Granzyme B-VEGF Receptor-binding Peptide (GraB-VRB)Fusion Protein Expressed In B. Longum Induces Apoptosis Of KDR Positive Cells

1OBJECTIVEVascular endothelial growth factor (VEGF) is an important signal protein in blood that stimulates vasculogenesis and angiogenesis. All members of the VEGF family stimulate cellular responses by binding to their receptors which called VEGFRs on the cell surface. VEGFR-2which also called kinase domain receptor (KDR), appears to mediate almost all of the known cellular responses to VEGF during vasculogenesis and angiogenesis in tumour. The region of110amino acid residue polypeptide (VEGF110) which located in N-terminal of VEGF mature peptide chain, known as VEGFR-binding peptide (VEGFR the binding peptide VRB), recognizes and binds to VEGFR. Growth and metastasis of malignant tumors depend on angiogenesis. VEGFR is one of specific molecular markers of tumor angiogenesis, thus it become a major target for antiangiogenic therapy. Granzyme B (GRABS) is one of cytotoxic factors in lymphocytes which plays an effector molecules in anti-tumor and antiviral. GraB can enter the target cells through cell membranes by the assistance of perforin or other cell membrane molecules which can destruct cell menbrance and activate multiple signaling pathways to induce cell apoptosis.According to the characteristics of tumor angiogenesis and the molecular mechanisms of cellular immunity, it is possible that GraB has Target effect on the tumor vasculature by using the guiding of VRB. We cloned the human VRB gene cDNA and GraB gene cDNA, and built a pBADA-VRB, pBADA-GraB and pBADA-GraB-VRB expression vector by recombinant DNA technology. In our experiment, these recombinant expression vectors were transformed into Bbifidobacteria and then L-arabinose was used to induce the expression of the recombinant protein. Our aim is to know whether the GraB-VRB fusion protein have the effect on KDR-positive vascular endothelial cells and tumor cells. Engineering recombined can express GraB-VRB in the intestine after being taking in and target to neovascular endothelial cells. These will provide some experimental basis in treat the intestinal tumors.2METHODS2.1Bacteria and cell cultureBifidobacteria were seeded into MRS medium at37℃without oxygen. All cells were cultured in RPMI1640medium with10%NCS at37℃with5%CO2. Fresh medium was changed every2days. Experiments were performed during the logarithmic phase of the cells.2.2Transform of recombinant plasmidsAll three recombinant plasmids were transformed into bifidobacteriums using the optimal electric field strength (2.5kV,25μF,200Ω). Bacterium were cultured onto MRS medium with60μg/ml ampicillin at37℃without oxygen. The milk white clones were picked and grew in MRS medium at37℃.2.3Expression of recombinant proteinsRecombinant plasmids transformed bifidobacteriums were seeded into PRMI1640medium and cultured at37℃. After the OD650nm got to0.6～1.0, bacteriumwere induced with0.2%L-arabia candy and incubated for24hours. After centrifugation, lysates and supernatants were collected and stored at-70℃.2.4ELISADetermine the content of rhVRB in the culture supernatant by VEFG ELISA kits, rhGraB and rhGraB-VRB in the culture supernatant by GraB ELISA kits according to the instructions of the ELISA kits. Culture supernatant of the recombinant protein concentration is converted into mol concentration according to the ELISA results and the calculation of the recombinant protein molecular mass.2.5Western blot24hours after L-arabia candy induced expression of recombinant plasmids, lysates and supernatants of the transformed bifidobacteriums were separated by12%SDS-PAGE followed by transferring onto PVDF membranes. After blocking with5%nonfat dried milk, blots were incubated with monoclonal antibody against GraB for2hours. Then the membranes were washed and incubated with HRP-rabbit anti mouse IgG.Good growth state of cells of each cell line were collected by centrifugation and prepared crude membrane protein samples of each cell lines by soluble membrane protein phase separation preparation kit. Membrane proteins were separated by SDS-PAGE (10%gel) and transferred to a membrane and anti-the KDR/Flk-1monoclonal antibody were added in.2.6Cell proliferation assaySRB (Sulforhodamine B) method was used in Cell proliferation assay. Good growth state of cells were collected and washed, then resuspended to the appropriate density of cell suspension, added to96well culture plates by105cells per hole. Then a certain final concentration of engineering products of Bifidobacterium culture supernatant was added and cultivate a certain time. To fix, dye and bleach inaccordance with the SRB cell proliferation and cytotoxicity assay kit instructions and then detect the OD515nm values on the the enzyme-linked instrument.2.7TUNEL apoptosis in situ detectionHuman umbilical vein endothelial cells (HUVEC) were collected and added to the culture plates by105cells per well. Culture supernatant which contain the rhGraB-VRB was added to the plates until the final concentration of8nmol and were cultured for24h, the cells were taken to coatedfilm. Using the TUNEL (TdT-mediated dUTP nick end labeling) apoptosis in situ detection kit to analyze cell apoptosis in accordance with instructions.2.8StatisticsAll data are presented as mean±SD, and the experiment number was indicated. SPSS13.0was used for statistical analysis. Significances between groups were determined by using one-way ANOVA or two-tailed Student’s t test and were considered statistically significant if the P value was less0.05.3RESULTS3.1Expression of recombinant proteinsEngineered Bifidobacteria expressed rhVRB, rhGraB and rhGraB-VRB induced by L-arabinose for24h. The recombined proteins were separated from the supernatant and were determined by ELISA. The recombinant proteins’ content is roughly0.5to1.0μg/ml. the GraB-VRB gene conversed bifidobacteria and the supernatant were separated annd tested by immune blotting. The result showed that the supernatant and cell samples were approximately showed protein band at38kD, indicating that in addition to the supernatant with rhGraB-VRB, bacteria in vivo also exist rhGraB-VRB.3.2Expression of KDR/Flk-1in several different types of cellsProtein samples extracted from cell membrane were analysed by Western blot. It was found that HUVEC expressed high levels of KDR/Flk-1, murine colon carcinoma cell line CT-26also expressed lower level of, but several other cell lines including human colon cancer cell line SW480, human Burkitt’s lymphoma cell lines Raji and mouse fibroblast cell line NIH3T3did not expressed KDR/Flk-1.3.3Inhibition of rhGraB-VRB on the HUVEC and CT-26cell growthThe cells were incubated with different concentrations of a variety of recombinant protein treatment for24h. The SRB method was used to determine the level of cell survival. The results showed that rhGraB-VRB had significantly inhibited the proliferation of HUVEC compared with control while not seen any impact with rhGraB and slight growth promoting effect with rhVRB. It was also found that low concentrations of rhGraB-VRB had good inhibition of HUVEC up to a certain dose (8nmol), and of Ct-26cells which with lower expression of VEGF receptors. It did not have any effect on the cell proliferation in those cells which did not express VEGF receptors.3.4rhGraB-VRB cytotoxic effects on HUVECHUVEC cells were treated by8nmol/L rhGraB-VRB and observed under light microscope. The result showed that rhGraB-VRB protein had fast and strong cytotoxic effects on the cells. It could be observed the dramatic changes of the cells after30minutes’ treatment under the light microscope and the majority of cell lysis which remaining cells traces. while rhGraB treated for12h cells could not be observed significant changes.3.5rhGraB-VRB induced apoptosis in HUVEC cellsfor24h and used the TUNEL method to analyse cell apoptosis. The result showed that the control group with the culture mudium only had a few nuclear was TUNEL labeled positive (the positive rate of13%), while the experimental group processing with rhGraB-VRB protein had the vast majority of TUNEL-positive nuclei (the positive rate of89%).4CONCLUSIONSPre-constructed recombinant expression vector pBADA-VRB, pBADA-GraB and pBADA-GraB-VRB were conversed to long type Bifidobacteria and expressed the rhVRB, rhGraB and rhGraB-VRB protein induced by L-arabinose. rhGraB-VRB can effectively inhibit the proliferation of KDR/Flk-1positive cells, but no effection on of KDR/Flk-1negative cells. Recombined protein rhGraB-VRB can quickly cause the death of KDR/Flk-1positive cells, and the death mechanism is apoptosis. The results showed that antiangiogenic effect of rhGraB-VRB protein can simulate the lymphocytes physiological mechanism and overcome the shortcomings of the general vascular inhibitors which only inhibit angiogenesis. The recombined protein can destroy the nascent vessels of tumors, directly attack the tumor the cell, and inhibit tumor growth and metastasis effectively. This kind of oral form engineering Bifidobacteria may express rhGraB-VRB protein in the intestine. The protein may have the effect on the neovascular endothelial cells and induce apoptosis in the tumor cells. The treatment of targeting on the intestinal tumors could hope to achieve greater breakthroughs in the prevention and treatment of colorectal and other gastro-intestinal tumors.