Study Of Antitumor Effects Of RmFⅦ-Gen Conjugate By Targeting Tissue Factor

Genistein (5,7,4'-trihydroxyisoflavone,Gen), a naturally occurring isoflavone and phytohormone founded chiefly in soybeans, has been reported to be a potent antitumor agent. Epidemiological data showed that consumption of soybean-containing diets was associated with a low incidence of certain human cancers in Asia populations, such as breast, coli and prostatic carcinomas, et al. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth, including inhibition of protein tyrosine kinase activity, topoisomerasⅡinhibition, induction of differentiation, and inhibition of angiogenesis. It has also been shown that genistein inhibits cell proliferation, exerts a specific G2/M arrest and induces cell apoptosis. But the effective dose can hardly reached because of its non-specific distribution in vivo and the weak estrogenic effect, which limits its dosage. One of the effective solutions of the problem is antibody-directed targeting to tumor.Tissue factor, a central component in the initiation of coagulation cascade, is normally not expressed on cells contacted with blood directly, but is highly expressed on most of tumor cells and endothelial cells of the solid tumor vasculature. This expression pattern made it possible that drugs could selectively targeting to tumor vasculature through TF pathway. It is reported that TF not only can accelerate the growth of tumor vasculature, but also is closely related with tumor infiltration and metastasis. If too many TF were abnormally entered into blood vessel, they would cause a severe complicating disease named disseminated intravascular coagulation (DIC). For those reasons, more and morescholars recognized that TF plays an important role in vasculogenesis, and tumor growth and metastasis. and, therefore, may serve as a promisingly potential target for biotherapy or chemotherapy. The mechanism of TF-induced tumor growth is still unclear. It is reported that it could be related to its acceleration of coagulation and the intracellular signaling transduction. The key step of TF- factor Ⅶ intracellular signaling transduction is the phosphorylation of the three ser amino at the end of TF intracellular domain. It's worth noticing that the intracellular signaling transduction is PKC dependent. PKC and inhibitor of tyrosine kinase can inhibit the TF-induced expression of many vascular grwoth factor, including VEGF. While PKC activator such as PKC-αhave effects on the opposite.Genistein is not only a potent anticancer medicine ,but also a specific inhibitor of protein tyrosine kinase.In this study we constructed a kind of recombinant mice coagulation factor Ⅶ(rmFⅦ), the nature ligand of mice TF by Site-Direct Mutagenesis(P10Q,K32E,K341),Pichia Pastoris expression technology. The recombinant could combine to TF without leading to blood coagulation. Furthermore, the recombinant was cross-linked with genistein. The aim of this study is to simultaneously inhibit the blood coagulation initiation activity of TF and its intracellular signaling transduction in order to inhibit the growth,metastasis and the angiogenesis of tumor by using the rmFⅦ- genistein conjugate.There are three parts in our study. ① The construction of rmFⅦ-pPIC9K yeast expression vector. Firstly, the full length cDNA encoding mFⅦ was amplified from a mouse liver by RT-PCR method. Then mutated the aim amino acids by Site-Direct Mutation,and verified by DNA sequence test. Finally, the mutant cDNA gene was subcloned into NotⅠand EcoR sites of pPIC9K. The recombinant expression vector bearing mutant mFⅦ cDNA gene was obtained by screening. ② The Pichia yeast expression , purification of rmFⅦ protein, and the primary test of its biological activity. The verified recombinant vector was transformed into Pichia strain GS115 by electroporation. The positive clones grown on the HIS dropout medium were selected and confirmed by PCR analysis. The transformants with multiple inserts were screened in vivo by their resistance to G418, and Mut+ phenotype by Mut screening. A time cou...