| Osteosarcoma is a common primary malignant bone tumor in children and adolescent, whose distinct characteristic is early metastasis. 80% of patients have already had pulmonary metastasis when the clinical diagnosis of osteosarcoma is made. During the recent 25 years, remarkable advance in the treatment of osteosarcoma has been made. Though nowadays nearly 80% of patients can have their invalid extremities preserved after treatment and the 5-year survival rate has been elevated from approximately 20% to nearly 80%, still more than 50% of the osteosarcoma patients would die as a result of metastasis and relapse. In recent 10 years, the rise of gene therapy, immunotherapy and molecular targeted therapy has shed new light to the treatment of osteosarcoma. The exploring of new effective treatment is now a significant direction in osteosarcoma research. We have successfully constructed a recombinant Caspase-6 fusion protein which carries the specific HER2 targeting gene, and conducted both in vitro and in vivo study to investigate its possible curing property for osteosarcoma.Caspase-6, also named mch2, is an essential member of caspases family. It is one of effectors. The gene encodes a 34-kD protein that is highly homologous to human CPP32(Caspase-3), mammalian Ich1/Nedd2 and mammalian interleukin-1-beta. Active Caspase-6 protein induces apoptosis by cleavage of poly(ADP-ribose) polymerase and lamin A. It is reported that recombinant Caspase-6 by rearrangement of its subunits is an active molecule. Recombinant Caspase-6 can recognize and cleave PARP and lamin A, and its expression in Hela cells.HER2/neu (also known as HER2 or c-erbB2) is a 185 KDa protein receptor with tyrosine kinase activity and extensive homology to the epidermal growth factor (EGF) receptor. Normally HER2/neu is expressed only in fetal period;and expressed in some epithelial tumors at a low level.However,HER2/neu for adults, is known to be overexpressed in approximately 25-30% of metastatic breast cancer, 50% of inflammatory breast cancer, 60-70% of metastatic ductal carcinoma , 13-55% of non-small cell lung cancer, 18-43% of oophoroma, 10-52% of endometrial cancer, 33-85% of colon and rectum carcinoma, 22-36% of renal carcinoma , 16-50% of head and neck cancer, 21-64% of gastric cancer, 10-26% of esophageal cancer, 5-46% of prostatic cancer, 40-44% of osteosarcoma and 58% of pulmonary metastasis of osteosarcoma. HER2/neu overexpression represents a marker of poor prognosis. Many studies have demonstrated that repression of HER2/neu overexpression suppresses the malignant phenotypes of cancer cells. The initial success of trastuzumab, HER2/neu monoclonal antibody indicates that HER2/neu is a promising target for cancer therapy.Immunocasp-6(e23sFv-PEⅡ(253-364)-caspse-6,)which can selectively recognize and kill HER2-overexpressed tumor cells was obtained by sequentially fusing the coding sequences of a signal peptide, a single-chain HER2 antibody (e23sFv), a PE translocation domain (PE253–364aa) and recombinant Caspase-6. We discovered that the targeted pro-apoptotic activity occurred in HER2 positive tumor cells, while no significant influence occurred in normal and HER2 negative tumor cells.Firstly, the immunocasp-6 were cloned into pCMV plasmids to construct a kind of eukaryotic expression vector,i.e. pCMV-e23sFv-PEⅡ(253-364)- caspase-6 (abbr. pCMV-6) and transfected into osteosarcoma SOSP-9607 cells with the method of cathodolyte liposome parking. The effect of target gene expression on morphology and growth status of osteosarcoma SOSP-9607 cells were observed by fluorescence and electron microscope and were analyzed by Annexin V-FITC staining and MTT assay respectivekly.Our study showed the target gene were detected in the cytoplasm of transfected osteosarcoma SOSP-9607 cells;the transfected cells presented the typical characteristics of apoptosis ,such as plasma membrane bubbling,cell nucleus condensing,chromatin concentrating and so on,as detected by electron microscope, while no-transfected cells presented normal constructure;Furthermore,Annexin V-FITC staining revealed that the percentages of apoptotic cells in the transfectants of pCMV-6 were 31.4%,compared to 6% in the control cells, and MTT assay discovered the proliferation of immunocasp-6 gene-transfected osteosarcoma SOSP-9607 cells were much slower than those of non- or mock-transfected cells. We subsequently tested the anti-tumor activity of immunocasp-6 in nude mouse models in vivo. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. It was observed that the administration of immunocasp-6 by intramuscular injection selectively suppressed the growth of HER2 positive osteosarcoma SOSP-9607 cells,the growth of the tumor in the treatment group was significantly slower than that of control group. HE and TUNEL analysis of the tumor and muscle tissues of nude mice in the treated group nude mouse showed the typical characteristics of apoptosis,while normal constructure were found in the control group. Furthermore, Caspase-6 was not found to locate in tumor tissue of rude mice of the control group,but only in the treatment group by means of immunohistochemistry staining.At the same time,Caspase-6 was not yet found in muscle tissues and other tissues of both treatment group and control group.Our study has proved that immunocasp-6 can selectively recognize and bind to HER2 positive osteosarcoma SOSP-9607 cells,after internalized into cells they can recongnize and cut cell substrate and carry out pro-apoptosis function,while they have no damage to HER2 negative tissues.In a word,in our constructed fusion protein,e23sfv still can selectively recognize and bind to HER2 antigen,and recombinant Caspase-6 still have high pro-apoptosis activity.In summary, immunocasp-6 can selectively recognize, bind to and kill HER2 positive osteosarcoma cells,which can offer some foundation for the gene therapy of osteosarcoma. |
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