Experimental Study Of Synthesis And Bioactivity Analysis Of EGF-Linker-TCSKDEL Fusion Proteins With Targeted Anti-tumor Effect

Tumor is such a kind of diseases that seriously threaten human health.To find effective anti-tumor drugs and methods is an important research project in world medicine field.Research indicates that many Chinese traditional medicines have anti-tumor effects,but the components of them are complicated and their anti-tumor effects are caused by many components and many action pathways.So fully understanding the anti-tumor mechanism and tumor targeted site is important to accelerate the development of anti-tumor Chinese traditional medicines.Although some progress has been achieved since recent 50 years,chemotherapy,radiotherapy and surgical operation are still the main method for tumor therapy.There are obvious reactions of toxicity in treatment because the selectivity of existing chemotherapy and radiotherapy drugs is low and these drugs injure normal cells when they kill tumor cells.As a new generation of tumor therapy method,immunotoxin therapy is different with traditional chemotherapy.There is relatively high concentration of immunotoxin therapy drugs in tumor location and the retention time of these drugs is longer than that of chemotherapy drugs.Because the killing activity of immunotoxin drugs on tumor cells is strong and the toxicity of these drugs on normal cells is none or little,immunotoxin therapy is an important way to improve curing effect on tumor. Called "biomissile",immunotoxins are representative drugs in tumor-targeted therapy, and are one of hotspots in immunology research.Diverse uses of immunotoxins are constructed these years.Immunotoxins are special cell-killed molecular,composed of molecular of target and molecular of toxicity.The molecular of target is composed of antibody,cytokines and hormones,and toxins are derived from bacteria or plants.The molecular of target can take toxins to tumor focus and toxins can kill tumor cells but few normal cells.Research indicates that the target character of immunotoxins makes toxins easier to bind tumor cells,and to improve rate of killing tumor cells by toxins, and to lower toxicity effects on normal cells.Human epidermal growth factor(EGF) is an important growth factor that activates the reproduction and differentiation of cells by receptors.EGF receptors are abnormally over-expressed on surfaces of many tumor cells especially for solid tumor cells;Trichosanthin(TCS) is a kind of natural plant single-chain toxin,and can inhibit tumor cells' growth.KDEL is a predominantly hydrophobic Sequence.PE toxin with a C-terminal KDEL can increase the activity of Killing Tumor cells.In this research,a fusion protein with biological functions(target killing tumor cells) was designed and synthesized in molecular level by use of the target character of EGF and the toxicity of TCS,favorable to tumor therapy.At first EGF-Linker-TCSKDEL gene fragment was amplified by PCR,and was digested by two enzymes and inserted into the expression vector Pet28a.The fusion protein EGF-Linker-TCSKDEL was expressed and purified by affinity chromatography.Then we estimated the biological activity of EGF-Linker-TCSKDEL and further explored its inhibition effect on tumor cell growth.Firstly,we detected the anti-proliferation effect of EGF-Linker-TCSKDEL on various tumor cells and normal cells.(4～5)×10⁵/ml logarithmic growth phase cell suspension was digested by 0.25%trypsin and then single-cell solution was prepared by the culture medium containing 10%fetal calf serum.Cells of 4×10⁴/ml were directly inoculated in 96-well plates with 100ul/hole;the same 100μl cultureMedium with protein of various concentration was appended into each hole after cells adhension.Cells were cultivated for 48hr after 12 parallel holes were set for control group and 6 parallel holes were set for fusion protein dose group.100μl PBS(pH6.8) containing 0.5mg/ml MTT was added into each hole after the medium was disposed of.PBS was disposed of by fast upturn method after 4hr cells cultivation and 100uL Dimethyl sulfoxide(DMSO) was added.The plate was vibrated 5min using micro-oscillation equipment.We then detected absorbance values (OD values) using 570nm enzyme detection equipment.The method to culture cell plate and to add fusion protein was the same as MTT method.Cells were cultivated for 48hr after the fusion protein was added,and then 20μl BrdU labeling solution was added into each hole and cultivated for continuous 18hr.After the medium was disposed of 200μl stationary liquid was added into each hole and was incubated for 30min in room temperature.The stationary liquid was disposed of by fast upturn method and residual liquid was soaked up by converting cell plate on the filter paper.Anti-BrdU-POD working solution was added and was incubated for 90min in room temperature.After the antibody conjugate solution was disposed of,we cleaned plate using 200μl cleaning solution 3 times and added 100μl substrate solution into each hole.Then we incubated it for 10min in room temperature and added 25ul 1M H₂SO₄ into each hole to stop the reaction.We detected absorbance values(A values) using 450nm micro-plate reading equipment.We calculated cell growth inhibition rate using following formula:Growth inhibition rate=(A value in normal control group-A value in drug group)/A value in normal control group)×100%We found the growth inhibition effect of EGF-Linker-TCSKDEL on both tumor cells and normal cells,but the inhibition effect on tumor cells is much stronger than that on normal cells.The IC50 valuses are 2.53μg/ml,5.56μg/ml and＞100μg/ml for human A549 lung adenocarcinoma,human hepatoma HepG2 and LO-2 normal liver cells respectively detected by MTT method.2.Secondly,we tried to find whether EGF-Linker-TCSKDEL could induce the apoptosis of tumor cells using flow cytometry equipment.After cultivating and grouping cells,we added drugs and collected cells after 3h cultivation.The cells were collected in centrifuge tube and centrifuged.The cell precipitation was washed by cold PBS twice and the supernatant was disposed of.To fossilize for more than 30min,we put cells in 500μl 70%ethanol solution(diluted by PBS).50μl cell suspension could be obtained after centrifuging and 10μl RNase (RNA enzyme,10mg/ml) was added.After 30min in 37℃,we added Iodization aziridine(final concentration 20μg/ml) for dyeing.We filtrated the resulting solution using 200 mesh strainer after storing it in darkness for 15 min in room temperature.It was found that EGF-Linker-TCSKDEL(0.5,1,2,4μg/ml) could induce the apoptosis of A549 cells in 3h,and the rate of apoptosis was 6.9%,11.7%,22.1%, 39.8%,respectively.It was found that EGF-Linker-TCSKDEL could obviously inhibit synthesis in cell S phase,and block tumor cells in the S phase detected by flow cytometry.3.Then,we performed anti-tumor experiment using animals.The LD50(Lethal Dose,50%) for EGF-Linker-TCSKDEL on male mice was 734.52μg/kg and on female mice was 733.31μg/kg,respectively.Tumor inoculation:A549 lung cancer cells were conventionally cultured and diluted to 5×10⁶/ml with physiological saline and were inoculated to mice right axillary subcutaneously with the dose of 0.2ml/mouse.Grouping and administration:we divided the inoculated mice into five groups based on their weights with five mice for each group.â‘ Tumor control group:was injected 100ug/kg/d physiological saline via tail vein;â‘¡High-dose fusion protein group:was injected 100μg/kg/d fusion protein via tail vein;â‘¢Median-dose fusion protein group:was injected 50μg/kg/d fusion protein via tail vein;â‘£Low-dose fusion protein group:was injected 25μg/kg/d fusion protein via tail vein;⑤Cyclophosphamide group:was injected intraperitoneally 100mg/kg/d cyclophosphamide.Administration of each group began in the day after tumor inoculation,and the administration volume was 0.1ml,once a day,continuously for 14 days,except that the cyclophosphamide group(the positive drug group) was injected intraperitoneally every other day.All mice were killed the day after the end administration.The weights of mice and tumors were all measured.Tumor tissues were conventionally dehydrated using 10%formaldehyde solution,clarified and waxed.Paraffin sections were then made for Immunohistochemistry.We calculated the growth inhibition rate using following formula:growth inhibition rate=(A value in normal control group-Avalue in drug group)/A value in normal control group)×100%It was found that the inhibition rate of tumor cells for therapy group(100,50, 25μg/kg) was 81.03%,80.85%and 50.38%,respectively.There was evident dose-dependent relationship.It was also showed that fusion protein could inhibit the formation of tumor blood vessels and had no large damage to the Liver,kidney, spleen and heart of mouse with no evident toxicity reactions by pathologic section and electron microscope combined with biochemical indicators.4.The EGF receptor expression on cell surfaces was studied by following three methods:flow cytometry detection,ELISA and western blotting.EGF RECEPTOR EXPRESSION ON CELL SURFACES DETECTED BY FLOW CYTOMETRYCells were collected in centrifugal tubes,and were washed twice using cold PBS after centrifuging(1000 rpm for 5min).Precipitation cells(5 105 -- 1 106 ) were then suspended in 50μl PBS(1.5ml centrifuge tube),and were added anti-EGFR antibody(10ul each tube) diluted in appropriate proportion.The cells solution was then incubated for 1h in 4℃temperature with intermittent shaking;the cells were washed thrice using PBS with 1%serum(fill centrifuge tube every time); Precipitation cells were suspended in 50μl PBS with FITC-tagged anti-EGFR antibody(2.5μl).The solution was then incubated for 40min in 4℃temperature;We then repeated step 3;Precipitation cells were suspended in 50μl PBS with 250μl 4% Multi-POM,and were blended and saved in 4℃temperature(can be saved for 1 week) for flow cytometry detection.AT least 10000 cells could be detected.The results showed that there was the highest EGFR expression rate in A549 cells,and the lowest EGFR expression rate in LO2 cells,which indicated that the inhibition effects of tumor cells growth for EGF-Linker-TCSKDEL were closely related to the levels of EGF receptor expression in tumor cells.EGF RECEPTOR EXPRESSION ON CELL SURFACES DETECTED BY ELISA METHODCulture supematant and coating solution(0.5mol/L NaHCO₃ buffer solution,pH 9.6) were mixed in the ratio of 1:1 and were added into ELISA plate(100μL each hole) for coating in 4℃for 24hr;The coating solution was disposed of,and the ELISA plate was closed overnight by PBS containing 5%calf serum;HGF antibody was then added(100μL each hole) and was incubated in 37℃for 60min;the first antibody was disposed of and unbound antibody was washed off by PBS with 0.05%TWEEN-20,3×5min;the second antibody was added 100ul each hole and Incubated for 45min and then was washed using above method;The ELISA plate was colored by OPD-H₂O₂ system and the optical absorption value was measured by 490nm wavelength.DETECTION OF EGF RECEPTOR EXPRESSIONS USING WESTERN BLOTTING METHODAnalysis using Western blotting:Cells were cultivated to 90%abundance using 10%Calf serum DMEM high glucose medium and washed by cold PBS thrice before 400μl lysis solution(60mmTris PH6.8,2%SDS,10%glycerol,0.1M DTT) was added to cell surfaces and shaken for 5rain on ice.Lysis solution was boiled to 100^C for 10min before centrifugated by 10000rcf.Total protein concentration in the supernatant was measured by Lowry method with BSA as the standard.Protein was separated through 15%SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane overnight in 4℃.Nitrocellulose membrane was then occluded by TBSâ… (100mmol Tris(PH7.4),0.01%NaN₃,5%skim milk) in room temperature for 1h before the occluded solution was disposed of.Nitrocellulose membrane was then incubated by TBSâ… (newly prepared,with 1:500 rabbit anti-EGFR monoclonal antibody and with 3%BSA replaced of skim milk) in room temperature for 1h.The membrane was then washed by 100mmol Tris(PH7.4) for 10min,3 times and TBSâ…¡(150mmol NaCl,50mmol Tris(PH7.5),5%skim milk) containing goat-anti-rabbit IgG-HRP(1:1000) was added and incubated for 1hr in room temperature.Then the membrane was washed by TBSâ…¡without skim milk for 3×10min before enzyme chemiluminescent substrate had been added for 5min, and the images could be obtained through 10min exposure on chemilmagerTM 5500 systemsWe studied the EGF receptor expression on cell surface by the three methods: flow cytometry detection,ELISA and Western blotting.The results showed that there was the highest EGFR expression rate in A549 cells,and the second highest EGFR expression rate in HepG2 cells,and the lowest EGFR expression rate in LO2 cells,which indicated that the growth inhibition effect on tumor cell for EGF-Linker-TCSKDEL were closely related to the levels of EGF receptor expression.5.To study the inhibition effect of EGF-Linker-TCSKDEL fusion protein on tumor angiogenesis,We performed in vitro experiment using endothelial cells ECV304,human umbilical vein endothelial cells(HUVEC) and chicken chorioallantoic membraneIMPACT ON THE GROWTH OF VASCULAR ENDOTHELIAL CELLS IN VITRO MEDIUMHUVEC was cultivated using 2%LSGS Medium200;ECV-304 cells were cultivated 10%FBS(Fetal Bovine Serum) RPMI1640.Cells on the logarithmic growth phase were digested by 0.25%trypsin.HUVEC were inoculated by corresponding medium in 96 plate with 100μl each hole in 5×10⁴/ml and ECV-304 was inoculated by corresponding medium in 96 plate with 100μl each hole in 3×10⁴/ml.Cells were cultivated with 5%CO₂ for 24hr,and then the same medium with different fusion protein concentrations(0,1,3,10,20,40,80,150,300μg) were added in 100μl each hole[control group(0μg fusion protein),12 parallel holes,test group,6parallel holes]for 48h,and cell activity was detected by MTT method.IMPACT ON THE angiogenesis OF CHICKEN CHORIOALLANTOIC MEMBRANEChicken eggs were hatched in 37℃for 3d and pinholes were sealed by paraffin after 2ml egg white was extracted aseptically from every egg by syringe. The eggs were hatched continuously for 4d.Fusion protein was pre-diluted by physiological saline to membranes(about 3mm width each piece,containing 0,50,100,200,400,800ng drug).We then aseptically opened a window(aboutφ1cm) on the top balloon of egg and put reactive-stop membrane on allantocherion and sealed the window with aseptic transparent tape.The eggs were then sent back to incubation box to be incubated for 48h.We then observed the chorioallantoic membrane angiogenesis and took photographs and evaluated angiogenesis based on relevant literature.5.Finally,we explored the effects of EGF-Linker-TCSKDEL on signaling pathway that Induces apoptosis using protein blot method,such as ERK1/2,AKT pathway.Although we could not preclude other mechanisms that might Induce cell apoptosis,we obtained clear evidence for proving that EGF-Linker-TCSKDEL can induce tumor cell apoptosis by lowering the phosphorylation of ERK1/2 and AKT signals.In summary,we believe that EGF-Linker-TCSKDEL has evident anti-tumor effect with high efficiency and low toxicity,and is well worthy of further studying and developing as a target anti-tumor drug.