Targeting Heat Shock Proteins on Cancer Cells:  Selection, Characterization, and Cell-Penetrating Properties of a Peptidic GRP78 Ligand

Abstract

Peptidic ligands can be used for specific cell targeting and the delivery of payloads into the target cell. Here we describe the screening of a pool of cyclic peptide phage display libraries using whole-cell panning against human melanoma cell line Me6652/4. This strategy resulted in the selection of the cyclic 13-mer Pep42, CTVALPGGYVRVC, which showed preferential internalization into melanoma cell line Me6652/4 versus the reference cell line Me6652/56. This translocation is a receptor-mediated process that does not require electrostatic interactions nor does it involve transfer to the lysosomal compartment. The cellular receptor for Pep42 was identified as the surface membrane form of glucose-regulated protein 78 (GRP78), a member of the heat shock protein family and a marker on malignant cancer cells. The cellular uptake and intracellular trafficking of Pep42-Quantum Dot conjugates was monitored by confocal laser microscopy, and colocalization within the endoplasmic reticulum was observed. The uptake of Pep42 could be blocked by a monoclonal antibody against the identified receptor. Furthermore, Pep42 was shown to target specifically GRP78-expressing cancer cells. The in vitro cytotoxicity of a Pep42-Taxol conjugate was evaluated by flow cytometry wherein the conjugate was shown to induce apoptosis and was more effective in promoting programmed cell death in Me6652/4 cells. In summary, the data presented suggest that cyclic peptide Pep42 might be a powerful tool in the construction of drug conjugates designed to selectively kill malignant cancer cells.

The Escherichia coli Subtilase Cytotoxin A Subunit Specifically Cleaves Cell-surface GRP78 Protein and Abolishes COOH-terminal-dependent Signaling

“…Previous studies using transmembrane prediction programs have suggested that GRP78 has four potential transmembrane domains at the cell surface: I (amino acids (aa) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17], II (aa 29 -45), III (aa 222-242), and IV (aa 414 -431) (34). However, the studies performed here demonstrate that at least residues 416 -417 are exposed on the cell surface, as this is the sole SubA cleavage site, and we observe cell-surface cleavage.…”

“…GRP78 at the cell surface is detected in multiple types of cancer cells both in vitro and in vivo and is usually not present on non-malignant cells (9,10). Furthermore, the expression of GRP78 on the cell surface is associated with tumor progression, metastasis, and a poor prognosis (11).…”

Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) Secretion and Cell Surface Binding Are Modulated by KDEL Receptors

“…Additional proteins with ER retention signals are also detected extracellularly, including GRP78 (BiP) and GRP94 (Hsp90␤), and there is evidence for the function of these proteins in the extracellular space. For example, GRP78 was first identified at the surface of malignant lymphocytes (17), and the elevated levels observed on the surface of malignant cells have been exploited to successfully target and kill these cells with small molecules (18,19). GRP94 is similarly detected at the membrane in the nervous system, where it was found to regulate cell migration (20).…”

Potential of phage-displayed peptide library technology to identify functional targeting peptides

“…A search of present literature published between 2005 and 2006 revealed > 30 papers that disclose the identification of peptides using phage display, which were demonstrated to specifically target proteins in vitro. The technology has been used in attempts to develop novel targeting therapeutics or diagnostic reagents for human cancers [23,[40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58], HIV infection [59,60], human papilloma virus infection [61], obesity [62], bacterial infection [63][64][65][66], parasitic infection [67], autoimmune disorders [68], thrombosis and inflammation [69,70], Alzheimer's disease [71] and increasing vaccine efficacy [72]. However, the in vivo therapeutic efficacy of these novel peptides remains to be determined.…”