Studies On Expression Of Targeted Toxin DLM,Anti-tumor Activity And The Possible Mechanism

Cancer is a serious disease, which threaten human health, but till now there are still no especially effective strategies and therapeutic drugs. The routinely used chemotherapy drugs due to its non-selectivity, high treatment dose, have obvious side effects on normal tiss and when using in longterm, the tumor cells will get tolerance to these drugs, so it can not get satisfactory therapeutic effect. With the development of molecular oncology, a deeper understanding of the tumor development of mechanisms makes tumor treatment method changed. Nowdays, cancer targeted therapy has became the main trends in tumor treatment, more and more research has been done in the field.Targeted toxins are used as a new generation of cancer therapies durg, it contains three parts:targeted carrier, cytotoxic molecules (warhead) and linker that link the carrier and warhead. It has the advantage of enriching the toxin at tumor-specific receptor, linker can be cleaved efficiently at targeted site and released the toxin to kill tumor. But till now, many trial targeted toxins did not present expected therapeutic efficacy in the application due to many reasons. Such as targeted carrier have no enough binding capacities to the receptor, the linker can not be cleaved efficiently or unstable in the cyclating system. For solving those problem in practice, we design a novel targeted toxin has the advantage of high binding specificity, low side effects and can release the cytotoxic molecules efficiently at tumor.Integrin is a class of cell surface receptor family, involved in the process of tumor formation, growth, invasion and metastasis. It changes with the development of tumor, affects the interaction between the tumor and its microenvironment. The memberαvβ3 integrin is highly expressed in the surface of many tumors and in the endothelial cells of tumor angiogenesis also strongly express. But in mature endothelial system and the majority of normal organs,αvβ3 is not expressed or low expression. These features make theαvβ3 to become an ideal target for cancer therapy, we selecteαvβ3 as the targeted site to design the targeted toxin DLM in this study.RGD (Arg-Gly-Asp) is the ligand recognition site of integrinαvβ3. Use it as a targeted drug carrier to target delivery of cytotoxic drugs to the tumor has become a promising technology for cancer targeted therapy. In this study, a RGD sequence containing snake disintegrin was used as the carrier of recombinant toxin targeted. The ability of disintrgrin binds toαvβ3 is more 2000 times than the RGD. The disintegrin can transport the targeted toxin to tumor, anchore the warhead to the tumor cell surface. Because of the low concentration in the normal and non-targeted tiss, this reduces the side effects on normal tissues.For the selection of cytotoxic molecules, we choose melittin. Melittin is a typical cationic peptide. It is a 2840 Da small molecular which comprised by 26 amino acids. The NH2-terminal region 20 amino acids of melittin are largely hydrophobic whereas the region at the COOH-terminus 4 amino acid residues is hydrophilic and the whole molecule has six positively charged. At low concentrations and low ionic strength melittin exists in the random coiled structure, but at higher concentrations and higher ionic strength melittin is able to assemble itself into tetrameric and amphipathic structures. In the different solutions, the angles between a helix structural area and helix of melittin are differentiated.The first 21 amino acids in the helix structure is polar, locating at the surface of helix, and the non-polar amino acids locate at the other side of helix. This amphiphilic property is the characteristic of membrane-bound peptidases and transmembrane helix in membrane proteins. It decides that melittin can be dissolved in water and bound with membrane naturally. Melittin kill cell without going through cell internalization, it can destroy the integrity of the cell membrane and makes the cell contents to leak out, cell death.Expect of the selection of carrier and toxin, how to choose an appropriate way to connect them is another critical factor, because of this can affect the stability of the system cycle and the efficiently release toxin. For effective release the melittin on tumor surface, we need to select a short peptide linker which is stable in the circuloationg systerm and can be cleaved by the tumor-specific protease at cell surface. uPA is a serine protease which is highly expressed in many tumors. uPA protein is 411 amino acid residues long, secreted as a 53 kD zymogen(pro-urokinase). It is activated to double uPA by plasmin cleavaging. uPA and integrinαvβ3 can bind together to form a complex concentration by its Kringle, so the uPA was enriched at the tumor receptorαvβ3. If an uPA cleave site is inserted between the carrior and the warhead, the tumor expressed uPA can cleavage the linker and release the warheads to kill tumor. So we used uPA cleavage short peptide as the linker of targeted toxin.As metion above, in the study we used the snake disintegrin as the targeted carrior, uPA cleavage short peptide as the linker and melittin as the warhed to construct targeted toxin DLM. We will verify the biological function of the recombinant toxin DLM at the follow aspects:1) Expression of DLM in Escherichia coliFirstly, we intended to express the fusion protein GST-DLM by pGEX-2T, a prokaryotic expression vector. The plasmid pGEX/DLM was transformed to BL21, induced with 0.8 mM IPTG at 37℃for 3 h. The cells were sonicated and purificated the soluble GST-DLM protein by GST affinity chromatography. Cut the GST-DLM protein by thrombin to remove the GST tag and obtained DLM.2) Expression of DLM in Pichia pastorisPichia pastoris expression vector pPIC9K/DLM was constructed,6×HIS was added to the N-terminal of DLM to facilitate downstream purification. The Sac I linearized plasmid pPIC9K/DLM was transformed to strain GS115 via electroporation. The genomic DNA of the transformants was extracted for PCR identification. Selected the positively transformants with high G418 resistant, induced with 0.5% methanol to express DLM. The supernatant of fermentation was identified by SDS-PAGE and Western Blot. The high express level strain was screened.Centrifuged the fermentation supernatant and concentrated it with sulfate ammonium, then used the gel filtration chromatography and affinity chromatography to obtain purity more than 95% of the recombinant protein DLM. The ESI mass spectrometry analysis show the molecular weight of DLM is about 12187.56. That is approximately the same with anticipated, means a successive expression in Pichia pastoris.3) Studies on pilot-scale fermentation and purification process of DLMWe performed pilot-scale fermentation of DLM in a 100 L fermentor, refering the results of shake flask fermentation to analysis the parameters, such as the fermentation pH, fermentation medium composition, dissolved oxygen, temperature of fermentation, methanol flow acceleration rate, the initial biomass. Finally the conditions of pilot-scale fermentation were determined as follows:fermentation pH 6.0, with FM 21 medium, when cell density reached 180～200 g/L, inducing with methanol, the value of dissolved oxygen through the process control maintained at 20～40%, after 30 h induced with methanol stop fermentation.The fermentation supernatant was concentrated with 75% sulfate ammonium. Then, use Butyl Sepharose FF, Ni-Sepharose 6 FF and Sephadex G-50 chromatography at pH 7.4 to purify the DLM. A final purity of more than 95% and the yield of 55% were achived. Each liter of fermentation broth could eventually harvest 198.4 mg DLM. 4) Activity assay of purified DLMTo verify the active of purified DLM, a growth inhibition assay on the tumor cells A549, SKOV-3 and SMMC-7721 cell line with MTT. The result show that the lethal effect of DLM to the three tumor cell lines is obvious, that certify the recombinant protein DLM has a good biological activity.5) Preliminarily discussion the mechanism of DLM on MCF-7 cell lineThe hemolysis experiment results show, DLM obviously reduces the hemolytic activity of melittin.32μg/mL DLM almost has no hemolysis. Concentrations of 2μg/mL,4μg/mL, 8μg/mL,16μg/mL,32μg/mL of DLM incubated with 293 cells 48 h, the result shows that low concentrations, the DLM almost has no cytotoxic, even in a concentration 32μg/mL DLM only show litte cytotoxic instead of appearing obvious effect on the 293 cells. Those result show the DLM has low cytotoxicit to normal tissues.In vitro experiment, uPA can cleave DLM at the linker verify the linker used in this study can be cleavage. Western Blot result show the uPA expressed in MCF-7, which MCF-7 cell can used as the targeted cell for study the reseach of mechanism.MTT method was used to described effect of different concentrations of DLM incubate with the MCF-7 for 7 days, results showed that DLM has a significant killing effect on it and show a time and dose-dependent. The flow cytometry result shows that 2μg/mL,4μg/mL,8μg/mL,16μg/mL DLM have obvious cytotoxicit on MCF-7 cell. As concentration of DLM increased, the cytotoxicity on MCF-7 is significantly increased. The mechanism of DLM towards MCF-7 is necrosis rather than apoptosis pathway.Fluorescence microscopes observe the Hoechst 33342/PI stained MCF-7 cells. Control group MCF-7 nucleus showed a diffuse light blue fluorescent nucleus and can not see red fluorescence. The treated with different concentrations DLM MCF-7 cell dose not appear apoptotic body, but cells do not have complete cell membrane, appears red fluorescence, the cell has been necrosis. The degree of necrosis increased with the concentration increase. DNA electrophoresis showed that the after 4μg/mL,8μg/mL,16μg/mL DLM treatment, the cell DNA appear diffusion strand instead of step-shaped strands. The control and 2μg/mL dose group cells DNA appear a macromolecule fragment. Those indicated that cell occurres necrosis instead of apoptosis.We observe the chang of cell morphology after teated by DLM for 48 h by inverted microscope. The cell began to emerge obviously necrotic morphology change from the dose of 4μg/mL, high membrane permeability, cell swelling, membrane rupture, release of cell contents. At the dose 8μg/mL, most of the cells showed a cell membrane rupture, lower light transmission, cell contents spill. It is difficult to find complete cellular morphology at the dose of 16μg/mL.The influence of DLM on MCF-7 cell ultrastructure is observed under transmission electron microscope. In control, MCF-7 cell is larger, the cell surface microvilli are clearly visible, the nuclei are larger and irregular and mitochondria, endoplasmic reticulum and other organelles can be seen in the cytoplasm. After 4μg/mL DLM treatment, MCF-7 cell membrane were damaged, cell cavitation, organelles disappear, and cell contents overflow. At dose of 8μg/mL, the cells are difficult to find the integrivitive cell morphology, cell cavitation, organelles disappear. The above results indicate that the action mechanism towards MCF-7 cells is necrosis instead of apoptosis.In summary, we first express the DLM in E. coli. For the purpose of highly expression, we constructed a pPIC9K/DLM eukaryotic expression vector, screened and selected the DLM high level and steady expression P. pastoris engineering strains. We performed the pilot-scale fermentation of DLM in fermentor and built a new method for large-scale purification of it. Observed lethal effect of DLM on tumor and studies its possible mechanisms preliminarily. The result shows that DLM has advantage of stability in circulating system, high targeting specifity and low side effect. The accomplishment of this study provides a new cancer treatment way and have important scientific and practical significance.