RGDechi-hCit: αvβ3 Selective Pro-Apoptotic Peptide as Potential Carrier for Drug Delivery into Melanoma Metastatic Cells

Abstract

αvβ3 integrin is an important tumor marker widely expressed on the surface of cancer cells. Recently, we reported some biological features of RGDechi-hCit, an αvβ3 selective peptide antagonist. In the present work, we mainly investigated the pro-apoptotic activity of the molecule and its ability to penetrate the membrane of WM266 cells, human malignant melanoma cells expressing high levels of αvβ3 integrin. For the first time we demonstrated the pro-apoptotic effect and the ability of RGDechi-hCit to enter into cell overexpressing αvβ3 integrin mainly by clathrin- and caveolin-mediated endocytosis. Furthermore, we deepened and confirmed the selectivity, anti-adhesion, and anti-proliferative features of the peptide. Altogether these experiments give insight into the biological behavior of RGDechi-hCit and have important implications for the employment of the peptide as a new selective carrier to deliver drugs into the cell and as a therapeutic and diagnostic tool for metastatic melanoma. Moreover, since the peptide shows a pro-apoptotic effect, a great perspective could be the development of a new class of selective systems containing RGDechi-hCit and pro-apoptotic molecules or other therapeutic agents to attain a synergic action.

Deciphering RGDechi peptide‐α5β1 integrin interaction mode in isolated cell membranes

“…RGDechi encompasses a cyclic portion, containing the RGD triad for integrin binding, and a linear sequence derived from the C‐terminal fragment of the echistatin protein conferring specificity for β 3 subunit, as demonstrated by mutation and photoaffinity cross linking experiments and NMR conformational analysis combined with docking studies . In vitro and in vivo studies proved the selective binding of this peptide to α v β 3 integrin and a recent structural analysis performed in presence of isolated cell membranes has detailed the key structural determinants of this interaction . Noteworthy, the structural data confirmed that the selectivity of the peptide derives from its combined nature: while the RGD motif recognizes the extracellular domain of α v β 3 (but also other integrins), the C‐terminal fragment provides specificity for α v β 3 .…”

“…The effect of RGDechi (synthesized as previously reported) on the adhesion of K562 cells seeded onto fibronectin (extracellular matrix recognizing a range of different integrins, including α v β 3 and α 5 β 1 receptors) or, alternatively, on α 5 β 1 antibody was studied in the specific cell adhesion buffer as reported in a previous manuscript …”

“…As shown in Figure A, RGDechi decreases the α 5 β 1 mediated adhesion of K562 cells plated onto fibronectin having similar effect of cRGDfK, a α v β 3 /α v β 5 /α 5 β 1 antagonist used as positive control and the adhesion inhibition by RGDechi occurs in a concentration‐dependent mode (Figure B). Incubation with scrambled peptide (Ac‐KPGRGHNDPDPG hCit DeMHAT‐OH), utilized as negative control, does not decrease K562 adhesion onto matrix protein or specific antibody. To highlight the binding to α 5 β 1 integrin, a further adhesion assay was carried out onto anti‐α 5 β 1 antibody coated plate (Figure C).…”

“…Successively, a blocking step of 1 h at room temperature for nonspecific binding was done with 1.5% BSA in PBS. K562 cells were suspended and mixed for 30 min at 4°C to prevent receptor internalization, in Hank's balanced salt solution with peptides (50 μM) or anti‐α 5 β 1 antibody (10 μg mL −1 ). Afterward, cells were plated on precoated plates (1.5 × 10 4 cells/well).…”

A selective αvβ5 integrin antagonist hidden into the anophelin family protein cE5 from the malaria vector Anopheles gambiae

“…The effect of peptides on cell adhesion on different matrices or specific antibodies was tested on 96 multiplates (NUNC MaxiSorp from Dasit Sciences, Milano, Italy) coated with vitronectin, fibronectin or collagen (10 μg mL −1 ) (Millipore, USA) or α v β 3 and α v β 5 antibodies (10 μg mL −1 ) (Millipore, USA) . About 1.5 × 10 4 cells/well were seeded and incubated for 1 hr in the presence (50 μM) of peptides or cE5 (expressed and purified as described elsewhere) .…”