Knockdown of COPA, Identified by Loss-of-Function Screen, Induces Apoptosis and Suppresses Tumor Growth in Mesothelioma Mouse Model

AbstractMalignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes—COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, and RPL18A—would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma

Anticancer Effect of Dihydroartemisinin (DHA) in a Pancreatic Tumor Model Evaluated by Conventional Methods and Optical Imaging

Background: Dihydroartemisinin (DHA) inhibits the growth of certain cancer cells and xenograft tumors. Further understanding of the molecular mechanisms and genetic participants that govern the antineoplastic effects of DHA is necessary. The anticancer effects of DHA and its underlying mechanisms in pancreatic cancer and the efficacy in animal models by noninvasive optical imaging were evaluated. Materials and Methods: Combined with cell/tumor growth assays, flow cytometric analysis, and Hoechst staining, the effect of DHA was investigated using the pancreatic cancer cell line BxPc3-RFP stably expressing red fluorescence protein and in vitro/in vivo optical imaging. Proteins that regulate proliferation (PCNA), apoptosis (Bax and Bcl-2), and angiogenesis (vascular endothelial growth factor (VEGF)) were evaluated in cell and tumor samples by Western blotting and immunohistochemical analyses. Results: DHA inhibited the proliferation and viability of cells in a dose-dependent manner and induced apoptosis. We observed down-regulation of PCNA and Bcl-2, and up-regulation of Bax. VEGF was down-regulated by DHA in cells under normoxic, but not hypoxic, conditions. Fluorescence intensity emitted from cells and tumors correlated linearly with cell count and tumor burden, respectively. Conclusion: DHA inhibits cell and tumor growth by interfering with cell proliferation and inducing apoptosis. The antiangiogenic effect of DHA appears to be a complicated process. Optical imaging supports the real-time assessment of DHA efficacy in a preclinical model and comprehensive analysis substantiates that DHA is a potential candidate for pancreatic cancer therapy

Assessment and Evaluation of the Anticancer Effect of Dihydroartemisinin (DHA) in a Pancreatic Tumor Model by Conventional Methods and Optical Imaging

Objective: Dihydroartemisinin (DHA), an active derivative of artemisinin which is now recognized as antimalarial drug, inhibits the growth of certain cancer cells and xenograft tumors. Further understanding of the molecular mechanisms and genetic participants that govern the antineoplastic effects of DHA is necessary. The anticancer effects of DHA and its underlying mechanisms in pancreatic cancer and the efficacy in animal models by noninvasive optical imaging, a suitable technique for real-time characterization of the therapeutic effects on tumor, were evaluated. Materials and Methods: Combined with conventional methods such as cell/tumor growth assays, flow cytometric analysis, and Hoechst staining for apoptosis, the effect of DHA was confirmed using the pancreatic cancer cell line BxPc3-RFP stably expressing red fluorescence protein and in vitro/in vivo optical imaging. Proteins that regulate proliferation (proliferating cell nuclear antigen (PCNA)), apoptosis (Bax and Bcl-2), and angiogenesis (vascular endothelial growth factor (VEGF)) were evaluated in cell and tumor samples by Western blotting and immunohistochemical analyses. Results: DHA inhibited the proliferation and viability of cells in a dose-dependent manner and induced apoptosis. We observed down-regulation of PCNA and Bcl-2, and up-regulation of Bax. VEGF was down-regulated by DHA in cells under normoxic, but not hypoxic, conditions. Fluorescence intensity emitted from cells and tumors correlated linearly with cell count and tumor burden, respectively. Conclusion: DHA inhibits cell and tumor growth by interfering with cell proliferation and inducing apoptosis. The antiangiogenic effect of DHA may be partial and appears to be a complicated process. Optical imaging allows convenient and reliable real-time evaluation of DHA efficacy in a preclinical model and comprehensive analysis substantiates that DHA is a potential candidate for pancreatic cancer therapy.2011 World Molecular Imaging Congress (WMIC

A Loss of Function Screening for Radiation Susceptibility Genes

　Carcinogenesis is thought to be a multistep process that occurs through the accumulation of mutations in multiple genes required for the maintenance of normal growth control. Genomic instability is considered the earliest cellular event in the process of carcinogenesis. Although genomic instability is induced by ionizing radiation, the molecular mechanisms underlying this process are poorly understood. Cell cycle checkpoints play a key role in cell survival following DNA damage. The failure of DNA repair and cell cycle regulation in response to DNA damage are thought to be important factors in the early stages of genomic instability. Several known genes are reportedly associated not only with DNA damage repair and cell cycle regulation but also with radiation susceptibility. Thus, the identification of novel radiation susceptibility genes will likely help elucidate the molecular mechanisms underlying DNA damage–induced genomic instability.　By the large-scale expression profiling of 15 human cell lines and three mouse strains having varying degrees of susceptibility to ionizing radiation, we identified 200 genes correlated with radiation susceptibility. We constructed the shRNA library of these 200 genes and screened this library using a 96-well format and measuring the cell survivals, as determined by a sulforhodamine B–based cell proliferation assay, after X-irradiation. We identified 12 genes involved in cellular proliferation after irradiation. Three genes, ATM, ATR and CDKN1A, were known to be radiation susceptibility genes and other nine genes have not been reported to be associated with radiation susceptibility. Eight of the 12 genes were reported to directly or potentially regulate cell cycle, and the remaining four genes have not been reported biological functions. We then performed cell cycle analysis of cells transfected with shRNA vectors against thee four genes following X-irradiation, and found that knockdown cells of one gene did not accumulate at G2/M phase. This result suggests that one gene is associated with G2/M checkpoint after DNA damage. Further study of these 12 genes would help elucidate the molecular mechanism for genomic instability and carcinogenesis, and develop novel drugs for effective tumor radiotherapy and early diagnosis.１３ｔｈ International Congress of Radiation Reserc

Evaluation of a Novel Human Anti-EGFR Monoclonal Antibody as an Imaging Probe.

The epithelial growth factor receptor (EGFR) is over-expressed in many epithelial cancers, and is an attractive target for cancer imaging and therapy. Here we report a novel anti-EGFR human monoclonal antibody which was isolated from a human phage-display antibody library by comprehensive screening using living cancer cells. In this study, we selected one antibody from nine anti-EGFR antibody clones by cell binding assay and further assessed the selected antibody in vitro and in vivo regarding the potential as an imaging probe.Material and Methods: Nine anti-EGFR antibodies and cetuximab, a well-defined anti-EGFR antibody, were radiolabeled with 125I using chloramine-T method. Cell binding assay using human epidermal cancer cell line A431 highly expressing EGFR was performed to select an antibody showing the highest binding. The selected antibody was further radiolabeled with 111In by conjugating antibody with a bifunctional chelate, N-[(R)-2-Amino-3-(p-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N\u27,N",N"-pentaacetic acid (CHX-A"-DTPA), and assessed by in vitro cell binding, competitive inhibition and internalization assays. The xenografted tumor was made by subcutaneous inoculation of 2x106 A431 cells into BALB/c-nu/nu mice, and in vivo biodistribution study, planar imaging and SPECT were performed after intravenous administration of 111In-labeled selected antibody.Results: The monoclonal antibody (048-006) was selected showing the highest binding to A431 cells among seven antibodies tested. 125I- and 111In-labeled 048-006 showed almost equivalent affinity constant to 125I- and 111In-labeled cetuximab (3.8x109M-1 and 1.8x109M-1 for 048-006 vs. 3.3x109M-1 and 2.3x109M-1 for cetuximab, respectively), and both antibodies were internalized after binding. 111In-048-006 showed high uptake in A431 tumors (highest tumor uptake of 15.98+/-3.65 %ID/gram obtained at 48 hours after injection), and the xenografted tumor was clearly visualized by planar and SPECT using 111In-048-006.Conclusion: Radiolabeled human anti-EGFR monoclonal antibody 048-006 would be promising for the imaging tumors with EGFR over-expression.World Molecular Imaging Congress 201

Molecular Imaging of Hepatocellular Carcinoma using Human Anti-EGFR Monoclonal Antibody

Introduction: Over-expression or mutation of EGFR is identified in several cancers including hepatocellular carcinoma (HCC). We are trying to develop a novel imaging method to detect EGFR over-expression at an early stage of cancers including HCC. In this study, we evaluated the radiolabeled human anti-EGFR monoclonal antibody for this purpose.\nMaterial and Methods: High EGFR-expressing HCC cell lines were selected from four cell lines (Hep-G2, HuH-7, SK-Hep1 and Li-7) by western blot analysis and immunohistochemistry. Cancer cells (2x106) were inoculated subcutaneously into BALB/c-nu/nu mice with Matrigel. Anti-EGFR monoclonal antibody (048-006, IgG1 subclass) were labeled with 125I using the chloramine-T or 111In using CHX-A"-DTPA as a bifunctional chelate. The in vitro character of the radiolabeled 048-006 was evaluated by cell binding, competitive inhibition and internalization assays, and then in vivo character was assessed by biodistribution and planar imaging in tumor model mice. \nResults: Western blotting analysis revealed that HuH-7 showed the highest EGFR expression among four cell lines tested. EGFR was expressed on the cell membrane of HuH-7 by immunohistochemistry. After subcutaneous inoculation with Matrigel, HuH-7 cells successfully formed tumor xenografts showing membranous EGFR expression. In vitro studies showed that 125I-048-006 and 111In-048-006 specifically bound to HuH-7 with the affinity constants of 9.3x108M-1 and 8.0x108M-1, respectively. This antibody was internalized after binding to EGFR. 111In-048-006 highly accumulated in xenografted tumors (highest tumor uptake of 21.24+/-5.84 %ID/gram obtained at 48 hours after injection), and the tumor xenograft was clearly visualized by planar imaging. \nConclusion: This radiolabeled human anti-EGFR monoclonal antibody 048-006 would be promising for the imaging HCC.World Federation of Nuclear Medicine and Biology 201

18F-FDG PET for Semiquantitative Evaluation of Acute Allograft Rejection and Immunosuppressive Therapy Efficacy in Rat Models of Liver Transplantation

Acute allograft rejection remains a major complication after liver transplantation. We report a semiquantitative imaging method of detecting acute allograft rejection with 18F-FDG PET. Methods: Syngeneic and allogeneic transplanted rats, with or without immunosuppressive treatment, were subjected to serial PET. Autoradiography of the liver was conducted in both the syngeneic and the allogeneic rats. Results: A significant increment of 18F-FDG accumulation in liver allografts was observed byPET on day 2. The 18F-FDG signal was concentrated in the area where inflammatory cells around the vessels were detected by autoradiography. Allotransplanted rats treated with an immunosuppressive agent displayed a marked decrease in hepatic 18F-FDG uptake, compared with allotransplanted rats that were not treated. Conclusion: 18F-FDG PET may be a valid method for facilitating the development of protocols to diagnose graftrejection and to monitor the efficacy of immunosuppressive therapy

Basic Studies on Radioimmunotargeting of CD133-Positive HCT116 Cancer Stem Cells

As cancer stem cells (CSCs) are postulated to play critical roles in cancer development, including metastasis and recurrence, CSC imaging would provide valuable information for cancer treatment and lead to CSC-targeted therapy. To assess the possibility of in vivo CSC targeting, we conducted basic studies on radioimmunotargeting of cancer cells positive for CD133, a CSC marker recognized in various cancers. Antibodies against CD133 were labeled with 125 I, and their in vitro cell binding properties were tested. Using the same isotype IgG as a control, in vivo biodistribution of the labeled antibody retaining immunoreactivity was examined in mice bearing an HCT116 xenograft in which a population of the cancer cells expressed CD133. Intratumoral distribution of the labeled antibody was examined and compared to the CD133 expression pattern. The 125 I-labeled anti-CD133 antibody showed a modest but significantly higher accumulation in the HCT116 xenograft compared to the control IgG. The intratumoral distribution of the labeled antibody mostly overlapped with the CD133 expression, whereas the control IgG was found in the area close to the necrotic tumor center. Our results indicate that noninvasive in vivo targeting of CSCs could be possible with radiolabeled antibodies against cell membrane markers

CNP Receptor Gene (Npr2) is Involved in the Regulation of Blood-Testis Barrier and Spermatogenesis in Mouse

C-type natriuretic peptide (CNP) exerts its main biological effects by binding to natriuretic peptide receptor B (NPR-B), a membrane-bound guanylyl cyclase receptor that produces cyclic guanosine monophosphate (cGMP). It was previously reported that cGMP induced by nitric oxide via soluble guanylyl cyclase regulates Sertoli cell tight junction in vitro and permeability of the blood-testis barrier (BTB) which separates the seminiferous epithelium into the adluminal and basal compartments during spermatogenesis. Furthermore, it was also reported that CNP regulates BTB dynamics in adult rat testes. We recently reported that the short-limbed dwarfism (SLW) mouse has a mutation in Npr-2 gene, which encodes NPR-B and this mutation results in deletion of the intracellular domain of NPR-B. However, the effect of the flaw in the NPR-B-induced cGMP production through CNP signal against spermatogenesis has not yet been demonstrated. Therefore, the aim of this study is to elucidate the function of NPR-B in the progression of spermatogenesis in mouse. We first examined the immunohistochemical distribution of NPR-B in the seminiferous epithelium of the normal mouse testis during the first-wave of spermatogenesis. NPR-B was initially expressed within a spermatogonium, and subsequently located to the cytoplasm of a spermatocyte and a round spermatid predominantly in the normal testes. We next examined the phenotype of testes in SLW mice. It was revealed that the testis of slw/slw homozygotes showed retardation of spermatogenic development in 2w and vacuolation in the seminiferous epithelium in 3w. Immunohistochemical staining of ZO-1, a tight junction protein revealed that BTB was located in a boundary between the basement and the lumen in the seminiferous epithelium of 2w normal testis, whereas BTB formation was disordered and misaligned in the 2w slw/slw. These results suggest that NPR-B mediated cGMP production through CNP signal has a role important for BTB regulation which is required for spermatogenesis.58th/60th NIBB Conferen