Expression Of Anti-prostate Cancer Polypeptide In E.coli And Its Active In Vitro And In Vivo

Most drugs for chemotherapy lack selective specificity to malignant cells in cancer treatment, which usually brings damage to normal tissues simultaneously while targeting on tumor cells instantaneously because of cytotoxicity. So in recent years one of the most important research topics on tumor chemotherapy is to build high selective directed gene therapy.Prostate cancer, one of the most prevalent malignant tumors in older males, takes the first place for American old man on the list of male tumor incidence rate. The incidence rate has been increasing year by year with the change of average life span and dietary structure in China. Except operation, there is no optimal protocol to treat the prostatic carcinoma and it's very necessary to explore and find some new strategies for this malignant disease. In recent years, increasing attention for prostate-specific antigen activated prodrug, a peptide-cytotoxic drugs conjugate, was designed to deliver the conventional cytotoxic drugs selectively toprostate cancer cells. The peptide portion of the drug is cleaved by prostate specific antigen (PSA), a protease expressed by prostate cells. PSA-mediated cleavage of the peptide allows the active metabolites cytotoxic drugs to enter the cells and exert cytotoxic effects.In recent year, apoptosis and cell grows factor was become important in the tumor gene therapy. Structure/function studies revealed that BH3 domains function as uniquely important "death domains" in proapoptotic Bcl-2 family members. BH3 was deliveried on mitochondrion in tumor by protein transduction domain(PTD) and induce tumor apoptosis. vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) is a specific mitogen for endothelial cells that has been shown to be expressed in many tumor cell lines and is important not only in angiogenesis, but also in tumor cell proliferation through interaction with its receptors. Inhibition of VEGF and bFGF interaction with its receptors that restrain tumor-induced angiogenesis and induced tumor apoptosis simultaneously may be a successful approach to treat human Prostatic carcinoma.We designed, expressed a novel polypeptide prodrug for anti-prostate cancer which activation in the tumor by PSA-specific hydrolysis and determinate its activity in vitro and vivo, to provid a new strategy and agent for specific treatment of prostate cancer. The polypeptide drug was designed based on the amino sequences of BH3, K237, DG2 domain and peptide that could be digested by PSA. The coding sequence of the drug was obtained by back-translation using preferred codons of E.coli and chemically synthesized in nine oligonucleotides.Then the whole sequence was constructed by phosphorylation, annealing and ligating into EcoRI and BamHI site of pGEX-4T-2 expression vector, and confirmed by restriction enzyme digestion and DNA sequencing. DNA sequencing result verified that the sequence of pGEX 4T-2-APP216 are consistent with that we had designed. SDS-PAGE analysis demonstrated that protein was expressed in E.coli. The protein band amounted to30% of total bacteria protein through the computer scan analysis. The dissolvable protein was purified by GSTrap FF column and was determined activity in vitro and vivo. MTT test suggest 22%, 34% cell livability afer APP216 (270ug/ml) treatment 48h and 9.8%, 8.2% cell livability afer APP216 (270ug/ml) treatment 72h in human prostate cancer cell line 22RV,, LNCaP which secrete PSA, spectively. 90%, 95% cell livability afer APP216 (270ug/ml) treatment 48h and 87%, 92% cell livability afer APP216(270ug/ml) treatment 72h in human prostate cancer cell line PC3, DU145 which no secrete PSA, spectively. Hoechst 33258 staining cells manifested the typical features of apoptosis, including cell shrinkage, chromatin condensation and hypodiploid genomic DNA content in human prostate cancer cell line LNCaP which secrete PSA and no change in human prostate cancer cell line PC3m which no secrete PSA. In human prostate tumor xenograft, 22RV1, statistically significant reduction in the serum PSA consentration of 71.1% (p<0.05) and an average tumor volum reduction 51%(p<0.05) in end-therapy compared with pre-therapy, an average tumor weight of 52% (p<0.05) in therapy group compared with vehicle group. In human prostate tumor xenograft, PC3 which no-secreting...