,and allowing co-administered drugs (DOX) to penetrate into extravascular tumortissue. The results revealed increased anticancer efficiency of these analogssuggesting clinical potential as specifically targeted anticancer agents.