The use of a combinatorial library method to isolate human tumor cell adhesion peptides

Abstract

Tumor cell progression is dependent in part on the successful adhesive interactions of the cells with the extracellular matrix. In this study, a new approach is described to isolate linear peptide ligand candidates involved in cellular adhesion. A synthetic combinatorial peptide library based on the 'one-bead-one-peptide' concept was incubated with live human prostate cancer cells for 90 min at 37 degrees C. The peptide bead coated with a monolayer of cells was then isolated for microsequencing. The DU145 (DU-H) cells were chosen since they have been previously characterized as containing elevated levels of a laminin receptor for cell adhesion, the alpha 6 beta 1 integrin on the cell surface. The use of a function-blocking antibody (GoH3) allows for the detection of peptides which are alpha 6-specific ligand candidates. From two different libraries (linear 9-mer and 11-mer) of a total of 1,500,000 beads, 68 peptide beads containing attached cells were isolated. These positive beads were then retested to determine the ability of the GoH3 antibody to block binding of the cells to the peptide beads. The alpha 6 integrin candidate peptide beads (five in total) were recovered and two of the beads were microsequenced. These two peptides, RU-1 (LNIVS-VNGRHX) and RX-1 (DNRIRLQAKXX), resemble the previously reported active peptide sequences (GD-2 and AG-73) from native laminin. The RU-1, RX-1 and AG-73 peptides were tested for their ability to support cell attachment and to bind the cell surface of DU-H prostate carcinoma cells in suspension using fluorescence-activated cell-sorting (FACS) analysis. Both RU-1 and AG-73 peptides supported cellular attachment within 1 h. In contrast, after 1 h, EHS laminin supported both cellular attachment and spreading. The RX-1 peptide exhibited only weak binding to the DU-H prostate carcinoma cells. FACS analysis indicated that AG-73 peptide attached to tumor cell surfaces over a range of concentrations, whereas the RU-1 peptide showed a homogeneous concentration required for attachment. The described strategy for screening a random peptide library offers three advantages: (i) ligands for conformationally sensitive receptors of adhesion can be isolated using live cells; (ii) specific binding can be selected for using function-blocking antibodies; and (iii) peptides supporting adhesion independent of spreading properties can be distinguished. In principle, specific adhesive peptides without prior knowledge of the sequence could be isolated for any epithelial cell surface receptor for which a function-blocking reagent is available.

Identification of novel targeting peptides for human ovarian cancer cells using “one-bead one-compound” combinatorial libraries

“…In addition, peptides containing D-amino acids and organic moieties are generally much more resistant to proteolysis, which is critical for effective cancer therapeutics. Using this approach, we have identified several unique ligands for lymphoma (17), prostate cancer (14,15), and lung cancers (16,18). Here, we report the identification of short cyclic peptide ligands that bind to cell surface receptors of four ovarian adenocarcinoma cell lines (CaOV-3, ES-2, SKOV-3, and OVCAR-3).…”

Advances in Diagnostic and Treatment Modalities for Intracranial Tumors

“…Random screening of peptide libraries is an efficient method to define tumor‐specific peptides,224 and has been done for several canine cancers including lymphoma, melanoma, and glioma 130, 225, 226. Tumor‐specific targeting of glioma using peptides recognizing alpha 3 beta 1 integrin has been demonstrated in human cancers 227.…”

Tumor-Targeting Peptides for Therapeutic Gene Delivery (97breast)

“…Many existing therapies could be made more effective if site-specific delivery could be achieved, particularly vectors for cancer gene therapy (42)(43)(44)(45)(46). The utility of adenovirus vectors for gene therapy is being intensely investigated, and there are a number of genetic approaches that are being explored for the treatment of cancer.…”

“…Here we have been exploring the homing ability of angiogenic vasculature-homing peptides by incorporating them as targeting devices for gene delivery systems. Of all the vector systems currently being explored, adenoviruses have the greatest potential to test the principles of cancer gene therapy (37,(42)(43)(44)(45)(46). Here we plan to develop a new approach for vector targeting.…”

“…Phage display of random peptide libraries has proven to be successful in the isolation of peptides capable of binding to integrins (38)(39)(40), growth factor receptors (41), and other tumor cell-associated proteins (42)(43)(44). Moreover, in vivo phage targeting has allowed us to identify several peptides that home to vasculature of specific organs as well as to tumor neovasculature (45)(46)(47).…”