The metal-binding domain of IGFBP-3 selectively delivers therapeutic molecules into cancer cells

Abstract

Conventional chemotherapy for cancer has limited specificity for cancer cells. Here, we investigate the possibility of improving the selectivity of chemotherapy by coadministering targeted biological modifier peptides. We show that the 22-amino acid metal-binding transporter domain (MBD) derived from insulin-like growth factor-binding protein-3 selectively targets cancer cells. The rate of MBD uptake by cells was measured using a panel of 54 human cancer cell lines and correlated with MBD cross-linking to cell surface transferrin receptor, caveolin 1, and integrin beta. Gene array data show that MBD uptake correlates with the expression of genes associated with cellular stress-coping mechanisms commonly upregulated in cancer (nuclear factor-kappaB, Hsp-70B). MBD-tagged peptides designed to inhibit such mechanisms have cytotoxic effects on a broad range of human cancer cell lines. The discriminant validity of these peptides as potential cotherapeutic agents was investigated by comparing their cytotoxicity to cancer cell lines versus normal human cell counterparts. Synergies between these peptides and marginally cytotoxic levels of 5-fluorouracil were demonstrated. Biodistribution data from in-vivo experiments in mice and rats confirm that MBD-tagged peptides and proteins preferably localize to specific tissues, such as kidney and pancreas. Intracardial injection of CCRF-CEM T-cell leukemia or MDA-MB-435 cells into Rag-2 mice establishes disseminated disease within 7 days. Twenty-five-day subcutaneous administration of a three-peptide cocktail (3 mg/kg) in combination with 5-fluorouracil in Rag-2 mice with established CCRF-CEM leukemia significantly reduces splenomegaly and bone marrow cancer cell burden. In a similar experiment using MDA-MB-435 cells, MBD-tagged peptides reduced human cell burden in bone marrow. Taken together, these data suggest that MBD-tagged molecules can be used as highly selective chemosensitizers in the treatment of hematological and disseminated malignancies.

Core Functional Sequence of C-terminal GAG-binding Domain Directs Cellular Uptake of IGFBP-3-derived Peptides

“…In our study, IGFBP-3 C-terminal peptides readily entered cells as CPPs in a GAG-dependent manner, extending previously published studies demonstrating their cell penetrating property [16,18,28]. GAGs exist in animals as a part of heavily glycosylated proteoglycans, the other component being the core proteins.…”

“…However, the upstream 8-mer sequence (KK-8) that contains 5 basic lysine residues was not able to permeate into the cells at all. Previously it was suggested that the 12-mer Cys-Cys loop in the peptide QW-14 may be important for its uptake activity [18], but in our study the replacement of the first cysteine in position 224 of the loop with glycine (QW-14mut) did not weaken the uptake much in both wt CHO K1 and the heparan sulfate-deficient pgs D-677 cells (Fig. 5-IIB and C).…”

“…As IGFBP-3 is a moderately abundant human protein in circulation, these peptides have enormous potential for applications in drug delivery as an alternative to the HIV-Tat derived cell penetrating peptide. Previous study indicates that IGFBP-3-derived peptides can selectively deliver therapeutic molecules into cancer cells, but the mechanism remains poorly understood [18]. There is some evidence that abnormal expression of glycosaminoglycans is present in cancer and found to correlate with clinical prognosis in several malignant neoplasms [39,40].…”

“…It was found that a 14-mer peptide present in the C-terminus of IGFBP-3 was sufficient for cellular uptake. Later, a longer 22-mer peptide expanded from the 14-mer was exploited as the carrier of therapeutic molecules selectively targeting cancer cells [18]. These two peptides encompass the Tf-binding domain, but not the complete caveolin-scaffolding domain.…”

“…It is not clear whether the GAG-binding domain in IGFBP-3 also uses cell surface GAGs as receptors to enter cells. In view of potential clinical application of IGFBP-3 and an increasing interest in using IGFBP-3 peptides for intracellular cargo delivery [18,20], we further characterized the role of the GAG-binding domain in cellular entry. We found that GAGs are indeed cell surface receptors for GAG-binding domain and within the GAG-binding domain the cell entry function and the GAG-binding function are physically separated.…”