Targeted In Vitro And In Vivo Tumor Suppression By A Secreted Fusion Protein Consisting Of Anti-erbB2 Antibody And Reversed Caspase-3

Although the application of gene therapy has brought new hopes to the cure of cancer, now available protocols are far from satisfying because of poor therapeutic efficiency or low tumor specificity. Apoptosis is a spontaneous cell death process that occurs during oncogenesis or in pathological status, while the cleavage and activation of caspases is a common downstream event in different pathways of apoptosis. It is generally believed that caspase-3 plays the key role during apoptosis of mammalian cells. Caspase-3 exists in cells as proenzyme, which includes a prodomain, a large subunit and a small subunit. After being activated it will causes intense and irreversible apoptosis. It was reported that active form of caspase-3 could be acquired when the large and small subunit of caspase-3 were reversed. Our colleagues constructed reversed caspase-3 (revcasp3) before-mentioned and fused the genes coding a leading peptide, single-chain anti-erbB2 antibody, translocation domain of PE and revcasp3 to generate a novel "immunocaspase-3" genes.In our study revcasp3 was transiently transfected into human mammarycarcinoma cells SKBr3 and stained with HE. Typical apoptotic cells were found two day after being transfected. At the same time the hypodiploid peak located was observed through FACS. The number of apoptotic cells gradually increased. On the fourth day, many apoptotic bodies were formed. Experiment on nude mice proved that expression of reversed caspased-3 gene can obviously induce apoptosis and inhibit growth of tumor in vivo and obvious apoptosis was detected by TUNEL. Thus it can be seen that revcaspS can induce cell to apoptosis in vitro and in vivo.Next "immunocaspase-3" gene was introduced into human lymphoma Jurkat cells. The culture media was collected. The targeted pro-apoptotic activity of immunocaspase-3 was confirmed by culture of Hela, SKBrS and SKOV3 cells with above media. The results of MTT and cell count displayed that the growth of SKBrS and SKOV3 cells was inhibited while that of Hela cells was not. Administration of such Jurkat cells to nude mouses bearing SKBr3 tumor through tail vein decreased the speed of tumor growth clearly. Targeted distribution of revcasp3 in tumor was detected by indirect immunofluence. The experiment proved that immunocaspase-3 can specifically kill cells expressing erbB2 antigen in vitro and in vivo.Introducing gene to cells is one of the core techniques in gene therapy, to which much importance is attached. At present retrovirus, adenovirus and liposome entrapment are most frequently used. So in this study immunocaspase-3 was subcloned into the above three vectors and different treatment approaches were tried to discuss their effect on tumors. Resulting displayed that immunocasp3 gene could be expressed by many means. Then immunocasp3 protein recognizes targeted and kill erbb2 positive cells. Forexample, administrating recombinant plasmids/ liposome both in the muscle and tumor can inhibit the growth of tumors and prolong the survival times of mice. The effect of the former is better (prolongation of survival time 72.09%) than that of the latter (prolongation of survival time 48.11%).The poor efficiency of transfecsion and the probable influence on survival of the nude mice limit the utilization of the liposome. So at present retrovirus and adenovirus are used in the gene therapy more and more. In the paper they were also used to introduce immunocaspase-3 gene to cells.Retrovirus is used to introduce gene to cells in gene therapy at the first and at the most. T cell modified with immunotoxin gene via retrovirus can secrete persistently immunotoxin, which recognize and kill tumor cells. In the paper recombinant retrovirus pLNCX-immunocaspase-3 was constructed and packaged. Monoclone cell line secreting high titer viruses was selected and media with viruses was collected to infect Hela> SKBr3 and SKOV3 cells. The hypodiploid peaks were located in the groups of SKBr3 and SKOV3 cells but not in that of Hela cells. T cells were administrated to nude...