ABSTRACT
Hepatoma-derived growth factor (HDGF) has been shown to correlate with increased malignancy of different types of tumors and could be an independent prognostic index for human cancers. We previously found that HDGF is overexpressed in glioma tissues and that its expression level may correlate with the clinical pathological grade. In the present study, we investigated the effects of HDGF downregulation on the biological behaviors of U87 glioma cells. Our results showed that HDGF knockdown significantly inhibited the malignant phenotype of U87 cells, including the colony formation, migration and invasion in vitro, as well as tumorigenesis in vivo. Our data also suggest that hepatocyte growth factor/scatter factor (HGF/SF) may contribute to the HDGF-associated aggressive behavior of glioma cells.
Subject(s)
Down-Regulation , Glioma/pathology , Intercellular Signaling Peptides and Proteins/genetics , Animals , Cell Line, Tumor , Cell Movement , Cell Transformation, Neoplastic , Glioma/metabolism , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, Nude , Neoplasm Invasiveness , Neoplasm Transplantation , Phenotype , RNA Interference , Tumor BurdenABSTRACT
Hepatoma-derived growth factor (HDGF) is closely related to aggressive tumor behavior and could be a broader biomarker for cancer prognosis and diagnosis. The goal of this study is to develop a sandwich ELISA system to test if HDGF can be detected in serum samples. We produced an anti-HDGF monoclonal antibody designated 2F12 using recombinant human HDGF protein. The specificity of 2F12 mAb was characterized by western blotting, immunohistochemistry (IHC), immunofluorescent staining (IF) and immunoprecipitation (IP). The results showed that 2F12 recognized HDGF in both native and denatured form, and can be used for multiple purposes. We have found that HDGF is also expressed in several cancers unreported previously by IHC staining on tumor cell array sections. In addition, we have developed a sandwich ELISA assay using mAb 2F12 and rabbit anti-HDGF polyclonal antibody, and validated the assay using normal serum and non-small cell lung cancer (NSCLC) serum samples. The sensitivity of this assay is 0.5 ng/ml and the linear range is 0.5-32 ng/ml. The HDGF average level in serum samples from lung cancer patients is significantly elevated relative to that from healthy controls, 9.43+/-6.13 ng/ml versus 4.36+/-2.50 ng/ml (p=1.12E-10).
Subject(s)
Antibodies, Monoclonal/chemistry , Carcinoma, Non-Small-Cell Lung/blood , Intercellular Signaling Peptides and Proteins/blood , Lung Neoplasms/blood , Animals , Antibodies, Monoclonal/immunology , Carcinoma, Non-Small-Cell Lung/immunology , Enzyme-Linked Immunosorbent Assay/methods , Female , Hep G2 Cells , Humans , Intercellular Signaling Peptides and Proteins/immunology , Lung Neoplasms/immunology , Male , Mice , NIH 3T3 Cells , Rabbits , Recombinant Proteins/immunology , Sensitivity and SpecificityABSTRACT
Phage display is a powerful tool in identifying specific antibody fragment binding to the target. Several strategies can be used to screen out a specific Fab from the phage library that binds to an antigen; it may be performed on immobilized targeted molecule, on the intact cells, or by other strategies. Antibodies with properties of recognizing tumor cell surface receptors in native conformation and ability to internalize to tumor cells through receptor binding are ideal carriers for targeted immuno-chemo and/or immuno-radiation therapy. This chapter describes a unique bio-panning method to screen out a Fab fragment against human epithelial growth factor receptor (EGFR) from a naive human Fab phage library for potential targeted therapeutic application on EGFR overexpressed cancers.
Subject(s)
Endocytosis/immunology , Immunoglobulin Fab Fragments/immunology , Molecular Biology/methods , Peptide Library , Blotting, Western , Cell Line, Tumor , Clone Cells , Enzyme-Linked Immunosorbent Assay , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Escherichia coli , Eukaryotic Cells/metabolism , Helper Viruses , Humans , Immobilized Proteins , Protein Structure, Tertiary , TitrimetryABSTRACT
The aims of this study were to generate a human Fab fragment against EGFR; conjugate it to paclitaxel (Taxol) as an immuno-chemotherapy agent; and investigate its in vitro anti-tumor efficacy on A431 epidermoid carcinoma cells. A431 cells (EGFR-positive), NIH 3T3 cells (EGFR-negative), and purified EGFR were used for subtractive panning on a human naïve Fab phage library to generate a human anti-EGFR Fab fragment that binds the EGFR extracellular domain in native conformation and subsequently internalizes it into the cytosol. The Fab was then conjugated with the chemotherapeutic Taxol, and cell proliferation inhibition and apoptosis (TUNEL) assays were conducted to determine the effect of this Fab-drug conjugate on A431 cells. The specificity and internalization property of this Fab were characterized by immunoprecipitation, fluorescence staining, flow cytometry, and Hum-Zap assay. The binding affinity to purified EGFR was 30 nM. The Fab-Taxol conjugate inhibited A431 cell proliferation at low concentrations and in a dose-responsive manner; more than 70% inhibition was observed at 52 pM. Furthermore, almost 100% of cells underwent apoptosis after treatment with Fab-Taxol at 26 pM for 48 hours. Our findings suggest that this Fab-Taxol conjugate could be a potential immuno-chemotherapeutic drug for clinical treatment of EGFR-overexpressing tumors.
