Tae-Sup Lee

Apoptosis-inducing antitumor efficacy of hexokinase II inhibitor in hepatocellular carcinoma

Hypoxia stimulates hepatocellular carcinoma (HCC) cell growth via hexokinase (HK) II induction, and alternatively, HK II inhibition induces apoptosis by activating mitochondrial signaling. This study was to investigate whether the induction of HK II by hypoxia is associated with enhanced mitochondrial stability and to confirm the apoptosis-inducing efficacy of HK II inhibitor in an in vivo model of HCC. Mitochondrial stability was examined by treating isolated mitochondria with deoxycholate, a permeability-enhancing agent. Alteration of permeability transition pore complex composition was analyzed by immunoprecipitation and immunoblotting. An in vivo model of HCC was established in C3H mice i.d. implanted with MH134 cells. The antitumor efficacy of i.p. given 3-bromopyruvate (3-BrPA), a HK II inhibitor, was evaluated by measuring tumor volumes and quantifying apoptosis using terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining and 99mTc-hydrazinonicotinamide-Annexin V scans. Hypoxia enhanced mitochondrial stability, and this was inhibited by 3-BrPA treatment. In particular, HK II levels in permeability transition pore complex immunoprecipitates were reduced after 3-BrPA treatment. In mice treated with 3-BrPA, mean tumor volumes and tumor volume growth were found to be significantly reduced. Moreover, percentages of terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive cells were significantly increased in 3-BrPA–treated mice, and this apoptosis-inducing efficacy was reflected in vivo by 99mTc-hydrazinonicotinamide-Annexin V imaging. Our results show that hypoxia enhances mitochondrial stability via HK II induction and that HK II inhibitor treatment exhibits an in vivo antitumor effect by inducing apoptosis. Therefore, HK II inhibitors may be therapeutically useful for the treatment of advanced infiltrative hypovascular HCCs, which are growing in a hypoxic environment. [Mol Cancer Ther 2007;6(9):2554–62]

Characterization and Cancer Cell Specific Binding Properties of Anti-EGFR Antibody Conjugated Quantum Dots

Synthesis of biologically active antibody conjugated quantum dots (QDs) has been of great importance in cellular imaging and diagnostics. Cetuximab (or Erbitux) is the first monoclonal antibody drug which targets the epidermal growth factor receptor (EGFR) overexpressed in most cancer cells. In the present work, we investigated three different conjugation strategies to obtain the biologically functional QD-cetuximab conjugates for the tumor-specific imaging. Successful conjugation of cetuximab to QDs was achieved using PEG conjugated polymer-coated QDs and two long-chain heterobifunctional linkers, sulfo-LC-SPDP and sulfo-SMCC. The dissociation constant of the QD-cetuximab conjugates to EGFR was determined to be 0.61 +/- 0.28 nM. The cancer cell-specific binding ability of the QD-cetuximab conjugates was evaluated in vitro, and the cellular internalization of the QD-cetuximab conjugates was clearly demonstrated in live cells by confocal microscopy. The cellular imaging experiments using the QD-cetuximab conjugates showed a clear endocytosis pathway, which was evidenced by the colocalization of the QD-cetuximab conjugates with dye-labeled transferrin. These results suggest that the QD-cetuximab conjugates as an imaging modality for tumor EGFR overexpression can be expected to provide important information on the expression levels of EGFR on the cancer cells.

Tumor targeting of humanized fragment antibody secreted from transgenic rice cell suspension culture

The tumor-associated glycoprotein 72 (TAG 72) has been shown to be expressed in the majority of human adenocarcinomas. In an effort to develop a technique for the safe and inexpensive production of large quantities of anti-TAG 72 humanized antibody fragments (hzAb) as a future source of clinical-grade proteins, we developed a transgenic rice cell suspension culture system. The in vivo assembly and secretion of hzAb were achieved in a transgenic rice cell culture under the control of the rice alpha amylase 3D (RAmy 3D) expression system, and the biological activities of plant-derived hzAb were determined to be quite similar to those of animal-derived antibody. Purified hzAb was shown to bind to the recombinant antigen, TAG 72, and to bind specifically to human LS 174T colon adenocarcinoma cells expressing the TAG 72 antigen, and this binding occurred to the same extent as was seen with animal-derived antibody. Plant-derived hzAb proved as effective as animal-derived antibody in targeting tumors of xenotransplanted LS 174T cells in nude mice. The results of this study indicate that the hzAb derived from plant cell suspension cultures may have great potential for pharmaceutical applications in the development of future cancer therapeutic and diagnostic protocols.

Experimental condition and registration method for the tumor detection of lung metastasis small animal PET and CT whole body images

Small animal PET scanning with 18F-FDG is increasingly used in murine tumor model. Experimental condition of animal handling impacts on the biodistribution of 18F-FDG in mouse study. The aim of this study was to optimize experimental conditions of animal handling and registration method for imaging lung metastasis tumor by using 18F-FDG small animal PET and CT. The experimental conditions for 18F-FDG biodistribution and animal PET imaging were 20 plusmn 2 h fasting and warming kept at 30degC. Biodistribution, standard uptake value (SUV) image and glucose SUV (SUVG) of 18F-FDG were evaluated and blood glucose level was also measured. Female C57BL/6 mice were injected intravenously with 2 times 105 of B16-F10 melanoma cells which developed metastasis tumors in lung. In lung metastasis tumor model, 18F-FDG images was obtained in optimized anesthesia condition and registered with anatomical clinical CT image. The small animal PET and CT images were registered using the hardware fiducial markers and small animal contour point in both data sets to perform a point- based rigid registration. Isoflurane 0.5 condition was chosen for lung metastasis tumor imaging. Lung metastasis tumor was clearly visualized by 18F-FDG PET image with Iso 0.5 anesthesia. Tumor imaging in lung region with 18F-FDG small animal PET should be considered feeding, temperature, and especially choice and level of anesthetic for minimizing background radioactivity. Fused imaging with small animal PET and CT image could be useful for the detection of metastatic tumor. This method improves the quantitative accuracy and interpretation of the tracer.