靶向抗肿瘤EGF-Linker-TCSKDEL融合蛋白的研制及其生物活性研究
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摘要
肿瘤是严重威胁人类生命健康的重大疾病之一,寻找有效的抗肿瘤药物与方法是当今世界医学界的重要研究课题。研究表明,许多中药具有抗肿瘤作用,但其作用成分复杂,具有多种作用成分和通过多种作用途径产生抗肿瘤作用。弄清中药的抗肿瘤作用途径和靶位点对加速抗肿瘤中药的开发具有重要意义。近50年来尽管肿瘤治疗取得了一些进展,化疗、放疗、手术仍是治疗基本手段。但是由于现有的化疗药物和放疗的选择性不高,杀伤肿瘤细胞的同时也损害体内正常细胞,治疗中常出现较明显的毒副反应,研究、开发一种对肿瘤细胞选择性高、杀伤作用强且对正常组织副作用小的抗肿瘤药物非常有意义。免疫毒素是提高治疗效果的一条重要途径,也是新一代的癌症治疗方法,此疗法与传统的化疗不同,其药物能在肿瘤部位有相对较高的浓度,存留较长时间,对肿瘤靶细胞有较强的杀伤活性,而对正常细胞无作用或毒性很小。免疫毒素是靶向性治疗肿瘤的代表性药物,也是当前免疫学研究的热点之一,被称为“生物导弹”。近年来,利用基因工程技术构建了许多不同用途的免疫毒素。免疫毒素是具有导向能力的分子(载体)和具有细胞毒性的分子(毒素)构成的具有特异性细胞杀伤能力的杂合分子。载体一般由抗体、细胞因子和激素等组成,毒素一般为来自于细菌或植物。载体可以把毒素定向带到病灶部位,将癌细胞或病变细胞杀死,减少了对正常细胞的杀伤。研究表明,免疫毒素在体内具有靶向性,使毒素更易于与肿瘤细胞结合,大大提高了毒素对肿瘤细胞的杀伤率,降低了其对正常细胞的毒副作用。
人表皮生长因子(human epidermal growth factor,EGF)是一种通过受体起作用刺激细胞增殖和分化的重要生长因子,很多肿瘤尤其是实体瘤细胞表面有异常高水平的EGF受体表达;天花粉蛋白(Trichosanthin,TCS)是一种自然存在的植物单链毒素,具有抑制肿瘤生长的作用。KDEL是一段高疏水的序列,它曾应用于PE毒素的C末端可以显著的提高PE杀伤肿瘤细胞的活性。本研究旨在利用EGF的靶向性和TCS蛋白的细胞毒性,将KDEL序列应用于TCS的C末端以提高TCS的活性,在分子水平设计合成一个融合蛋白,使其具有组成部分各自的生物学功能,即能够靶向性杀伤肿瘤细胞,有利于临床肿瘤的治疗。
首先使用PCR方法扩增EGF-Linker-TCSKDEL基因片段,双酶切后插入表达载体Pet28a中,表达了EGF-Linker-TCSKDEL融合蛋白,使用亲合层析纯化了EGF-Linker-TCSKDEL融合蛋白。接下来,我们研究评估了EGF-Linker-TCSKDEL的生物活性,并进一步探索了其抑制肿瘤细胞生长的作用机制。
1.首先,我们检测了EGF-Linker-TCSKDEL对各种肿瘤细胞和正常细胞的抗增殖作用。选择对数生长期的细胞,(4～5)×10~5/mL的悬液细胞用0.25%的胰蛋白酶消化后用含10%小牛血清的培养液配成单细胞溶液,细胞直接以4×10~4/ml接种于96孔板100μl/孔;细胞贴壁后,添加含不同浓度目的蛋白的同样培养基100μl/孔,对照组(0ng/ml融合蛋白)12个平行孔,融合蛋白每个剂量组设6个平行孔,继续培养48h。吸弃培养基,加含MTT 0.5mg/ml的PBS(PH6.8)100μl/孔,培养4h后,快速翻板法除去PBS,加二甲基亚砜100μl/孔,微量振荡仪振荡5min,用酶标仪570nm测定各孔吸光度值(OD值)。
细胞种板、融合蛋白的加入同MTT法。融合蛋白加入后48h,加BrdU标记溶液20μl/孔,继续培养18h。吸弃标记培养基,加固定溶液200μl/孔,室温孵育30min。快速翻板法除去固定溶液,并将培养板倒扣于滤纸上吸干残余固定液后,加抗BrdU-POD工作溶液室温孵育90min。吸弃抗体偶联溶液,用清沈溶液200μl/孔×3洗板后,加底物溶液100μl/孔,室温孵育10min,加1M H_2SO_425ul/孔阻止反应,微板读数仪450nm测吸光度值(A值)。细胞生长抑制率按下列公式计算:
生长抑制率=((正常对照组A值-用药组A值)/正常对照组A值)×100%
结果表明:EGF-Linker-TCSKDEL对肿瘤细胞和正常细胞都具有一定的生长抑制作用,但对肿瘤细胞的抑制作用要远远大于对正常细胞的生长抑制作用。MTT法检测其对人肺腺癌A549,人肝癌HepG2和人正常肝L02细胞的IC50分别为2.53μg/ml、5.56μg/ml和大于100μg/ml。
2.接着,我们应用流式细胞仪研究检测EGF-Linker-TCSKDEL是否可以诱导肿瘤细胞的凋亡。
培养细胞、分组。然后加入药物作用3小时后收集细胞。收集各组细胞于离心管中,离心后用冰冷的PBS洗细胞2次弃去上清液;将细胞沉淀重悬于70%乙醇(用PBS稀释)500μl的PBS中,固定细胞30min以上。离心后,弃上清。留下约50μl细胞悬液加10μl的RNase即RNA酶(10mg/ml)。37℃水浴30min。加PBS约300μl后加入碘化丙啶(终浓度20μg/ml)染色,室温下避光15min,200目滤网过滤后,研究结果表明,EGF-Linker-TCSKDEL(0.5,1,2,4μg/ml)作用肿瘤细胞3小时具有诱导A549细胞凋亡的作用,凋亡率分别为:6.9%、11.7%、22.1%,39.8%;融合蛋白能明显抑制细胞S期的合成,流式细胞仪检测将肿瘤细胞增殖阻滞在DNA合成的S期。
3.随后我们进行了整体动物抗肿瘤实验。EGF-Linker-TCSKDEL对小鼠的半数致死剂量(LD_(50))为:雄性734.52μg/kg,雌性733.31μg/kg。肿瘤接种:常规培养A549肺癌细胞,用生理盐水稀释为5×10~6个/ml,以0.2ml/只接种于小鼠右侧腋窝皮下。分组与给药:将接种肿瘤后的小鼠按体重分为5组,每组5只。①肿瘤对照组:尾静脉注射生理盐水;②融合蛋白高剂量组:尾静脉注射100μg/kg/d;③融合蛋白中剂量组:尾静脉注射50μg/kg/d;④融合蛋白低剂量组:尾静脉注射25μg/kg/d:⑤环磷酰胺组:腹腔注射环磷酰胺100mg/kg/d;各组均于肿瘤接种后次日开始给药,注射容积为0.1ml/10g,每日1次,连续14d,阳性对照药环磷酰胺隔日腹腔注射一次。全程给药结束后次日,处死动物,称取鼠重、瘤重,肿瘤组织随后用10%的甲醛溶液固定常规脱水、透明、浸蜡并制成石蜡切片,供免疫组化实验用。按下列公式计算抑瘤率。
抑瘤率=((正常对照组瘤重均值-用药组瘤重均值)/正常对照组瘤重均值×100%抑瘤率＞40%,P＜0.05时为抗肿瘤有意义。
实验结果表明,治疗组(100,50,25μg/kg)肿瘤抑制率分别为81.03%,80.85%,50.38%,有明显剂量依赖关系。同时通过病理切片和电镜观察并结合生化指标研究表明,融合蛋白对小鼠的肝脏、肾脏、脾脏和心脏损伤不大,未见到明显的毒性反应,能抑制肿瘤血管的生成。
4.通过流式细胞术、ELISA和Western blotting三种方法,我们研究了各细胞表面的EGF受体表达情况。
流式细胞术检测细胞EGF受体的表达
收集个组细胞于离心管中,离心(1000转5min)后用冰冷的PBS洗细胞2次,将细胞(5×10~5-1×10~6)沉淀重悬于50μl的PBS(1.5ml离心管)中,加入适当比例稀释的抗EGFR抗体(10μl/管),4℃孵育1h,期间间歇摇晃细胞;用含1%血清的PBS洗细胞三次(每次加满离心管);将细胞沉淀重悬于50μl的PBS中,加入适当比例稀释的FITC标记的二抗(2.5μl),4℃孵育40min左右;重复步骤3;将细胞沉淀重悬于250μl的PBS中,加入等体积250μl的4%的多聚甲醛,混匀,保存于4℃,待流式检测用(通常可以保存1周左右)。至少检测10000个细胞。
ELISA法检测EGFR表达
将细胞的培养上清和包被液(0.5mol/L NaHCO_3缓冲液,pH 9.6)以1:1的比例混匀,加入酶标板中,每孔100μl,4℃包被24h;倒掉包被液,用含5%小牛血清的PBS封闭过夜;加入EGF抗体,100μl/孔,37℃孵育60min;弃去一抗,用含0.05%TWEEN-20的PBS洗去未结合的抗体,3×5min;加入二抗,每孔100μl,37℃孵育45min,清洗同上;用OPD-H202系统显色,490nm波长测定光吸收值。
Western blotting检测EGFR的表达
Western blotting分析:在25cm~2培养瓶中,用含10%小牛血清的DMEM高糖培养基将细胞培养至约90%丰度,冷PBS洗细胞3次,加400μl裂解液(60mmTrisPH6.8,2%SDS,10%甘油,0.1M DTT)到细胞表面,冰上晃动5min。将裂解液于100℃煮沸10min,10000rcf离心5min,上清液中的总蛋白浓度以BSA为标准用Lowry法定量。蛋白通过SDS-15%聚丙烯酰胺电泳分离,4℃过夜电转移至硝酸纤维素膜上。用TBSⅠ(100mmol Tris(PH7.4),0.01%NaN_3,5%脱脂奶粉)室温封闭硝酸纤维素膜1h,除去封闭液。用以3%BSA替代脱脂奶粉、且加入兔抗EGFR单抗(1:500)的新制TBSⅠ室温孵育硝酸纤维素膜1h。用100mmolTris(PH7.4)洗膜3×10min,加含羊抗兔IgG-HRP(1:1000)的TBSⅡ(150mmolNaCl,50mmol Tris(PH7.5),5%脱脂奶粉)室温孵育硝酸纤维素膜1h。用不含脱脂奶粉的TBSⅡ洗膜3×10min,加入酶化学发光底物孵育膜5min,于ChemilmagerTM5500系统上曝光10min获取图像。
实验结果表明,A549细胞EGFR含量最高,其次是HepG2细胞,L02细胞含量最低,说明EGF-Linker-TCSKDEL抑制肿瘤细胞生长的作用与EGFR表达高低密切相关的。
5.EGF-Linker-TCSKDEL融合蛋白能抑制肿瘤血管的生成,我们选用了血管内皮ECV304细胞和人脐带静脉内皮HUVEC细胞以及经典的鸡胚尿囊膜进行了体外实验。
对体外血管内皮细胞生长的影响
HUVEC,用2%LSGS的Medium200培养基培养;ECV-304细胞,用10%胎牛血清的RPMI1640培养。取对数生长期细胞,0.25%的胰蛋白酶消化后,HUVEC用相应培养基以5×10~4/ml接种于96孔板100μl/孔,ECV-304用相应培养基以3×10~4/ml接种于96孔板100μl/孔。细胞于5%CO_2 37℃培养24h后,添加含不同浓度融合蛋白(0,1,3,10,20,40,80,150,300μg)的同样培养基100μl/孔[对照组(0μg融合蛋白组)12个平行孔,反应每个剂量组设6个平行孔,继续培养48h,按MTT法检测细胞活性。
对鸡胚尿囊膜新生血管形成的影响
鸡受精卵于37℃孵育3d,用注射器每蛋无菌抽取约2ml蛋清后石蜡封闭针孔,继续孵育4d。将融合蛋白预先以生理盐水稀释成含药0、50、100、200、400、800ng/片的膜(φ约3mm)。于蛋气囊顶端无菌开一约φ1cm的窗口,将反应停药膜放于尿囊膜上,无菌透明胶带封闭窗口,放回孵育箱再孵育48h。用解剖显微镜观察尿囊膜新生血管生成情况并拍摄照片,按文献标准评价新生血管形成。
实验结果表明,EGF-Linker-TCSKDEL对体外血管内皮细胞具有明显的生长抑制作用,有明显剂量依赖关系。融合蛋白对HUVEC和ECV-304细胞的平均IC50分别是22.54μg/ml、31.84μg/ml;同时通过解剖显微镜观察及对照文献标准评价,融合蛋白能抑制鸡胚尿囊膜新生血管的形成,抑制作用随剂量增加而增强。
6.最后,我们用蛋白印迹实验的方法初步探讨了EGF-Linker-TCSKDEL对导致细胞凋亡的信号通路的影响。如:ERK1/2、AKT通路。
尽管我们不能排除EGF-Linker-TCSKDEL诱导肿瘤细胞凋亡的其它机制,但是我们有清晰的证据证明EGF-Linker-TCSKDEL可以下调ERK1/2、AKT信号的磷酸化从而导致肿瘤细胞的凋亡。
总结以上研究,我们认为EGF-Linker-TCSKDEL具有明显的抗肿瘤作用,高效低毒,是一个非常值得进一步深入开发研究的靶向抗肿瘤药物。
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)×10~5/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 4×10~4/ml were directly inoculated in 96-well plates with 100ul/hole;the same 100μl culture
Medium 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 H_2SO_4 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 5×10~6/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.①Tumor control group:was injected 100ug/kg/d physiological saline via tail vein;②High-dose fusion protein group:was injected 100μg/kg/d fusion protein via tail vein;③Median-dose fusion protein group:was injected 50μg/kg/d fusion protein via tail vein;④Low-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 CYTOMETRY
Cells 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 METHOD
Culture supematant and coating solution(0.5mol/L NaHCO_3 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-H_2O_2 system and the optical absorption value was measured by 490nm wavelength.
DETECTION OF EGF RECEPTOR EXPRESSIONS USING WESTERN BLOTTING METHOD
Analysis 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 TBSⅠ(100mmol Tris(PH7.4),0.01%NaN_3,5%skim milk) in room temperature for 1h before the occluded solution was disposed of.Nitrocellulose membrane was then incubated by TBSⅠ(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 TBSⅡ(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 TBSⅡwithout 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 systems
We 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 membrane
IMPACT ON THE GROWTH OF VASCULAR ENDOTHELIAL CELLS IN VITRO MEDIUM
HUVEC 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 5×10~4/ml and ECV-304 was inoculated by corresponding medium in 96 plate with 100μl each hole in 3×10~4/ml.Cells were cultivated with 5%CO_2 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 MEMBRANE
Chicken 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.
人表皮生长因子(human epidermal growth factor,EGF)是一种通过受体起作用刺激细胞增殖和分化的重要生长因子,很多肿瘤尤其是实体瘤细胞表面有异常高水平的EGF受体表达;天花粉蛋白(Trichosanthin,TCS)是一种自然存在的植物单链毒素,具有抑制肿瘤生长的作用。KDEL是一段高疏水的序列,它曾应用于PE毒素的C末端可以显著的提高PE杀伤肿瘤细胞的活性。本研究旨在利用EGF的靶向性和TCS蛋白的细胞毒性,将KDEL序列应用于TCS的C末端以提高TCS的活性,在分子水平设计合成一个融合蛋白,使其具有组成部分各自的生物学功能,即能够靶向性杀伤肿瘤细胞,有利于临床肿瘤的治疗。
首先使用PCR方法扩增EGF-Linker-TCSKDEL基因片段,双酶切后插入表达载体Pet28a中,表达了EGF-Linker-TCSKDEL融合蛋白,使用亲合层析纯化了EGF-Linker-TCSKDEL融合蛋白。接下来,我们研究评估了EGF-Linker-TCSKDEL的生物活性,并进一步探索了其抑制肿瘤细胞生长的作用机制。
1.首先,我们检测了EGF-Linker-TCSKDEL对各种肿瘤细胞和正常细胞的抗增殖作用。选择对数生长期的细胞,(4～5)×10~5/mL的悬液细胞用0.25%的胰蛋白酶消化后用含10%小牛血清的培养液配成单细胞溶液,细胞直接以4×10~4/ml接种于96孔板100μl/孔;细胞贴壁后,添加含不同浓度目的蛋白的同样培养基100μl/孔,对照组(0ng/ml融合蛋白)12个平行孔,融合蛋白每个剂量组设6个平行孔,继续培养48h。吸弃培养基,加含MTT 0.5mg/ml的PBS(PH6.8)100μl/孔,培养4h后,快速翻板法除去PBS,加二甲基亚砜100μl/孔,微量振荡仪振荡5min,用酶标仪570nm测定各孔吸光度值(OD值)。
细胞种板、融合蛋白的加入同MTT法。融合蛋白加入后48h,加BrdU标记溶液20μl/孔,继续培养18h。吸弃标记培养基,加固定溶液200μl/孔,室温孵育30min。快速翻板法除去固定溶液,并将培养板倒扣于滤纸上吸干残余固定液后,加抗BrdU-POD工作溶液室温孵育90min。吸弃抗体偶联溶液,用清沈溶液200μl/孔×3洗板后,加底物溶液100μl/孔,室温孵育10min,加1M H_2SO_425ul/孔阻止反应,微板读数仪450nm测吸光度值(A值)。细胞生长抑制率按下列公式计算:
生长抑制率=((正常对照组A值-用药组A值)/正常对照组A值)×100%
结果表明:EGF-Linker-TCSKDEL对肿瘤细胞和正常细胞都具有一定的生长抑制作用,但对肿瘤细胞的抑制作用要远远大于对正常细胞的生长抑制作用。MTT法检测其对人肺腺癌A549,人肝癌HepG2和人正常肝L02细胞的IC50分别为2.53μg/ml、5.56μg/ml和大于100μg/ml。
2.接着,我们应用流式细胞仪研究检测EGF-Linker-TCSKDEL是否可以诱导肿瘤细胞的凋亡。
培养细胞、分组。然后加入药物作用3小时后收集细胞。收集各组细胞于离心管中,离心后用冰冷的PBS洗细胞2次弃去上清液;将细胞沉淀重悬于70%乙醇(用PBS稀释)500μl的PBS中,固定细胞30min以上。离心后,弃上清。留下约50μl细胞悬液加10μl的RNase即RNA酶(10mg/ml)。37℃水浴30min。加PBS约300μl后加入碘化丙啶(终浓度20μg/ml)染色,室温下避光15min,200目滤网过滤后,研究结果表明,EGF-Linker-TCSKDEL(0.5,1,2,4μg/ml)作用肿瘤细胞3小时具有诱导A549细胞凋亡的作用,凋亡率分别为:6.9%、11.7%、22.1%,39.8%;融合蛋白能明显抑制细胞S期的合成,流式细胞仪检测将肿瘤细胞增殖阻滞在DNA合成的S期。
3.随后我们进行了整体动物抗肿瘤实验。EGF-Linker-TCSKDEL对小鼠的半数致死剂量(LD_(50))为:雄性734.52μg/kg,雌性733.31μg/kg。肿瘤接种:常规培养A549肺癌细胞,用生理盐水稀释为5×10~6个/ml,以0.2ml/只接种于小鼠右侧腋窝皮下。分组与给药:将接种肿瘤后的小鼠按体重分为5组,每组5只。①肿瘤对照组:尾静脉注射生理盐水;②融合蛋白高剂量组:尾静脉注射100μg/kg/d;③融合蛋白中剂量组:尾静脉注射50μg/kg/d;④融合蛋白低剂量组:尾静脉注射25μg/kg/d:⑤环磷酰胺组:腹腔注射环磷酰胺100mg/kg/d;各组均于肿瘤接种后次日开始给药,注射容积为0.1ml/10g,每日1次,连续14d,阳性对照药环磷酰胺隔日腹腔注射一次。全程给药结束后次日,处死动物,称取鼠重、瘤重,肿瘤组织随后用10%的甲醛溶液固定常规脱水、透明、浸蜡并制成石蜡切片,供免疫组化实验用。按下列公式计算抑瘤率。
抑瘤率=((正常对照组瘤重均值-用药组瘤重均值)/正常对照组瘤重均值×100%抑瘤率＞40%,P＜0.05时为抗肿瘤有意义。
实验结果表明,治疗组(100,50,25μg/kg)肿瘤抑制率分别为81.03%,80.85%,50.38%,有明显剂量依赖关系。同时通过病理切片和电镜观察并结合生化指标研究表明,融合蛋白对小鼠的肝脏、肾脏、脾脏和心脏损伤不大,未见到明显的毒性反应,能抑制肿瘤血管的生成。
4.通过流式细胞术、ELISA和Western blotting三种方法,我们研究了各细胞表面的EGF受体表达情况。
流式细胞术检测细胞EGF受体的表达
收集个组细胞于离心管中,离心(1000转5min)后用冰冷的PBS洗细胞2次,将细胞(5×10~5-1×10~6)沉淀重悬于50μl的PBS(1.5ml离心管)中,加入适当比例稀释的抗EGFR抗体(10μl/管),4℃孵育1h,期间间歇摇晃细胞;用含1%血清的PBS洗细胞三次(每次加满离心管);将细胞沉淀重悬于50μl的PBS中,加入适当比例稀释的FITC标记的二抗(2.5μl),4℃孵育40min左右;重复步骤3;将细胞沉淀重悬于250μl的PBS中,加入等体积250μl的4%的多聚甲醛,混匀,保存于4℃,待流式检测用(通常可以保存1周左右)。至少检测10000个细胞。
ELISA法检测EGFR表达
将细胞的培养上清和包被液(0.5mol/L NaHCO_3缓冲液,pH 9.6)以1:1的比例混匀,加入酶标板中,每孔100μl,4℃包被24h;倒掉包被液,用含5%小牛血清的PBS封闭过夜;加入EGF抗体,100μl/孔,37℃孵育60min;弃去一抗,用含0.05%TWEEN-20的PBS洗去未结合的抗体,3×5min;加入二抗,每孔100μl,37℃孵育45min,清洗同上;用OPD-H202系统显色,490nm波长测定光吸收值。
Western blotting检测EGFR的表达
Western blotting分析:在25cm~2培养瓶中,用含10%小牛血清的DMEM高糖培养基将细胞培养至约90%丰度,冷PBS洗细胞3次,加400μl裂解液(60mmTrisPH6.8,2%SDS,10%甘油,0.1M DTT)到细胞表面,冰上晃动5min。将裂解液于100℃煮沸10min,10000rcf离心5min,上清液中的总蛋白浓度以BSA为标准用Lowry法定量。蛋白通过SDS-15%聚丙烯酰胺电泳分离,4℃过夜电转移至硝酸纤维素膜上。用TBSⅠ(100mmol Tris(PH7.4),0.01%NaN_3,5%脱脂奶粉)室温封闭硝酸纤维素膜1h,除去封闭液。用以3%BSA替代脱脂奶粉、且加入兔抗EGFR单抗(1:500)的新制TBSⅠ室温孵育硝酸纤维素膜1h。用100mmolTris(PH7.4)洗膜3×10min,加含羊抗兔IgG-HRP(1:1000)的TBSⅡ(150mmolNaCl,50mmol Tris(PH7.5),5%脱脂奶粉)室温孵育硝酸纤维素膜1h。用不含脱脂奶粉的TBSⅡ洗膜3×10min,加入酶化学发光底物孵育膜5min,于ChemilmagerTM5500系统上曝光10min获取图像。
实验结果表明,A549细胞EGFR含量最高,其次是HepG2细胞,L02细胞含量最低,说明EGF-Linker-TCSKDEL抑制肿瘤细胞生长的作用与EGFR表达高低密切相关的。
5.EGF-Linker-TCSKDEL融合蛋白能抑制肿瘤血管的生成,我们选用了血管内皮ECV304细胞和人脐带静脉内皮HUVEC细胞以及经典的鸡胚尿囊膜进行了体外实验。
对体外血管内皮细胞生长的影响
HUVEC,用2%LSGS的Medium200培养基培养;ECV-304细胞,用10%胎牛血清的RPMI1640培养。取对数生长期细胞,0.25%的胰蛋白酶消化后,HUVEC用相应培养基以5×10~4/ml接种于96孔板100μl/孔,ECV-304用相应培养基以3×10~4/ml接种于96孔板100μl/孔。细胞于5%CO_2 37℃培养24h后,添加含不同浓度融合蛋白(0,1,3,10,20,40,80,150,300μg)的同样培养基100μl/孔[对照组(0μg融合蛋白组)12个平行孔,反应每个剂量组设6个平行孔,继续培养48h,按MTT法检测细胞活性。
对鸡胚尿囊膜新生血管形成的影响
鸡受精卵于37℃孵育3d,用注射器每蛋无菌抽取约2ml蛋清后石蜡封闭针孔,继续孵育4d。将融合蛋白预先以生理盐水稀释成含药0、50、100、200、400、800ng/片的膜(φ约3mm)。于蛋气囊顶端无菌开一约φ1cm的窗口,将反应停药膜放于尿囊膜上,无菌透明胶带封闭窗口,放回孵育箱再孵育48h。用解剖显微镜观察尿囊膜新生血管生成情况并拍摄照片,按文献标准评价新生血管形成。
实验结果表明,EGF-Linker-TCSKDEL对体外血管内皮细胞具有明显的生长抑制作用,有明显剂量依赖关系。融合蛋白对HUVEC和ECV-304细胞的平均IC50分别是22.54μg/ml、31.84μg/ml;同时通过解剖显微镜观察及对照文献标准评价,融合蛋白能抑制鸡胚尿囊膜新生血管的形成,抑制作用随剂量增加而增强。
6.最后,我们用蛋白印迹实验的方法初步探讨了EGF-Linker-TCSKDEL对导致细胞凋亡的信号通路的影响。如:ERK1/2、AKT通路。
尽管我们不能排除EGF-Linker-TCSKDEL诱导肿瘤细胞凋亡的其它机制,但是我们有清晰的证据证明EGF-Linker-TCSKDEL可以下调ERK1/2、AKT信号的磷酸化从而导致肿瘤细胞的凋亡。
总结以上研究,我们认为EGF-Linker-TCSKDEL具有明显的抗肿瘤作用,高效低毒,是一个非常值得进一步深入开发研究的靶向抗肿瘤药物。
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)×10~5/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 4×10~4/ml were directly inoculated in 96-well plates with 100ul/hole;the same 100μl culture
Medium 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 H_2SO_4 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 5×10~6/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.①Tumor control group:was injected 100ug/kg/d physiological saline via tail vein;②High-dose fusion protein group:was injected 100μg/kg/d fusion protein via tail vein;③Median-dose fusion protein group:was injected 50μg/kg/d fusion protein via tail vein;④Low-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 CYTOMETRY
Cells 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 METHOD
Culture supematant and coating solution(0.5mol/L NaHCO_3 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-H_2O_2 system and the optical absorption value was measured by 490nm wavelength.
DETECTION OF EGF RECEPTOR EXPRESSIONS USING WESTERN BLOTTING METHOD
Analysis 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 TBSⅠ(100mmol Tris(PH7.4),0.01%NaN_3,5%skim milk) in room temperature for 1h before the occluded solution was disposed of.Nitrocellulose membrane was then incubated by TBSⅠ(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 TBSⅡ(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 TBSⅡwithout 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 systems
We 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 membrane
IMPACT ON THE GROWTH OF VASCULAR ENDOTHELIAL CELLS IN VITRO MEDIUM
HUVEC 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 5×10~4/ml and ECV-304 was inoculated by corresponding medium in 96 plate with 100μl each hole in 3×10~4/ml.Cells were cultivated with 5%CO_2 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 MEMBRANE
Chicken 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.
引文
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[2].Tadeusz Lewin'ski , Maciej Z ul awski.Small cell lung cancer survival: 3 years as a minimum for predicting a favorable outcome.[J].Lung Cancer, 2003, 40: 203-213
[3]Strome SE, Sausville EA, Mann D.A mechanistic perspective of monoclonal antibodies in cancer therapy beyond target-related effects[J] .Oncologist. 2007 Sep; 12(9): 1084-95
[4].Yang ZR, Wang HF, Zhao J, _et al .Recent developments in the use of adenoviruses and immunotoxins in cancer gene therapy [J]. Cancer Gene Therapy. 2007 Jul;14(7):599-615
[5]. Pastan I, Hassan R, FitzGerald DJ et al .Immunotoxin treatment of cancer[J].Annu Rev Med. 2007;58:221-37
[6]. Kreitman RJ. Immunotoxins for targeted cancer therapy[J].AAPS 2006 Aug 18;8(3):E532-551.
[7]. Chiron MF. Recombinant immunotoxins and chimeric toxins for targetedtherapy in oncology[J] .Bull Cancer , 1997 , 84 (12) :1135-1140.
[8]. Mansfield E , Amlot P , Pastan L et al. Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD222bearing cells and tumors[J]. Blood , 1997 , 90(5) :2020-2026.
[9].Chen SY.Novel genetic immunotoxins and intra-cellular antibodies for cancer therapy[ J ] . Seminars in Oncology, 1996, 23:48
[10].Panchal RG.Novel therapeutic strategies to selectively kill cancer cells [ J ] .Biochem Pharmacol, 1998, 55(3):247
[11].Kreitman RJ, Wilson WH, Robbins D et al. Responses in refractory hairg cell leukemia to a recombinant immunotoxin[J] .Blood, 1999, 94(10):3340
[12].Khayat D, Antoine EC Coeffic D.Taxol in the management of cancers of the breast and the ovary[J] .Cancer Invest, 2000, 32:1401
[13].Keppler H A , Brinkmann U , Pastan I. Role of Caspases inimmunotox-in- induced apoptosis of cancer cells[J] .Biochemistry , 1998 , 37:16934-42.
[14]. Bozzetti C, Tiseo M, Lagrasta C, Nizzoli R et al .Comparison between epidermal growth factor receptor (EGFR) gene expression in primary non-small cell lung cancer (NSCLC) and in fine-needle aspirates from distant metastatic sites[J] . Thorac Oncol. 2008, 3(1): 18-22.
[15]. Papageorgio C, Perry MC.Epidermal growth factor receptor-targeted therapy for pancreatic cancer. Cancer Invest[J].2007, 25(7):647-57
[16].Tzeng CW, Frolov A, Frolova N et al . EGFR genomic gain and aberrant pathway signaling in pancreatic cancer patients[J].Surg Res. 2007 , 143(1):20-26
[17].Sok JC, Coppelli FM, Thomas SM_et al . Mutant epidermal growth factor receptor (EGFRvIII) contributes to head and neck cancer growth and resistance to EGFR targeting[J]. Clin Cancer Res. 2006, 12(17):5064-73
[18].Coley HM, Shotton CF, Ajose-Adeogun A et al. Receptor tyrosine kinase (RTK) inhibition is effective in chemosensitising EGFR-expressing drug resistant human ovarian cancer cell lines when used in combination with cytotoxic agents[J].Biochem Pharmacol. 2006, 72(8):941-948
[19]. Foss FM. DAB3892IL22 (ONTAK) :a novel fusion toxin therapy for lym2 phoma[J]. Clin Lymphoma , 2000 , 1 (2) :110-116.
[20]. Lo Russo PM, Lomen PL, et al .Phase I study of monoclonal antibody-ricin A Chain immunoconjugate Xomazyme-791 in patients with matastatic colon cancer [J]. Am J Clin Oncol , 1995, 18(4):307-312.
[21].Kuan C, Pastan I. Recombinant immunotoxin Containing a Disulfide Stabilized Fv Directed at erbB2 that does not require proteolytic activity[J].Biochemisty,1996,36:2872-2877.
[22]Huang S,Trujillo JM,Chakraharty S.Proliferation of human colon cancer cells:role of epidemal growth factor and transforming growth factor alpha.Int[J].Cancer,1992,52:978
[23]时伟红,宋今丹.含天花粉蛋白的抗人大肠癌的免疫毒素制备及其细胞毒性研究[J].中国医科大学学报,1996,25(2):111.
[24]鲁继斌,李亚明.抗人肺腺癌单克隆抗体-天花粉蛋白免疫毒素对荷人肺腺癌裸鼠模型的导向治疗[J].中国医科大学学报,1995,24(3):243.
[25]Staehler M,Rohrmann K,Haseke N et al.Targeted agents for the treatment of advanced renal cell carcinoma[J].Cancer.2005 Dec 1;104(11):2323-33
[26]Srikala S.Sridhar,Frances A.Shepherd.Targeting angiogenesis:a review of angiogenesis inhibitors in the treatment of lung cancer[J].Lung Cancer,2003,42:S81-S91.
[27]李恒,熊旭林.人肾癌组织EGFR-mRNA原位杂交分析及其意义.中华泌尿外科杂志,2000,21(7):409-410.
[28]Sakaeda T,Okamura N,Gotoh A et al.EGFR mRNAis upregulated,but somatic mutations of the gene are hardly found in renal cell carcinoma in Japanese patients[J].Pharm Res.2005 Oct;22(10):1757-61
[29]Wels W,Biburger M,Muller T,et al.Recombinant immunotoxins and retargeted killer cells:employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors[J].Cancer Immunol Immunother.2004,53(3):217-26
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[31]Ute Wagner,Eberhard Burkhardt,Klaus Failing.Evaluation of canine lymphocyte proliferation:comparison of three different colorimetric methods with the 3H-thymidine incorporation assay.Veterinary Immunology and Immunopathology,1999,70:151-159
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[33]Young S.Lew,Andrew Kolozsvary,Stephen L.Brown,Jae Ho Kim.Stnergistic interaction with arsenic trioxide and ffactionated radiation in locally advanced murine tumor.Cancer Research 2002 62(15):4202-4205
[34]细胞生物学实验技术 章静波 黄东阳主编 化学工业出版社98页
[35]细胞培养 司徒镇强 吴军正 世界图书出版社9-12页
[36]Kreitman RJ.Recombinant immunotoxins for the treatment of haematological malignancies[J].Expert Opin Biol Ther,2004,4(7):1115-1128.
[37]Rosinol L,Cibeira MT,Segarra M et al.Response to thalidomide in multiple myeloma:impact of angiogenic factors.Cytokine,2004,26:145-148.
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