Soon-Kyung Hwang

Body Distribution of Inhaled Fluorescent Magnetic Nanoparticles in the Mice

Body Distribution of Inhaled Fluorescent Magnetic Nanoparticles in the Mice: Jung‐Taek Kwon, et al. Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Korea—Reducing the particle size of materials is an efficient and reliable tool for improving the bioavailability of a gene or drug delivery system. In fact, nanotechnology helps in overcoming the limitations of size and can change the outlook of the world regarding science. However, a potential harmful effect of nanomaterial on workers manufacturing nanoparticles is expected in the workplace and the lack of information regarding body distribution of inhaled nanoparticles may pose serious problem. In this study, we addressed this question by studying the body distribution of inhaled nanoparticles in mice using approximately 50‐nm fluorescent magnetic nanoparticles (FMNPs) as a model of nanoparticles through nose‐only exposure chamber system developed by our group. Scanning mobility particle sizer (SMPS) analysis revealed that the mice were exposed to FMNPs with a total particle number of 4.89 × 105± 2.37 × 104/cm3(low concentration) and 9.34 × 105± 5.11 × 104/cm3(high concentration) for 4 wk (4 h/d, 5 d/wk). The body distribution of FMNPs was examined by magnetic resonance imaging (MRI) and Confocal Laser Scanning Microscope (CLSM) analysis. FMNPs were distributed in various organs, including the liver, testis, spleen, lung and brain. T2‐weighted spin‐echo MR images showed that FMNPs could penetrate the blood‐brain‐barrier (BBB). Application of nanotechnologies should not produce adverse effects on human health and the environment. To predict and prevent the potential toxicity of nanomaterials, therefore, extensive studies should be performed under different routes of exposure with different sizes and shapes of nanomaterials.

Aerosol delivery of urocanic acid-modified chitosan/programmed cell death 4 complex regulated apoptosis, cell cycle, and angiogenesis in lungs of K-ras null mice.

The low efficiency of conventional therapies in achieving long-term survival of patients with lung cancer calls for development of novel treatment options. Although several genes have been investigated for their antitumor activities through gene delivery, problems surrounding the methods used, such as efficiency, specificity, and toxicity, hinder application of such therapies in clinical settings. Aerosol gene delivery as nonviral and noninvasive method for gene therapy may provide an alternative for a safer and more effective treatment for lung cancer. In this study, imidazole ring-containing urocanic acid–modified chitosan (UAC) designed in previous study was used as a gene carrier. The efficiency of UAC carrier in lungs was confirmed, and the potential effects of the programmed cell death protein 4 (PDCD4) tumor suppressor gene on three major pathways (apoptosis, cell cycle, and angiogenesis) were evaluated. Aerosol containing UAC/PDCD4 complexes was delivered into K-ras null lung cancer model mice through the nose-only inhalation system developed by our group. Delivered UAC/PDCD4 complex facilitated apoptosis, inhibited pathways important for cell proliferation, and efficiently suppressed pathways important for tumor angiogenesis. In summary, results obtained by Western blot analysis, immunohistochemistry, and terminal deoxynucleotidyl transferase–mediated nick end labeling assay suggest that our aerosol gene delivery technique is compatible with in vivo gene delivery and can be applied as a noninvasive gene therapy. [Mol Cancer Ther 2006;5(4):1041–9]

AIMP2/p38, the scaffold for the multi-tRNA synthetase complex, responds to genotoxic stresses via p53

AIMP2/p38 is a scaffolding protein required for the assembly of the macromolecular tRNA synthetase complex. Here, we describe a previously unknown function for AIMP2 as a positive regulator of p53 in response to genotoxic stresses. Depletion of AIMP2 increased resistance to DNA damage-induced apoptosis, and introduction of AIMP2 into AIMP2-deficient cells restored the susceptibility to apoptosis. Upon DNA damage, AIMP2 was phosphorylated, dissociated from the multi-tRNA synthetase complex, and translocated into the nuclei of cells. AIMP2 directly interacts with p53, thereby preventing MDM2-mediated ubiquitination and degradation of p53. Mutations in AIMP2, affecting its interaction with p53, hampered its ability to activate p53. Nutlin-3 recovered the level of p53 and the susceptibility to UV-induced cell death in AIMP2-deficient cells. This work demonstrates that AIMP2, a component of the translational machinery, functions as proapoptotic factor via p53 in response to DNA damage.

Biodegradable poly(ester amine) based on glycerol dimethacrylate and polyethylenimine as a gene carrier

Polyethylenimine (PEI) vectors are widely used in gene delivery because of their high transfection efficiency owing to a unique proton sponge effect. An increase in molecular weight increases transfection efficiency, but simultaneously results in increased toxicity. Therefore, the design and synthesis of new degradable gene delivery carriers having high transfection efficiencies and reduced cytotoxicity are necessary.

Cancer-Associated Splicing Variant of Tumor Suppressor AIMP2/p38: Pathological Implication in Tumorigenesis

Although ARS-interacting multifunctional protein 2 (AIMP2, also named as MSC p38) was first found as a component for a macromolecular tRNA synthetase complex, it was recently discovered to dissociate from the complex and work as a potent tumor suppressor. Upon DNA damage, AIMP2 promotes apoptosis through the protective interaction with p53. However, it was not demonstrated whether AIMP2 was indeed pathologically linked to human cancer. In this work, we found that a splicing variant of AIMP2 lacking exon 2 (AIMP2-DX2) is highly expressed by alternative splicing in human lung cancer cells and patients tissues. AIMP2-DX2 compromised pro-apoptotic activity of normal AIMP2 through the competitive binding to p53. The cells with higher level of AIMP2-DX2 showed higher propensity to form anchorage-independent colonies and increased resistance to cell death. Mice constitutively expressing this variant showed increased susceptibility to carcinogen-induced lung tumorigenesis. The expression ratio of AIMP2-DX2 to normal AIMP2 was increased according to lung cancer stage and showed a positive correlation with the survival of patients. Thus, this work identified an oncogenic splicing variant of a tumor suppressor, AIMP2/p38, and suggests its potential for anti-cancer target.

Association of LETM1 and MRPL36 Contributes to the Regulation of Mitochondrial ATP Production and Necrotic Cell Death

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome, and was deleted in nearly all patients with the syndrome. LETM1 encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. Here, we describe LETM1-mediated regulation of mitochondrial ATP production and biogenesis. We show that LETM1 overexpression can induce necrotic cell death in HeLa cells, in which LETM1 reduces mitochondrial biogenesis and ATP production. LETM1 acts as an anchor protein and associates with mitochondrial ribosome protein L36. Adenovirus-mediated overexpression of LETM1 reduced mitochondrial mass and expression of many mitochondrial proteins. LETM1-mediated inhibition of mitochondrial biogenesis enhanced glycolytic ATP supply and activated protein kinase B activity and cell survival signaling. The expression levels of LETM1 were significantly increased in multiple human cancer tissues compared with normals. These data suggest that LETM1 serves as an anchor protein for complex formation with the mitochondrial ribosome and regulates mitochondrial biogenesis. The increased expression of LETM1 in human cancer suggests that dysregulation of LETM1 is a key feature of tumorigenesis.

The therapeutic efficiency of FP-PEA/TAM67 gene complexes via folate receptor-mediated endocytosis in a xenograft mice model

To circumvent carrier related obstacles, we developed a biodegradable, folate conjugated poly (ester amine) (FP-PEA) that mediates high level folate receptor (FR) mediated endocytosis in vitro as well as in vivo. We report the efficacy of a therapeutic strategy that combines the potency of FP-PEA based on polycaprolactone (PCL) and low molecular weight polyethylenimine (LMW-PEI) with the tumor targeting potential of receptor mediated endocytosis. When tested on cells in culture, FP-PEA was found to retain high affinity for FR-positive cells compared with PEA without folate moiety (P-PEA). The FR specific activity of FP-PEA was drastically decreased in the presence of an excess free folic acid and very less significant transfection was detected against FR-negative cells. FP-PEA showed marked anti-tumor activity against FR-positive human KB tumors in nude mice with no evidence of toxicity during and after therapy using TAM67 gene. Furthermore, the therapeutic effect occurred in the apparent absence of weight loss or noticeable tumor apoptosis. In contrast, no significant anti-tumor activity was observed in P-PEA treated mice which were co dosed with an excess of FR, thus demonstrating the target specific gene delivery. Furthermore, anti-tumor activity with PEA without folic acid moiety (P-PEA) proved not to be effective against xenograft mice model with KB cells when administered at the same dose to that of FP-PEA. Taken together, these results indicate that FP-PEA is highly effective gene carrier capable of producing therapeutic benefit in xenograft mice model without any sign of toxicity.

Inhaled Fluorescent Magnetic Nanoparticles Induced Extramedullary Hematopoiesis in the Spleen of Mice

Inhaled Fluorescent Magnetic Nanoparticles Induced Extramedullary Hematopoiesis in the Spleen of Mice: Jung‐Taek Kwon, et al. Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Korea

Tumor suppressor PDCD4 inhibits NF-κB-dependent transcription in human glioblastoma cells by direct interaction with p65

PDCD4 is a tumor suppressor induced by apoptotic stimuli that regulates both translation and transcription. Previously, we showed that overexpression of PDCD4 leads to decreased anchorage-independent growth in glioblastoma (GBM)-derived cell lines and decreased tumor growth in a GBM xenograft model. In inflammatory cells, PDCD4 stimulates tumor necrosis factor-induced activation of the transcription factor NF-κB, an oncogenic driver in many cancer sites. However, the effect of PDCD4 on NF-κB transcriptional activity in most cancers including GBM is still unknown. We studied the effect of PDCD4 on NF-κB-dependent transcriptional activity in GBM by stably overexpressing PDCD4 in U251 and LN229 cells. Stable PDCD4 expression inhibits NF-κB transcriptional activation measured by a luciferase reporter. The molecular mechanism by which PDCD4 inhibits NF-κB transcriptional activation does not involve inhibited expression of NF-κB p65 or p50 proteins. PDCD4 does not inhibit pathways upstream of NF-κB including the activation of IKKα and IKKβ kinases or degradation of IκBα, events needed for nuclear transport of p65 and p50. PDCD4 overexpression does inhibit localization of p65 but not p50 in the nucleus. PDCD4 protein interacts preferentially with p65 protein as shown by co-immunoprecipitation and confocal imaging. PDCD4 overexpression inhibits the mRNA expression of two NF-κB target genes in a p65-dependent manner. These results suggest that PDCD4 can significantly inhibit NF-κB activity in GBM cells by a mechanism that involves direct or indirect protein-protein interaction independent of the expected mRNA-selective translational inhibition. These findings offer novel opportunities for NF-κB-targeted interventions to prevent or treat cancer.

Aerosol Delivery of Small Hairpin Osteopontin Blocks Pulmonary Metastasis of Breast Cancer in Mice

Background Metastasis to the lung may be the final step in the breast cancer-related morbidity. Conventional therapies such as chemotherapy and surgery are somewhat successful, however, metastasis-related breast cancer morbidity remains high. Thus, a novel approach to prevent breast tumor metastasis is needed. Methodology/Principal Finding Aerosol of lentivirus-based small hairpin osteopontin was delivered into mice with breast cancer twice a week for 1 or 2 months using a nose-only inhalation system. The effects of small hairpin osteopontin on breast cancer metastasis to the lung were evaluated using near infrared imaging as well as diverse molecular techniques. Aerosol-delivered small hairpin osteopontin significantly decreased the expression level of osteopontin and altered the expression of several important metastasis-related proteins in our murine breast cancer model. Conclusion/Significance Aerosol-delivered small hairpin osteopontin blocked breast cancer metastasis. Our results showed that noninvasive targeting of pulmonary osteopontin or other specific genes responsible for cancer metastasis could be used as an effective therapeutic regimen for the treatment of metastatic epithelial tumors.