Experimental Study On The Targeting Therapy Of Epithelial Ovarian Cancer With TRAIL-Transduced EPCs

TNF-related apoptosis-inducing ligand (TRAIL) is a novel member of TNF family, it preferentially induces apoptosis in tumor cell lines, but not in normal cells. TRAIL could potentially use as a powerful cancer therapeutics.Endothelial progenitor cell (EPC) is a kind of directional cell that is able to differentiate to endothelial cell .The role of EPC is not only with vasculogenesis during embryonic development but also with physiological organ maintenance and angiogenesis during postnatal and adult period. The fact that EPCs home to foci of angiogenesis suggests potential utility as autologous vectors for gene therapy. There is a good clinical therapeutic prospect for EPC in the treatment of tumor.1. Antitumor Effect of rhTRAIL in Human Ovarian cancer cellObjective: To investigate the Induction of apoptosis in ovarian cancer cell (3AO) by rhTRAIL. Methods: TRAIL of different concentration was observed for its in vitro direct cytotoxic effects in ovarian cancer cell by MTT, Apoptosis rate was analyzed by flow cytometry. Results: The concentration of TRAIL from 25 -100 ng/ml had marked apoptosis in ovarian cancer cell line 3AO, and with the increasing of concentration, the apoptosis rate increased too. Conclusions: rhTRAIL has distinguished apoptosis in ovarian cancer , and there is positive relative between the apoptosis rate and the concentration of TRAIL.2. Isolation and Differentiation of Endothelial Progenitor Cells from Cord BloodObjective: To explore biological characteristics of the EPCs, endothelial progenitor cells from cord blood were isolated and induced to differentiate in optimal medium. Methods: CD 133+ cells were selected from fresh umbilical cord blood mononuclear cells (MNC) by MiniMACS system, grown on fibronectin-coated flasks in IMDM medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor( VEGF), basic Fibroblast Growth Factor (bFGF ) , stem cellfactor( SCF) . In addition , we tested the effect of VEGF, SCF and bFGF on cell differentiation , expansion kinetics. EPCs were determined and quantified by immunocytochemistry , flow cytometry, and electronic microscope. Result: Attached spindle-like cells appeared after 4 day's culture and formed tube-like strcture by 16 days. VEGF, SCF and bFGF could promote EPCs expansion and differentiation. These differentiated EPCs express endothelial-specific markers, including CD34, von Willebrand factor (vWF) and flk-1. Conclusions: There are endothelial progenitor cells in cord blood, which can differentiate into endothelial cells.3. Construction of GFP -hTRAIL Fusion Gene Expression VectorObjective To constructe fusion gene expression vector , EGFP-hTRAIL. Methods To constructe fusion gene expression vector, by inserting hTRAIL gene into GFP gene N-terminal. The recombinant had been differentiated by the technology of restriction digest and electrophoresis. GFP-hTRAIL plasmid was transfected into ovarian cancer cells by Lipofectamine? 2000. Gene transfer efficiency was determined by fluorescent microscope and flow cytometry. Results Restriction analysis showed that the structure of the EGFP -hTRAIL plasmid was exactly the same as anticipated. Further results indicated that both GFP and hTRAIL gene were simultaneously expressed in ovarian cancer cells. Conclusions A new plasmid has been established as the vector for studying the gene therapy of ovarian cancer. GFP gene expression can be used to monitor gene expression in living cells. This experiment is the basic part of the next experiment.4. Expression of TRAIL Protein in cell culture supernatant after two different tansfection methodObjective To determine the sTRAIL level in cell culture supernatant after TRAIL gene transfected into EPC by electroporation and liposome method. Method Immunoassay was used for the quantitative determination of sTRAIL concentration in cell culture supernatant. Result 24h after the processes of electroporation transfection and liposome transfection, The expression of sTRAIL increased, and could last 72h despite changing culture medium. The efficiency of liposome method seemd slightly higher than that of electroporation method. Conclusions There is basic physiological secretion of TRAIL in EPC. The sTRAIL level increase after TRAIL gene transfection. The efficiency of liposome method is better than that ofelectroporation method.5. Antitumor activity of EPC culture supernatant after TRAIL transfectionObjective To express TRAIL protein using endothelial progenitor cells (EPCs) and observe antitumor activity of sTRAIL in cell culture supernatant. Methods EPCs were isolated by immunomagnetic sorting. hTRAIL plasmid was transfected into EPC by LipofectamineTM 2000 and electroporation method. The cell culture supernatant was harvested and the secreted protein was measured by Immunoassay method. Antitumor activity of sTRAIL was measured by MTT method, apoptosis rate was analyzed by flow cytometry. Results EPC could develop from the culture of cord blood CD 133+ cells. EPC could be transiently transfected hTRAIL. The secreted protein could be harvested in the cell culture supernatant. MTT method and apoptosis detection showed that sTRAIL had high antitumor activity in 3AO cell. Conclusions In vitro, TRAIL EPC gene transfer enhances EPC secreting TRAIL protein, and inducing apoptosis of ovarian cancer cell. There is a good clinical prospective therapeutic use for EPC in the gene therapy of ovarian cancer.6. Inhibitory effect of EPC transfected with TRAIL on the subcutaneous tumors transplanted with human ovarian cancer in nude miceObjective TNF related apoptosis inducing ligand (TRAIL) could induce apoptosis in various cancer cell lines with little toxicity toward normal cells. It offers a promising therapeutic method against ovarian cancer. Endothelial progenitor cells (EPCs) homed to foci of angiogenesis suggested that potential utility could be a method as autologous vectors for gene therapy. In the current study, we observed antitumor activity of Endothelial Progenitor Cell Transfer with TRAIL gene on the human ovarian epithelial cancer transplanted subcutaneously in nude mice. Methods EPCs were isolated from human cord blood by magnetic bead selection on the basis of cell surface antigen CD 133. hTRAIL plasmid was transfected into EPC by Lipofectamine? 2000. And then the EPCs transfected with TRAIL were injected by caudal vein into immunodeficiency mice model with transplanted hypodermic 3AO. The changes of tumor volume were observed and the tumor growth inhibitory rate was calculated. Results The tumor growth of the treated group was inhibited ,and the maximum tumor growth inhibitory rate was 66.9 %( P < 0. 05) ; Conclusions The studies suggest that TRAIL transfection can inhibit the growth of ovarian...