ABSTRACT
<p><b>OBJECTIVE</b>To investigate the mechanism of paclitaxel-induced apoptosis in MCF-7 human breast carcinoma cells.</p><p><b>METHODS</b>In this study, the proteins extracted from paclitaxel-induced apoptotic MCF-7 cells were analyzed by 2-dimentional gel electrophoresis (2-DE), and compared with those from untreated MCF-7 cells. The differential proteins were identified by mass spectrometry.</p><p><b>RESULTS</b>At 24 hour after paclitaxel (100 nmol/L) treatment, MCF-7 cells were collected and extracted the whole proteins. Seventeen up-regulated or down-regulated proteins were found by analysis of the differential proteomic 2-DE map. Six of them were identified by mass spectrometry. They were enolase 1, chloride intracellular channel 1, keratin 8, ribosomal protein S12, galectin-1 and histidine triad nucleotide binding protein, respectively.</p><p><b>CONCLUSION</b>We effectively found the changed proteins in the process of paclitaxel-induced apoptosis in MCF-7 human breast carcinoma cells by proteomic techniques. These up-regulated or down-regulated proteins are important molecules for our further research about the mechanism of paclitaxel-induced apoptosis.</p>
Subject(s)
Female , Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Breast Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Electrophoresis, Gel, Two-Dimensional , Galectin 1 , Metabolism , Keratin-8 , Metabolism , Paclitaxel , Pharmacology , Phosphopyruvate Hydratase , Metabolism , Proteomics , Methods , Ribosomal Proteins , MetabolismABSTRACT
Functional heavy-chain antibodies (HCAbs) lacking light chains occur naturally in camels. The variable domain of heavy chain of heavy-chain antibody is referred to VHH. The VHH gene family is homologous to human VH subgroup III. The single-domain VHH antibodies are constructed by cloning the variable domains of HCAbs. Compared to human VHs, VHH germ-line sequences contain some hallmark substitutions in framework region 2, including V37F(Y), G44 E, L45 R, W47G. The substitutions at positions 44, 45, 47 are often used to camelise the human VHs. Being a small binders, VHH antibodies are well expressed, extremely stable and very soluble. Camelised human VHs are proved to exhibit the same qualities as those of VHH antibodies. The single-domain VHH antibodies will be useful in the drug development and basic research.
Subject(s)
Animals , Humans , Binding Sites, Antibody , Camelus , Allergy and Immunology , Metabolism , Genes, Immunoglobulin , Immunoglobulin Heavy Chains , Genetics , Immunoglobulin Variable Region , Genetics , Protein Engineering , Recombinant Proteins , Genetics , Allergy and ImmunologyABSTRACT
The aim of this study is to construct a phage display single-chain variable fragment (scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the library. The BALB/C mice were immunized with MCF-7 cells. Total RNA of spleens was isolated. The heavy-chain (VH) and light-chain variable region genes (VL) of the antibodies were amplified by RT-PCR and joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E) and the recombinant phagemids were used to transform competent E. coli TG1. The transformed TG1 cells were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The scFv fusion proteins were displayed on the surfaces of the recombinant phages. A phage display antibody library of repertoire of 1.2 x 10(6) clones was constructed. The specific antibodies against MCF-7 cells were enriched by 75 times after five rounds of affinity selection. Ten recombinant phages clones that exhibited specific binding to MCF-7 cells were identified. The specificity of those phage clones was analyzed by reactivity against HepG2 cells and Hela cells by ELISA. One of the selected phage clones against MCF-7cells was used to infect E. coli TOP10 to produce the soluble scFv antibodies after induction with IPTG. The strategy of construction and screening of antibody library directed against the whole tumor cells described in this report should be generally applicable to generate tumor cell-specific antibodies.
Subject(s)
Animals , Female , Humans , Mice , Antibodies, Neoplasm , Genetics , Allergy and Immunology , Breast Neoplasms , Allergy and Immunology , Therapeutics , Cell Line, Tumor , HeLa Cells , Hep G2 Cells , Immunoglobulin Heavy Chains , Genetics , Immunoglobulin Light Chains , Genetics , Immunoglobulin Variable Region , Genetics , Mice, Inbred BALB C , Peptide Library , Single-Chain Antibodies , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To develop toxin targeting vascular endothelial growth factor receptor II (VEGF-II/KDR) fused with a KDR-binling peptide screened from peptide library.</p><p><b>METHODS</b>By affinity to KDR molecular which expressed specifically by new born vascular endothelial cell, peptides to KDR were screened from C7 peptide library by phage display. Among them, a peptide binding to KDR with high affinity termed as P5 was selected and fused to the N-terminal of Shiga toxin subunit A (StxA). The protein (P5-StxA) was expressed in E. coli.</p><p><b>RESULTS</b>ELISA and Western blot were applied to characterize the binding interaction between the fusion protein, P5-StxA and KDR. Cytotoxicity assay showed that P5-StxA maintained similar toxicity to cell as StxA. In the model of angiogenesis, P5-StxA inhibited selectively VEGF-induced growth of preexisting vessels of the chick chorioallantoic membrane.</p><p><b>CONCLUSION</b>These studies demonstrate the small peptide, P5, maybe be used as carrier of toxin targeting to KDR.</p>