[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor-binding protein 4 (IGFBP-4/IBP-4) has potent IGF-independent anti-angiogenic and antitumorigenic effects. In this study, we demonstrated that these activities are located in the IGFBP-4 C-terminal protein fragment (CIBP-4), a region containing a thyroglobulin type 1 (Tg1) domain. Proteins bearing Tg1 domains have been shown to inhibit cathepsins, lysosomal enzymes involved in basement membrane degradation and implicated in tumor invasion and angiogenesis. In our studies, CIBP-4 was shown to internalize and co-localize with lysosomal-like structures in both endothelial cells (ECs) and glioblastoma U87MG cells. CIBP-4 also inhibited both growth factor-induced EC tubulogenesis in Matrigel and the concomitant increases in intracellular cathepsin B (CatB) activity. In vitro assays confirmed CIBP-4 capacity to block recombinant CatB activity. Biodistribution analysis of intravenously injected CIBP-4-Cy5.5 in a glioblastoma tumor xenograft model indicated targeted accumulation of CIBP-4 in tumors. Most importantly, CIBP-4 reduced tumor growth in this animal model by 60%. Pleiotropic anti-angiogenic and anti-tumorigenic activities of CIBP-4 most likely underlie its observed therapeutic potential against glioblastoma.
Neoplasia (New York, N.Y.) 05/2013; 15(5):554-67. DOI:10.1593/neo.13212 · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Soricidin is a 54-amino acid peptide found in the paralytic venom of the northern short-tailed shrew (Blarina brevicauda) and has been found to inhibit the transient receptor potential of vallinoid type 6 (TRPV6) calcium channels. We report that two shorter peptides, SOR-C13 and SOR-C27, derived from the C-terminus of soricidin, are high-affinity antagonists of human TRPV6 channels that are up-regulated in a number of cancers. Herein, we report molecular imaging methods that demonstrate the in vivo diagnostic potential of SOR-C13 and SOR-C27 to target tumor sites in mice bearing ovarian or prostate tumors. Our results suggest that these novel peptides may provide an avenue to deliver diagnostic and therapeutic reagents directly to TRPV6-rich tumors and, as such, have potential applications for a range of carcinomas including ovarian, breast, thyroid, prostate and colon, as well as certain leukemia's and lymphomas.
PLoS ONE 03/2013; 8(3):e58866. DOI:10.1371/journal.pone.0058866 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several reports have shown that secreted clusterin (sCLU) plays multiple roles in tumor development and metastasis. Here, we report on a 12-mer sCLU binding peptide (designated P3378) that was identified by screening a phage-display peptide library against purified human sCLU. Differential resonance perturbation nuclear magnetic resonance using P3378 and a scrambled control peptide (designated P3378R) confirmed the P3378-sCLU interaction and demonstrated that it was sequence specific. P3378 and P3378R peptides were conjugated to an Alexa680 near infrared fluorophore (NIRF) and assessed for their tumor homing abilities in in vivo time-domain fluorescence optical imaging experiments using living 4T1 tumor bearing BALB/c mice. When injected in separate animals, both peptides accumulated at the tumor site, however the NIRF-labeled P3378 peptide was retained for a significant longer period of time than the P3378R peptide. Similar observations were made after simultaneously injecting the same tumor-bearing animal with a peptide mixture of P3378 DyLight (DL)680 and the P3378R-DL800. Coinjection of P3378-DL680 with excess unlabeled P3378 blocked tumor accumulation of fluorescent signal while excess P3378R control peptide did not confirming the sequence specificity of the tumor accumulation. Finally, ex vivo fluorescence microscopy of these tumors confirmed the presence of P3378-DL680 in the tumor and its colocalization with CLU. These results confirm the tumor targeting specificity of the P3378 CLU-binding peptide and suggest its usefulness for the in vivo monitoring of solid tumors secreting detectable levels of CLU.
International Journal of Cancer 09/2012; 131(5):E681-92. DOI:10.1002/ijc.27368 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Noninvasive investigation of cellular and molecular processes becomes possible through the novel techniques, one of which is molecular imaging, where enhanced sensitivity is a key component for clinic translation of the technique. In this presentation, spontaneously forming, small unilamellar vesicles (ULVs) (30 nm in diameter) were used as a platform to build a bi-modal [i.e., optical and Magnetic Resonance Imaging (MRI)] targeted contrast agent for the molecular imaging of brain tumors. Small ULVs were loaded with a gadolinium (Gd) chelated lipid (Gd-DPTA-BOA), functionalized with targeting antibodies (anti-EGFR monoclonal and anti-IGFBP7 single domain), and incorporated a near infrared dye (Cy5.5). The resultant ULVs were characterized in vitro using small angle neutron scattering (SANS), in phantom MRI and dynamic light scattering (DLS). Targeted (with antibodies) and nontargeted-Gd loaded sULVs labeled with Cy5.5 were assessed in vivo in a mice brain tumor model using both optical imaging and MRI. The results demonstrated that a spontaneously forming, nanosized ULV loaded with a high payload of Gd can selectively target and image, using MR and optical imaging, brain tumor vessels when functionalized with antibodies. The unique features of these targeting ULVs make them promising molecular MRI contrast agents.
[Show abstract][Hide abstract] ABSTRACT: The posttranslational modification of therapeutic proteins with terminal sialic acids is one means of improving their circulating half-life, thereby improving their efficiency. We have developed a two-step in vitro enzymatic modification of glycoproteins, which has previously only been achieved by chemical means [Gregoriadis G, Jain S, Papaioannou I, Laing P (2005) Int J Pharm 300:125-130). This two-step procedure uses the Campylobacter jejuni Cst-II α2,8-sialyltransferase to provide a primer on N-linked glycans, followed by polysialylation using the Neisseria meningitidis α2,8-polysialyltransferase. Here, we have demonstrated the ability of this system to modify three glycoproteins with varying N-linked glycan compositions: the human therapeutic proteins alpha-1-antitrypsin (A1AT) and factor IX, as well as bovine fetuin. The chain length of the polysialic acid addition was optimized by controlling reaction conditions. After demonstrating the ability of this system to modify a variety of proteins, the effect of polysialylation on the activity and serum half-life of A1AT was examined. The polysialylation of A1AT did not adversely affect its in vitro inhibition activity against human neutrophil elastase. The polysialylation of A1AT resulted in a significantly improved pharmacokinetic profile when the modified proteins were injected into CD-1 mice. Together, these results suggest that polysialylated A1AT may be useful for improved augmentation therapy for patients with a deficiency in this protein and that this modification may be applied to other therapeutic proteins.
Proceedings of the National Academy of Sciences 05/2011; 108(18):7397-402. DOI:10.1073/pnas.1019266108 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor-binding protein 7 (IGFBP7) is an abundant, selective and accessible biomarker of glioblastoma multiforme (GBM) tumour vessels. In this study, an anti-IGFBP7 single-domain antibody (sdAb) was developed to target GBM vessels for molecular imaging applications.
Human GBM was modelled in mice by intracranial implantation of U87MG.EGFRvIII cells. An anti-IGFBP7 sdAb, isolated from an immune llama library by panning, was assessed in vitro for its binding affinity using surface plasmon resonance and by ex vivo immunobinding on mouse and human GBM tissue. Tumour targeting by Cy5.5-labelled anti-IGFBP7 sdAb as well as by anti-IGFBP7 sdAb conjugated to PEGylated Fe₃O₄ nanoparticles (NPs)-Cy5.5 were assessed in U87MG.EGFRvIII tumour-bearing mice in vivo using optical imaging and in brain sections using fluorescent microscopy.
Surface plasmon resonance analyses revealed a medium affinity (K(D)=40-50 nM) binding of the anti-IGFBP7 sdAb to the purified antigen. The anti-IGFBP7 sdAb also selectively bound to both mouse and human GBM vessels, but not normal brain vessels in tissue sections. In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour. Similarly, the anti-IGFBP7 sdAb-functionalised PEGylated Fe₃O₄ NP-Cy5.5 demonstrated enhanced tumour signal compared with non-targeted NPs. Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe₃O₄ NPs selectively in GBM vessels.
Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.
British Journal of Cancer 10/2010; 103(10):1606-16. DOI:10.1038/sj.bjc.6605937 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During winter 2004 and 2005, two field and glasshouse experiments were conducted to evaluate the response of 471 chickpea genotypes to Ascochyta rabiei, as Ascochyta blight (AB) disease in Chickpea (Cicer aurietimum). Frequent rainfall at flowering and pod formation stages made favorite conditions conducive for the infection and symptoms expression. So, the genotypes with high level of tolerance at seedling stage proved response to the pathogen under field condition. Disease at seedling and adult plant stage exhibited high association, although level of infection was higher at adult plant stage. In glasshouse 65 genotypes at seedling stage and in field experiment 14 genotypes at pod formation stage were resistant to the pathogen. Following green house and field screening methods, six genotypes FLIP98-229C, FLIP82-150C,NCS 950204, NCS 950219, NCS 9903 and PaidarxParbat from NARC and six lines (FLIP 00-20C, FLIP 02-18C, FLIP 02-44C, FLIP 97-120C, FLIP 02-39C and FLIP 97-102C) from ICARDA found resistant for multilocational / agronomic evaluation and use as resistant parent trials for high yielding AB resistance breeding varieties.