Noncationic Peptides Obtained From Azurin Preferentially Enter Cancer Cells

Abstract

Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, A-helical peptides are unique. Unlike the cationic cell-penetrating peptides, A-helical antennapedialike, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively. [Cancer Res 2009;69(2):537-46]

Structural studies on Laz, a promiscuous anticancer Neisserial protein

“…50-77 in the mature form, termed p28) functions as a putative protein transduction domain responsible for azurin's penetration into cancer cells 12 through endocytotic and nonendocytotic mechanisms. 13 Based on crystal structures, bacterial azurins show structural similarity to ephrinB2, a ligand for the receptor tyrosine kinase EphB2. 14,15 Cell signaling through Eph/ephrin is involved in cancer progression.…”

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy

“…Amino acids 50-77 of azurin form a 2.9 kDa peptide fragment that encompasses the azurin α-helix and retains the preferential penetration of the whole protein, but also its antitumor activity in vitro and in vivo. [21][22][23][24] The antiproliferative activity appears to result from aa residues 11-18 of p28 (61-69 of azurin) binding to p53 in a region that does not inhibit the binding of Mdm2 or subsequent ubiquitination. 23 Since the molecular details and the kinetics of its interaction with p53 and, more importantly, with what domain(s) have not yet been clarified, a detailed study of the p28-p53 interaction could provide significant information on p28 action at the molecular level.…”

p28-Mediated Activation of p53 in G2–M Phase of the Cell Cycle Enhances the Efficacy of DNA Damaging and Antimitotic Chemotherapy

“…p28, amino acids 50 to 77 of azurin, a cupredoxin secreted by Pseudomonas aeruginosa, is a non-toxic, amphipathic, anionic cellpenetrating peptide that preferentially enters a wide variety of cancer cells (18,19). Upon entry, p28 binds to a mutational "cold spot" (20) within the DNA-binding domain (DBD) of p53 wt,mut , where it blocks the binding of the E3 ligase Cop1, inducing a significant post-translational increase in the level and activity of wild-type and mutant p53 (21)(22)(23)(24)(25)(26).…”

“…Chemopreventive agents such as the antimitotic Vinca alkaloids that inhibit cellular proliferation also indirectly lead to apoptosis (6,30). As such, p28 appears as a true cell-cycle-specific cytostatic agent that induces cytotoxicity indirectly via a p53 wt,mut -mediated block at G 2 -M (18,26). In this study, we assessed the effects of p28 alone and in combination with IC 20 and IC 50 concentrations of standard DNA-damaging and antimitotic agents on the degree of cytotoxic effect in p53 wt,mut,null -matched human prostate, breast cancer, glioblastoma, melanoma, and neuroblastoma cell lines as a function of cell proliferation and p53 status.…”