Application Of Anthrax Toxin Targeting A Variety Of Tumor Cells In Drug-oriented System, A Preliminary Study

Improved understanding of the molecular mechanism of tumorgenesis, coupled with the improvement of molecular biology technology, make it possible to block the growth of cancer cells by interfering with specific targeted molecules, such as receptors, regulating factor, needed for carcinogenesis and tumor growth, which is called targeted cancer therapies. Targeted cancer therapies may be more effective than current treatments and less harmful to normal cells.In this research, we took advantage of the vast expression of different cell-surface MMPs or uPA in tumors to activate tumor cell-selective cytotoxicity and design a more broad-spectrum cell-surface MMP or uPA-activated anthrax toxin by replacing the furin protease cleavage site with different sequences selectively cleaved by MMPs or uPA in one PA mutant proteins. By mutating anthrax toxin protective antigen (PA) proteins in which the furin protease cleavage site is replaced by sequences selectively cleaved by MMPs or uPA, MMP or uPA-targeted PA proteins were activated rapidly and selectively on the surface of MMP or uPA-overexpressing tumor cells. The mutated PA proteins killed MMP or uPA overexpressing tumor cells while sparing nontumorigenic normal cells.We construct the pET-32a-PA plasmid. PA protein were expressed in E.coli BL21 (DE3), purified and renatured by dialysis. The purity of PA is 76%. The PA protein can be recognized and cleavage by Furin protease in vitro. The PA proteins can have cell cytotoxicity with the present of LF.We constructed different recombinant PA proteins by replacing the furin protease cleavage site with different sequences selectively cleaved by MMPs or uPA in PA mutant proteins. By replacing the furin protease cleavage site of PA with sequences selectively cleaved by MMP2 we constructed PA (MMP2). Smilarly we constructed PA (MMP9), PA (MMP2+MMP9), PA (uPA), PA (MMP2+uPA) and PA (MMP2+MMP9+uPA).The recombinant PA proteins can be cleavaged by corresponding MMPs or uPA. Cytotoxicity analysis demonstrate that recombinant PA have tumor cell toxicity to fibrosarcoma HT1080, breast cancer MDA-MB-231 cells with the present of LF.Immunofluorescence experiment showed that recombinant PA and LF can cause obvious morphology changes of fibrosarcoma HT1080 cells. This method of achieving more broad-spectrum cell-type specificity may provide more efficient new therapeutic agents for cancer treatment.