Biotinylated polyacrylamide-based metal-chelating polymers and their influence on antigen recognition following conjugation to a trastuzumab fab fragment

We report the synthesis and characterization of metal-chelating polymers (MCPs) with a terminal biotin and a polyacrylamide backbone harboring multiple diethylenetriaminepentaacetic acid (DTPA) chelating sites. These polymers are conjugated to a streptavidin (SAv)-modified Fab fragment of trastuzumab (tmFab) and subsequently complexed with (111)In through DTPA. Trastuzumab has specific targeting ability toward human epidermal growth factor receptor-2 (HER2), which is overexpressed on some types of breast cancer cells and ovarian cancer cells. (111)In can generate Auger electrons which cause lethal DNA double strand breaks. The radioimmunoconjugates (RICs) were designed to target HER2 overexpressing cancer cells and carry multiple copies of (111)In to these cells. The mole maximum specific activities of these polymers were investigated by loading the polymers with (111)In at an increasing (111)In to polymer ratio. The polymers show 55-fold to 138-fold higher maximum specific activity than DTPA modified tmFab-SAv. Moreover, the HER2 immunoreactivities of these RICs were evaluated by measuring their specific binding ability toward HER2 overexpressing SKOV-3 ovarian cancer cells. The results demonstrate that although in the presence of polymer there is increased nonspecific binding, HER2 targeting ability was retained, ensuring the radionuclide delivery ability of these RICs.     

Evaluation of a maleimido derivative of NOTA for site-specific labeling of affibody molecules

Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMA-NOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule Z(HER2:2395) resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys(61)]-Z(HER2:2395) with (111)In gave a yield of >95% after 20 min at 60 °C. In vitro cell tests demonstrated specific binding of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) was 8.2 ± 0.9% IA/g and the tumor-to-blood ratio was 31 ± 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) and [(111)In-MMA-DOTA-Cys(61)]-Z(HER2:2395) demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.     

Identification of novel nuclear localization signal within the ErbB-2 protein

ErbB2, a member of the receptor tyrosine kinase family, is frequently over-expressed in breast cancer. Proteolysis of the extracellular domain of ErbB2 results in constitutive activation of ErbB2 kinase. Recent study reported that ErbB2 is found in the nucleus. Here, we showed that ErbB2 is imported into the nucleus through a nuclear localization signal（NLS）-mediated mechanism. The NLS sequence KRRQQKIRKYTMRR （aa655-668） contains three clusters of basic amino acids and it is sufficient to target GFP into the nucleus. However, mutation in any basic amino acid cluster of this NLS sequence significantly affects its nuclear localization. Furthermore, it was found that this NLS is essential for the nuclear localization of ErbB2 since the intracellular domain of Erb2 lacking NLS completely abrogates its nuclear translocation. Taken together, our study identified a novel nuclear localization signal and reveals a novel mechanism underlying ErbB2 nuclear trafficking and localization.     

Immunocytochemical visualization of P185HER2 receptor using antibodies fused with dibarnase and conjugate of barstar with colloidal gold

The localization of the P185^HER2 transmembrane receptor in SKOV-3 and BT-474 cancer cells was studied by fluorescence, confocal, and electron immunomicroscopy. The P185^HER2 receptor is a marker of breast and ovarian tumors; it is also considered to be a target for anticancer therapy. It is extremely important to choose a universal immunicytotoxic agent applicable, firstly, to study the distribution of P185^HER2 in cancer cells, secondarily, to remove P185^HER2 from the cell surface and, thirdly, to eliminate target cells. In this study, for visualization P185^HER2 we propose an immunocytotoxic system, which consists of monoclonal miniantibody 4D5 scFv to the extracellular P185^HER2 domain fused with two molecules of barnase (cytotoxic RNAase from Bacillus amyloliquefaciens) and its specific inhibitor, barstar. Fluorescent microscopy showed that the module 4D5 scFc-dibarnase:barstar is efficient for identifying P185^HER2 on the surfaces of cancer cells. It was found by confocal microscopy that interaction with 4D5 scFc-dibarnase results in the internalization of P185^HER2. The localization of P185^HER2 in human ovarian carcinoma cells (SKOV-3) and breast carcinoma cells (BT-474) was compared by electron microscopy using 4D5 scFv-dibarnase:barstar-Au and 4D5 scFv-dibarnase-Au complexes. P185^HER2 is distributed unequally on the cell surface with preferential localization on protrusions or close to their bases and at contacts between protrusions and the cell membrane. At 37°C, P185^HER2 is internalized through coated pits and vesicles and concentrates in endosomes and multivesicular bodies in the cells of both cell lines, as well as in lysosomes in BT-474 cells.     

Targeted delivery in breast cancer cells via iodine: nuclear localization sequence conjugate

The nonviral vector with iodine-nuclear localization sequence (namely, NLS-I) targeting breast cancer cells was fabricated. Ternary complexes were formed via charge interactions among NLS-I peptides, PEI 1800, and DNA, and we investigated their cellular internalization, nuclear accumulation as well as transfection efficiency. All the experiments were assessed by employing MCF-7 cells that express sodium/iodide symporter and HeLa cells that lack the expression of the symporter. In MCF-7 cells, cell internalization and nuclear accumulation of NLS-I was markedly increased compared to that in NLS. In addition, compared to that of the PEI1800/DNA complex, PEI1800/DNA/NLS-I complexes exhibited much enhanced luciferase reporter gene expression by up to 130-fold. By contrast, in HeLa cells, the evident improvements of cellular internalization, nuclear accumulation, and transfection efficiency by NLS-I were not observed. This study demonstrates an alternative method to construct a nonviral delivery system for targeted gene transfer into breast cancer cells.     

Akt inhibition promotes hexokinase 2 redistribution and glucose uptake in cancer cells

Hexokinase II (HK2), the enzyme that catalyzes the first committed step of glycolysis, is overexpressed in many cancers, as is the central signaling kinase Akt. Akt activity promotes HK2 association with the mitochondria, as well as glucose uptake by cancer cells. In HeLa cervical cancer cells, Akt inhibitor IV (Ai4) increased nuclear HK2 localization, while in MDA‐MB‐231 breast cancer cells, Ai4 merely induced cytoplasmic redistribution without increased nuclear accumulation. Small interfering RNA (siRNA) directed against Akt confirmed the effect in HeLa cells. Next, we treated the cells with clotrimazole (CTZ), which detaches HK2 from the mitochondria, or leptomycin B (LMB), which promotes HK2 nuclear accumulation, and determined the effect on HK2 subcellular distribution. In both cell lines, CTZ detached HK2 from the mitochondria, without substantially increasing nuclear HK2, while LMB increased nuclear HK2, without redistributing cytoplasmic HK2. Contrary to expectations, Akt inhibition promoted glucose uptake in both cell lines, suggesting that Akt inhibition may increase glucose uptake by detaching HK2 from the mitochondria. In both cell lines, CTZ and LMB increased glucose uptake. However, the results in the HeLa cells showed greater effects: CTZ increased glucose uptake to a similar degree to Ai4, while LMB was far more effective than either. These data suggest that both detachment of HK2 from the mitochondria and increased nuclear HK2 are important for Ai4‐induced increased glucose uptake. J. Cell. Physiol. 228: 1943–1948, 2013. © 2013 Wiley Periodicals, Inc.     

Factors influencing the ability of nuclear localization sequence peptides to enhance nonviral gene delivery

Nonviral gene delivery is limited by inefficient transfer of DNA from the cytoplasm to the nucleus. Nuclear localization sequence (NLS) peptides have been widely used to exploit intracellular transport mechanisms and promote nuclear uptake of DNA. However, the exact conditions to successfully utilize the properties of NLS peptides are still unclear. In the present study a panel of NLS peptides that bind different transport receptors were compared for their ability to enhance nonviral gene transfer. Several factors such as method of incorporating the NLS peptide, type of NLS peptide, DNA morphology, and proper characterization of NLS peptide/DNA conjugates were identified as important considerations in utilizing NLS peptides to enhance gene transfer. In particular, it was shown that a peptide derived from human T cell leukaemia virus type 1 (HTLV) was able to effectively condense DNA into discrete particles and mediate levels of transgene expression up to 32-fold greater than polylysine-based polyplexes. This is the first study to demonstrate efficient transfection mediated by an importin beta-binding peptide based on the HTLV sequence. Promising results were also achieved with a 7-fold increase in gene expression using a NLS peptide/DNA conjugate formed by site-specific linkage of an extended SV40 peptide via a peptide nucleic acid (PNA) clamp. Altogether, the results from this study should help to define the requirements for successful NLS-enhanced transfection.     

Oncogene activation induces metabolic transformation resulting in insulin-independence in human breast cancer cells

Normal breast epithelial cells require insulin and EGF for growth in serum-free media. We previously demonstrated that over expression of breast cancer oncogenes transforms MCF10A cells to an insulin-independent phenotype. Additionally, most breast cancer cell lines are insulin-independent for growth. In this study, we investigated the mechanism by which oncogene over expression transforms MCF10A cells to an insulin-independent phenotype. Analysis of the effects of various concentrations of insulin and/or IGF-I on proliferation of MCF10A cells demonstrated that some of the effects of insulin were independent from those of IGF-I, suggesting that oncogene over expression drives a true insulin-independent proliferative phenotype. To test this hypothesis, we examined metabolic functions of insulin signaling in insulin-dependent and insulin-independent cells. HER2 over expression in MCF10A cells resulted in glucose uptake in the absence of insulin at a rate equal to insulin-induced glucose uptake in non-transduced cells. We found that a diverse set of oncogenes induced the same result. To gain insight into how HER2 oncogene signaling affected increased insulin-independent glucose uptake we compared HER2-regulated gene expression signatures in MCF10A and HER2 over expressing MCF10A cells by differential analysis of time series gene expression data from cells treated with a HER2 inhibitor. This analysis identified genes specifically regulated by the HER2 oncogene, including VAMP8 and PHGDH, which have known functions in glucose uptake and processing of glycolytic intermediates, respectively. Moreover, these genes specifically implicated in HER2 oncogene-driven transformation are commonly altered in human breast cancer cells. These results highlight the diversity of oncogene effects on cell regulatory pathways and the importance of oncogene-driven metabolic transformation in breast cancer.     

Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains

Gold nanoparticles modified with nuclear localization peptides were synthesized and evaluated for their subcellular distribution in HeLa human cervical epithelium cells, 3T3/NIH murine fibroblastoma cells, and HepG2 human hepatocarcinoma cells. Video-enhanced color differential interference contrast microscopy and transmission electron microscopy indicated that transport of nanoparticles into the cytoplasm and nucleus depends on peptide sequence and cell line. Recently, the ability of certain peptides, called protein transduction domains (PTDs), to transclocate cell and nuclear membranes in a receptor- and temperature-independent manner has been questioned (see for example, Lundberg, M.; Wikstrom, S.; Johansson, M. (2003) Mol. Ther. 8, 143-150). We have evaluated the cellular trajectory of gold nanoparticles carrying the PTD from HIV Tat protein. Our observations were that (1) the conjugates did not enter the nucleus of 3T3/NIH or HepG2 cells, and (2) cellular uptake of Tat PTD peptide-gold nanoparticle conjugates was temperature dependent, suggesting an endosomal pathway of uptake. Gold nanoparticles modified with the adenovirus nuclear localization signal and the integrin binding domain also entered cells via an energy-dependent mechanism, but in contrast to the Tat PTD, these signals triggered nuclear uptake of nanoparticles in HeLa and HepG2 cell lines.     

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10?mol%, 50?mol% and 90?mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90?mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.     

Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit

Although insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 are known to modulate cell growth by reversibly sequestering extracellular insulin-like growth factors, several reports have suggested that IGFBP-3, and possibly also IGFBP-5, have important insulin-like growth factor-independent effects on cell growth. These effects may be related to the putative nuclear actions of IGFBP-3 and IGFBP-5, which we have recently shown are transported to the nuclei of T47D breast cancer cells. We now describe the mechanism for nuclear import of IGFBP-3 and IGFBP-5. In digitonin-permeabilized cells, where the nuclear envelope remained intact, nuclear translocation of wild-type IGFBP-3 appears to occur by a nuclear localization sequence (NLS)-dependent pathway mediated principally by the importin beta nuclear transport factor and requiring both ATP and GTP hydrolysis. Under identical conditions, an NLS mutant form of IGFBP-3, IGFBP-3[(228)KGRKR --> MDGEA], was unable to translocate to the nucleus. In cells where both the plasma membrane and nuclear envelope were permeabilized, wild-type IGFBP-3, but not the mutant form, accumulated in the nucleus, implying that the NLS was also involved in mediating binding to nuclear components. By fusing wild-type and mutant forms of NLS sequences (IGFBP-3 [215-232] and IGFBP-5 [201-218]) to the green fluorescent protein, we identified the critical residues of the NLS necessary and sufficient for nuclear accumulation. Using a Western ligand binding assay, wild-type IGFBP-3 and IGFBP-5, but not an NLS mutant form of IGFBP-3, were shown to be recognized by importin beta and the alpha/beta heterodimer but only poorly by importin alpha. Together these results suggest that the NLSs within the C-terminal domain of IGFBP-3 and IGFBP-5 are required for importin-beta-dependent nuclear uptake and probably also accumulation through mediating binding to nuclear components.     

Pharmacokinetics of (111)In-labeled triplex-forming oligonucleotide targeting human N-myc gene

The radiolabeled triplex-forming oligonucleotide (TFO) demonstrated the potential for sequence-specific DNA binding and destruction. In this study, by selecting the polypurine-polypyrimidine stretch (2950-2978) in the human N-myc gene as a target, the (111)In-labeled TFO targeting human N-myc gene (N-mycTFO(111)In) was tested for its cellular uptake and nuclear localization in vitro and in vivo. This is because the deregulated N-myc expression is strongly implicated in the pathogenesis of several important human malignancies, including breast carcinoma and neuroblastoma. N-mycTFO(111)In was bound selectively to the N-myc sequence in vitro. The total cellular uptake of TFO after the incubation of various normal and cancer cells with TFO for 24 h was 20-54.8% of the injected dose (%ID), and the nuclear localization was 6.59-30.0%ID, depending on cell lines. The highest cellular uptake was found in the human neuroblastoma SK-N-DZ (54.8%ID), human mammary ductal carcinoma T47-D (54%ID), human acute T cell leukemia Jurkat (54%ID), and multidrug-resistant human breast adenocarcinoma MCF7/TH (49.5%ID). The lowest was in the human normal mammary epithelium MCF10A (20.0%ID). The highest nuclear localization was found in MCF7/TH (30%ID) and SK-N-DZ (28.7%ID). The lowest was in MCF11A (6.59%ID). We next injected TFO into human mammary tumor-xenografted Balb/c nude mice. Tumor targeting of TFO in vivo reached its maximum peak 5 h after the intravenous injection in three types of tumor models. They are 21.0 +/- 3.23%ID per gram of tissue (%ID/g) for MCF7/TH, 7.77 +/- 2.11%ID/g for MCF7, and 4.53 +/- 1.20%ID/g for MCF10A. The TFO blood level decreased from 8.00 +/- 0.90%ID/g 15 min after the injection, to 1.30 +/- 0.30%ID/g after 19 h. The kidney TFO level increased rapidly from 5.93 +/- 0.94%ID/g after 15 min, to 25.1 +/- 5.60%ID/g after 19 h. A high TFO level (19.7-24.5%ID/g) in the liver was maintained until 19 h after the injection. Therefore, we suggest that the (111)In-labeled N-myc-targeting TFO, a promising modality for nanoexplosive gene therapy, could effectively target the nucleus of the multidrug-resistant breast carcinoma MCF7/TH in vitro and in vivo. It has approximately 130 min of half-life of blood TFO.     

In vivo spectroscopic photoacoustic imaging and laser-induced nanoparticle vaporization for anti-HER2 breast cancer

This study reports on the development and application of theragnostic agents targeting the HER2 receptors in breast tumors. The agent was constructed by loading silica-coated gold nanorods (GNRs) and a perfluorohexane liquid into PLGA-PEG nanoparticles, followed by surface conjugation with antibody Herceptin. The particle uptake in human breast cancer MDA-MB-231 (HER2-negative) and BT474 (HER2-positive) cell lines was tested. A proof of principle in vivo study was also performed using a xenograft mouse bilateral tumor model (16 mice, 32 tumors). Photoacoustic imaging was performed using a VevoLAZR device at 720/750/850 nm illuminations and 21 MHz central frequency. The relative concentrations of GNRs in the tumor were quantified using a linear spectral unmixing technique. The therapeutic efficacy of these nanoparticles was evaluated through optical droplet vaporization, and cell damage was confirmed using tissue immunofluorescence and histology. Our results demonstrate the potential of PLGA-GNRs as theragnostic agents for anti-HER2 breast cancer therapy.     

Overlapping signals for protein degradation and nuclear localization define a role for intrinsic RAG-2 nuclear uptake in dividing cells

Expression of the recombinase proteins RAG-1 and RAG-2 is discordant: while RAG-1 is relatively long lived, RAG-2 is degraded periodically at the G(1)-S transition. Destruction of RAG-2 is mediated by a conserved interval in the recombination-dispensable region. The need for RAG-2 to reaccumulate in the nucleus at each cell division suggested the existence of an intrinsic RAG-2 nuclear localization signal (NLS). RAG-1 or RAG-2, expressed individually, is a nuclear protein. A screen for proteins that bind the recombination-dispensable region of RAG-2 identified the nuclear transport protein Importin 5. Mutation of residues 499 to 508 in RAG-2 abolished Importin 5 binding, nuclear accumulation, and periodic degradation of RAG-2. The Importin 5 binding site overlaps an NLS, defined by mutagenesis. RAG-1 rescued the localization of degradation-defective, RAG-2 NLS mutants; this required an intact RAG-1 NLS. Mutations in RAG-2 that abolish intrinsic nuclear accumulation but spare periodic degradation impaired recombination in cycling cells; induction of quiescence restored recombination to wild-type levels. Recombination defects were correlated with a cell cycle-dependent defect in the ability of RAG-1 to rescue localization of the RAG-2 mutants. These results suggest that the intrinsic RAG-2 NLS functions in the nuclear uptake of RAG-2 following its reexpression in cycling cells.