Reconstruction And Biological Activities Of Granzyme B

Cancer therapy is a strategic problem for community health along with increase of cancer mortality.Traditional actinotheraphy and chemotherapeutics are not effect enough for progressive tolerances of tumor cells,and targeted therapy is in front of this field now. However,the severe side effects and the strong immunogenicity are obstacles for the clinic application of a large number of targeted drugs getting from previous studies.The "Clever drug" for carcinoma therapy is appearing to be novel star in the twenty-first century.Such kind of drugs could comparatively accurately target cancer cells with minimized side effect.Under these circumstances,we focus on re-construction for a novel anti-cancer drug which should have potent cytotoxic activity to tumor cells with minimized side effect.Human endogenesis Granzyme B(GrB) is a serine esterase produced by cytotoxic lymphocyte cells and nature killer lymphocyte cells.Within the target cells,granzyme B rapidly activates caspase-dependent apoptosis and/or caspase-independent apoptosis and induces cytotoxicity by directly cleaving a variety of death substrates.Granzyme B concerns autoimmune diseases and anti-virus infection very nearly.It is increasingly attracting attention for its application value on cancer therapy.On the other hand,human endogenesis gonadotrophin releasing hormone(GnRH) is a peptide containing ten amino acids.It is secreted by hypothamus and regulats the incretion of hypophysis gonad.A lot of adenocarcinoma cells overexpress GnRH receptors(GnRHR) on their surfaces.And the GnRHR amount on normal cell surfaces is far lower than that of tumor cells.So, GnRH is a prefect target for adenocarcinoma therapy.In this project,we utilized knowledge and techniques derived from molecular biology to reconstruct human GrB and to design targeting fusion protein so as to concentrate the lethal effect of mutated GrB on GnRHR+ adenocarcinoma cells.A site-directed mutagenesis was first made on GrB to eliminate its original cell binding activity through the electrostatic exchange-adsorption model and to retain its enzymatic activity.Then the mutant mGrB was fused with G4S linker and GnRH gene to reconstruct a fusion encoding sequence so as to express mGrB-G4S-GnRH(mGrBLG) fusion protein.This design increases the binding accuracy of mGrBLG to GnRHR+ cancer cells and decreases the side effect caused by unspecific binding and killing,but has no negative effect on its cytotoxic activity.GnRH and GrB are both human endogenesis proteins,so they have not the immunogenicity like heterogeneous proteins at all.G4S is a linker to link two functional domains of mGrBLG for the sake of maintaining their own structures and preventing possible immunoreaction.It also conduces to minimize the immunogenicity.We successively followed two different strategies expressing the chimeric mGrBLG molecules as soluble protein and secreted proteins respectively in the E.coli and Pichia pastoris yeast.At the same time,we also designed and expressed GrB,mGrB and GrBLG(GrB-G4S-GnRH) as the controls to compare their biological activities with mGrBLG.We got four different kinds of proteins purified from yeast culture supernatants by single-step Ni²⁺-affinity chromatography with about 90%purity.Various assays were carried out to analyze the biological functions of GrB,mGrB, GrBLG and mGrBLG.Colorimetric assays were performed using the GrB-specific peptide substrate Ac-IETD-pNA.Similar to unmodified standard GrB,the four proteins cleaved this substrate in a concentration-dependent manner with almost equivalent activity.It indicated that the binding site mutation and GnRH fusion did not obstruct enzymatic activity of mGrB,GrBLG and mGrBLG.Chloroquine accumulates in acidic compartments such as endosomes leading to osmotic rupture of the vesicles.We tested whether chloroquine could release internalized GrB series proteins from intracellular vesicles,thereby allowing access to cytosolic GrB substrates and induction of cells death.In contrast to treatment in the absence of the endosomolytic reagent,incubation of the MCF-7(human breast cancer cell line,GnRHR +) with mGrBLG in the presence ofchloroquine resulted in potent and dosage-dependent cytotoxicity(IC50 of 34.1ng/ml),the cell killing rate was 96.35%on the highest protein dose of 10μg / ml.To HEK293(human fetal kidney cell,GnRHR⁺)control cells, mGrBLG displayed a killing rate of 5.9%on the highest dosage of 10μg / ml,but lower than that of GrBLG which killing rate was 13.2%at 10μg / ml dosage.To GnRHR-negative CCC-ESF-1(human fetal skin fibroblast),mGrBLG almost remained unaffected status at the mGrBLG dosage applied,.the cell survival rate was higher than 99%on the highest mGrBLG dosage of 10μg / ml.The results of cytotoxic effect assay in vitro indicated that mGrBLG was not sensitive to human normal GnRHR^-cells,and it had some side effect to human normal GnRHR⁺ cells.However,the killing rate of mGrBLG to HEK293(GnRHR⁺) was reduced 55.3%compared with that of GrBLG on dosage of 10μg / ml.The mutations towards binding sites of GrB effectively increase the killing accuracy targeting GnRHR⁺ cancer cells and decrease side effect of mGrBLG.Moreover,the killing rate of mGrBLG to carcinoma MCF-7 cell(GnRHR⁺) was remarkably higher than that to normal HEK293 cell(GnRHR⁺),and this is depended on the GnRHR amount on cell surfaces.It can be believed that once mGrBLG is transferred into patient blood circulation in vivo,the biological behaviors of the protein should be different from that in vitro.The circulation in a flowing system,and the competitive binding with GnRHR between tumor cells carrying rich GnRHR and normal cells carrying poor GnRHR,will lead most mGrBLG to bind to tumor cells carrying rich GnRHR,so that to exert its lethal effect on such kind of tumor cells.The side effect on non-tumor cells and on normal cells should be reduced markedly.For the functional analysis of two mGrBLG domains and confirming that even under conditions of effective endosome release cell killing activity of mGrBLG was strictly dependent on specific recognition of the GnRHR,MCF-7 cells were incubated with mGrBLG and chloroquine in the presence of an excess of GrB-specific mAb,or GnRH protein encompassing the GnRH-binding eptitope.Addition of the competitors markedly reduced mGrBLG-induced cell killing rate.It was indicated that the mGrB is the key domain for the cytotoxic activity of mGrBLG,and GnRH guides mGrBLG to binding target cells.Moreover,G₄S short linker ensures right fold and relative independence of two domains,so as to maintain their own biological activities.In the synergy cytotoxic effects assay,mGrBLG and choloroquine resulted in selective and rapid tumor cell MCF-7 death,accompanied by clear signs of apoptosis such as chromatin condensation,membrane blebbing,formation of apoptotic bodies and activation of endogenous initiator and effector caspases.Importantly,even in the presence of the caspase inhibitor,MCF-7 cells were killed by mGrBLG.This ability of activating caspase-dependent and/or caspase-independent apoptosis and other direct cell death pathways is significant for confronting the general apoptosis-escape of tumor cells.In the presence of an endosomolytic eagent,mGrBLG displayed high and selective cytotoxicity towards tumor cells with GnRH target receptors in vitro,and its side effect and immunogenicity were minimized.The adenocarcinomas bearing overexpressed GnRH receptor involve pancreatic cancer,mammary cancer,ovary tumor,endometrioid carcinoma,prostate carcinoma,colon cancer,hepatocarcinoma,gastric cancer,lung cancer and pituitary tumor etc.,which are usually resistant to actinotheraphy and chemotherapeutics.And therefore it is especially worth to search clever drugs for certain adenocarcinoma,mGrBLG is a better candidate for targeting killing GnRHR⁺ tumor cells, and this novel research approach is worthy of taking more explorations at animal model level for pre-clinical pharmacological and toxicological trials and at patient level for clinical trials,so as to make final conclusion on the therapeutical efficiency and safety of this drug.