Recombinant Construct, Expression And Purification Of Bacterial Redox Protein AZURIN And Preliminary Study Of The Molecule Mechanisms In Induced-U2OS Cells Apoptosis

Osteosarcoma is the most common primary malignant bone tumor occurring mainly in children and young adolescents, Owing to frequent systemic metastases, adjuvant chemotherapeutic treatment is mandatory to achieve a disease-free state in addition to appropriate surgical treatments. Applications of multidrug intensive chemotherapy have substantially improved the prognosis for patients with no detectable metastases at diagnosis, and the long-term survival rate of nearly 70% is noted at specialized sarcoma centers. A considerable portion of similar patients has a poor response to any kind of chemotherapeutic protocols. In patients with detectable metastases at diagnosis (approximately 10 - 20% of all cases) or with unrespectable pelvic or axial skeleton tumors, the prognosis remains poor with 3-year survival rates ranging between 11 and 56%. Precise molecular mechanisms underlying the drug-resistance haveremained to be investigated. Mutations of two tumor suppressor genes, the p53 and retinoblastoma (Rb) genes are molecular hallmarks of osteosarcomas, and several studies have shown the relationship of these genes with drug resistance, some of which were related to the induction of apoptosis, but definite conclusions have not been reached. The development of new antitumor agents that can enhance or replace the effects of current chemotherapeutic drugs will be expected to improve the prognosis.Various candidates for osteosarcoma therapy include differentiating agents, angiogenesis inhibitors, vaccines, monoclonal antibodies, and approaches using dendritic cells. Recently, interest was in using live or attenuated pathogenic bacteria or their products in the treatment of cancer. The infection of tumor-bearing mice with live attenuated cells of Salmonella typhimurium has been reported to produce tumor regression. A significant regression of subcutaneous tumors in mice was observed by combining anaerobic bacteria with various chemotherapeutic agents. The antitumor activity of these pathogens has been attributed to the activation of an immune response against tumor antigens, to the preferential growth of certain bacteria within the tumor), or to the inhibition of angiogenesis. However, live microorganisms with or without chemotherapeutic agents produce significant morbidity and mortality.Hence, studies are in progress to identify pure metabolites of microbial origin or any other components of the microbial cell that might have antitumor activity. One such example is the identification of secondary metabolites, epothilones A and B of the myxobacterium Sorangium cellulosum, which have shown both in vitro and in vivo cytotoxicity invarious cancer cells.AZURIN, a cupredoxin type of electron transfer agent elaborated by the bacterium Pseudomonas aeruginosa, triggers cell death in melanoma cells, In vitro studies revealed a direct physical interaction between azurin and p53. azurin forms a complex with the tumor suppressor protein p53, stabilizes it, and enhances its intracellular level, thereby inducing apoptosis via caspase-mediated mitochondrial pathways. The role of azurin as a potent inducer of apoptosis in melanoma prompted us to examine if azurin could trigger cell death in osteosarcoma, such as U2OS cells.In the present study, we constructed the recombinant vector containing AZURIN gene in Ecoli (PQE30/AZURIN) and get soluble fusion protein that a purified redox protein, azurin, which has been reported to be secreted by Pseudomonas aeruginosa enters human osteosarcoma U2OS cells and induces apoptosis in vitro. The molecule mechanisms of apoptosis were investigated by kinds of methods. The experiment was carried out at two steps.Part one: Cloning , expression and purification of soluble fusion protein of bacterial redox protein AZURIN and exprement of induced-U20S cells apoptosisThe primers containing specific enzyme-cutting sites were designed according to AZURIN gene sequence of the Genbank. Gene group of Pseudomonas aeruginosa were extracted from Pseudomonas aeruginosa. and the AZURIN cDNA were amplified by PCRCpolymerase ch...