ABSTRACT
Pancreatic adenocarcinoma is an aggressive disease with a high mortality rate. In this study, we have newly generated a monoclonal antibody (mAb), Pa65-2, which specifically binds to pancreatic cancer cells and tumor blood vessels. The target protein of Pa65-2 is identified as human clathrin heavy chain (CHC). In vitro and In vivo study showed that suppression of CHC either by shRNA or by Pa65-2 inhibited tumor growth and angiogenesis. One of the key functions of CHC was to bind with the hypoxia-inducing factor (HIF)-1α protein, increasing the stability of this protein and facilitating its nuclear translocation, thereby regulating the expression of VEGF. Taken together, our findings indicate that CHC plays a role in the processes of tumorigenesis and angiogenesis. Pa65-2 antibody or CHC shRNA can potentially be used for pancreatic cancer therapy.
Subject(s)
Adenocarcinoma/pathology , Cell Cycle Proteins/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neovascularization, Pathologic , Nuclear Proteins/metabolism , Pancreatic Neoplasms/pathology , Vascular Endothelial Growth Factor A/metabolism , Adenocarcinoma/blood supply , Adenocarcinoma/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Apoptosis , Blotting, Western , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Proliferation , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Female , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/genetics , Humans , Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Immunoenzyme Techniques , Immunoprecipitation , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, SCID , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Pancreatic Neoplasms/blood supply , Pancreatic Neoplasms/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A/geneticsABSTRACT
The utility of capillary electrophoresis (CE) has been demonstrated for the analysis of secretory immunoglobulin A (sIgA) in human saliva. The amount of sIgA in saliva correlates with immune status. For detecting salivary sIgA, laser-induced fluorescence was conducted in this report for signal amplification. sIgA and anti-sIgA antibody were labeled with cyanine fluorescence (Cy5) for competitive immunoassay and non-competitive analysis, respectively. Cy5 was excited by He-Ne laser with a wavelength of 635 nm, with maximum emission at 670 nm. Migration time during electrophoresis depended on whether sIgA-Cy5 was mixed with antibody or anti-sIgA-Cy5 mixed with sIgA to form Ag-Ab complex. The results indicated that CE competitive immunoassay was effective for analyzing serum sIgA, but not for salivary sIgA. However, salivary sIgA can be analyzed by complex formation assay. The peak area of the complex was proportional to the amount of sIgA added. A standard linear regression curve was generated using purified sIgA. From this standard curve, the amount of sIgA from saliva of either normal or immunocompromised patients can be calculated from the Ag-Ab complex peak area.
