靶向性抗肿瘤药物EGF-E4orf4的研究
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摘要
肿瘤是一类严重威胁到人类健康与生命的疾病。近年来尽管抗肿瘤药物已经有了很大的发展,但仍存在着针对性差,杀伤正常细胞的毒副作用。因此研制能够靶向性杀伤肿瘤细胞的基因工程药物有重大的理论和实践意义。
本组前期工作中利用EGF与EGFR结合的靶向性及腺病毒E4orf4蛋白的细胞毒性,通过基因重组及酵母表达技术,已设计并表达了一种新的融合蛋白EGF-E4orf4。前期工作表明,该融合蛋白对人乳腺癌细胞MDA-MB-231,人脑神经胶质瘤细胞BT325和人骨肉瘤细胞Saos-2都有抑制作用,但尚存在重组蛋白EGF-E4orf4表达量偏低,纯化效率不高等问题。本论文在此基础上,对融合蛋白EGF-E4orf4的表达,纯化和功能三个方面进行了进一步的探索。
本论文的第一部分工作是提高融合蛋白的表达量和纯度。利用5L发酵罐进行高密度发酵表达,与摇瓶相比较,提高了融合蛋白的表达量。优化纯化流程,通过超滤结合离子交换和凝胶过滤两步柱层析纯化,提高了目的蛋白的纯度。经鉴定,重组融合蛋白EGF-E4orf4是以蛋白聚合体形式存在,为进一步提高纯化效率带来了困难。为此,对该蛋白进行了改造:(1)在融合蛋白的羧基端添加(His)_6标签,以便于提高纯化效率。(2)以可以与EGFR结合的环七肽替代融合蛋白中的EGF,构建环七肽-E4orf4-His融合蛋白,减少二硫键的数目,并且可以排除E4orf4蛋白的细胞毒性作用受到EGF的促生长作用干扰的可能性。通过亲和层析和凝胶过滤层析,对上述两种蛋白进行纯化,得到纯度较高的目的蛋白。
本论文的第二部分工作是进一步研究融合蛋白EGF-E4orf4的生物学功能。主要以人乳腺癌细胞MDA-MB-231和人胃癌细胞BGC823为实验对象。通过Westernblotting检测EGFR磷酸化程度,发现融合蛋白EGF-E4orf4可以诱导EGFR发生磷酸化,并且呈时间和剂量依赖性。通过激光共聚焦显微镜观察FITC标记的融合蛋白和EGF孵育不同时间后的细胞,发现:FITC-EGF进入细胞后信号逐渐减弱,并且与溶酶体共定位;而FITC-EGF-E4orf4则随着孵育时间延长,信号在细胞内累积增强,并且在24 h不与溶酶体共定位。融合蛋白EGF-E4orf4的抗肿瘤作用则表现在抑制BGC823细胞的克隆形成能力以及抑制MDA-MB-231和BGC823细胞的裸鼠移植肿瘤的生长,最大抑瘤率分别为79%和49%(P＜0.05)。在急毒实验中未发现EGF-E4orf4蛋白对裸鼠有任何毒副作用。
最后一部分工作是构建了四环素可诱导表达E4orf4蛋白的真核细胞稳定表达株,并通过MTT实验证明诱导表达E4orf4蛋白可以抑制细胞生长。以未诱导细胞为对照,对四环素诱导E4orf4蛋白表达6 h和60 h的细胞进行全基因组芯片扫描,作了初步分析,为今后研究E4orf4蛋白在细胞内的作用机制奠定了基础。
综合上述研究结果表明,融合蛋白EGF-E4orf4确实可以通过EGFR这个分子靶点的介导,进入肿瘤细胞,体内实验证明,融合蛋白EGF-E4orf4可以特异地抑制肿瘤细胞增殖,并具有较为可靠的安全性。为了将融合蛋白EGF-E4orf4真正应用于临床的肿瘤治疗中,还有更多的对于制备工艺、细胞水平以及动物实验的艰巨工作有待完成。
Cancer is a kind of serious disease that jeopardizes the health and life of human beings.Although anti-tumor pharmaceuticals have been developed in recent years,their use is limited because of their low efficacy and adverse effect of toxicity.Therefore,the development of cytotoxic drugs that can specifically target tumors with minimal toxicity to normal cells continues to be a challenge in cancer therapy.
In our laboratory,we have constructed and expressed a new recombinant protein, EGF-E4orf4,based on the targeting of EGF and cytotoxicity of adenovirus E4orf4.In the previous experiments,it was indicated that this fusion protein inhibited human breast adenocarcinoma cell line MDA-MB-231,human Glioblastoma BT325 and human osteosarcoma cell line Saos-2 growth.There are some problems waiting for overcome such as how to increase the fusion protein's expression level in Pichia pastoris yeast and low-purity with existing purification procedure.The present study focused on the expression,purification and function of the fusion protein,EGF-E4orf4.
Firstly,the experiments were carried out on increasing the expression level of in Pp yeast and the purity of EGF-E4orf4.Improvement of EGF-E4orf4 expression level was done in bioreactor by fed-batch fermentation.The expression level of EGF-E4orf4 was increased compared with the shaking incubator.The fusion protein was purified by anion-exchange chromatography and gel exclusion chromatography.As EGF-E4orf4 was identified as polymer,we used two methods to reconstruct the fusion proteins:(1) adding 6xHis tag to the C terminal of EGF-E4orf4 to facilitate the purification process.(2) constructing cycling 7 peptides-E4orf4-His protein to reduce the number of disulfide bonds. The two fusion proteins were purified by His-tag affinity chromatography and gel exclusion chromatography.
Secondly,important part was to investigate the possible biological functions of the fusion protein EGF-E4orf4 using MDA-MB-231 and BGC823 cells that were characteristics for EGFR expression.The fusion protein stimulated EGFR phosphorylation in a dose- and time-dependent manner by western blotting analysis.Likewise,the results obtained from confocal microscopy confirmed the internalization and showed the differences between EGF-E4orf4 and EGF afterwards,EGF-E4orf4 significantly inhibited the ability of colony formation of BGC823.In vivo,EGF-E4orf4 suppressed tumor growth in a dose-dependent fashion with an inhibition ratio of 79%in MDA-MB-231 and 49%in BGC 823,respectively(P＜0.05).No adverse effects were observed in the nude mice even at the dose of 10mg/kg of EGF-E4orf4.
Final part was to create stable cell lines expressing E4orf4 and by MTT colorimetric assays,it was found that inducible expressing E4orf4 could inhibit cell growth. The first batch of microarray was carried out to identify changes in RNA levels resulting from E4orf4 expressing and the further independent experiments are still needed.
Our experiment results demonstrate that EGF-E4orf4 could be successfully introduced into the tumor cells by interaction between EGF and EGFR,and the antitumor efficacy of EGF-E4orf4 was evaluated involving clonogenic-cell-growth inhibition and the EGF-E4orf4-treated xenografts in nude mice.The fusion protein,EGF-E4orf4,with its high killing cancer cell potency and special selectivity,could be a promising drug for cancer therapy.
本组前期工作中利用EGF与EGFR结合的靶向性及腺病毒E4orf4蛋白的细胞毒性,通过基因重组及酵母表达技术,已设计并表达了一种新的融合蛋白EGF-E4orf4。前期工作表明,该融合蛋白对人乳腺癌细胞MDA-MB-231,人脑神经胶质瘤细胞BT325和人骨肉瘤细胞Saos-2都有抑制作用,但尚存在重组蛋白EGF-E4orf4表达量偏低,纯化效率不高等问题。本论文在此基础上,对融合蛋白EGF-E4orf4的表达,纯化和功能三个方面进行了进一步的探索。
本论文的第一部分工作是提高融合蛋白的表达量和纯度。利用5L发酵罐进行高密度发酵表达,与摇瓶相比较,提高了融合蛋白的表达量。优化纯化流程,通过超滤结合离子交换和凝胶过滤两步柱层析纯化,提高了目的蛋白的纯度。经鉴定,重组融合蛋白EGF-E4orf4是以蛋白聚合体形式存在,为进一步提高纯化效率带来了困难。为此,对该蛋白进行了改造:(1)在融合蛋白的羧基端添加(His)_6标签,以便于提高纯化效率。(2)以可以与EGFR结合的环七肽替代融合蛋白中的EGF,构建环七肽-E4orf4-His融合蛋白,减少二硫键的数目,并且可以排除E4orf4蛋白的细胞毒性作用受到EGF的促生长作用干扰的可能性。通过亲和层析和凝胶过滤层析,对上述两种蛋白进行纯化,得到纯度较高的目的蛋白。
本论文的第二部分工作是进一步研究融合蛋白EGF-E4orf4的生物学功能。主要以人乳腺癌细胞MDA-MB-231和人胃癌细胞BGC823为实验对象。通过Westernblotting检测EGFR磷酸化程度,发现融合蛋白EGF-E4orf4可以诱导EGFR发生磷酸化,并且呈时间和剂量依赖性。通过激光共聚焦显微镜观察FITC标记的融合蛋白和EGF孵育不同时间后的细胞,发现:FITC-EGF进入细胞后信号逐渐减弱,并且与溶酶体共定位;而FITC-EGF-E4orf4则随着孵育时间延长,信号在细胞内累积增强,并且在24 h不与溶酶体共定位。融合蛋白EGF-E4orf4的抗肿瘤作用则表现在抑制BGC823细胞的克隆形成能力以及抑制MDA-MB-231和BGC823细胞的裸鼠移植肿瘤的生长,最大抑瘤率分别为79%和49%(P＜0.05)。在急毒实验中未发现EGF-E4orf4蛋白对裸鼠有任何毒副作用。
最后一部分工作是构建了四环素可诱导表达E4orf4蛋白的真核细胞稳定表达株,并通过MTT实验证明诱导表达E4orf4蛋白可以抑制细胞生长。以未诱导细胞为对照,对四环素诱导E4orf4蛋白表达6 h和60 h的细胞进行全基因组芯片扫描,作了初步分析,为今后研究E4orf4蛋白在细胞内的作用机制奠定了基础。
综合上述研究结果表明,融合蛋白EGF-E4orf4确实可以通过EGFR这个分子靶点的介导,进入肿瘤细胞,体内实验证明,融合蛋白EGF-E4orf4可以特异地抑制肿瘤细胞增殖,并具有较为可靠的安全性。为了将融合蛋白EGF-E4orf4真正应用于临床的肿瘤治疗中,还有更多的对于制备工艺、细胞水平以及动物实验的艰巨工作有待完成。
Cancer is a kind of serious disease that jeopardizes the health and life of human beings.Although anti-tumor pharmaceuticals have been developed in recent years,their use is limited because of their low efficacy and adverse effect of toxicity.Therefore,the development of cytotoxic drugs that can specifically target tumors with minimal toxicity to normal cells continues to be a challenge in cancer therapy.
In our laboratory,we have constructed and expressed a new recombinant protein, EGF-E4orf4,based on the targeting of EGF and cytotoxicity of adenovirus E4orf4.In the previous experiments,it was indicated that this fusion protein inhibited human breast adenocarcinoma cell line MDA-MB-231,human Glioblastoma BT325 and human osteosarcoma cell line Saos-2 growth.There are some problems waiting for overcome such as how to increase the fusion protein's expression level in Pichia pastoris yeast and low-purity with existing purification procedure.The present study focused on the expression,purification and function of the fusion protein,EGF-E4orf4.
Firstly,the experiments were carried out on increasing the expression level of in Pp yeast and the purity of EGF-E4orf4.Improvement of EGF-E4orf4 expression level was done in bioreactor by fed-batch fermentation.The expression level of EGF-E4orf4 was increased compared with the shaking incubator.The fusion protein was purified by anion-exchange chromatography and gel exclusion chromatography.As EGF-E4orf4 was identified as polymer,we used two methods to reconstruct the fusion proteins:(1) adding 6xHis tag to the C terminal of EGF-E4orf4 to facilitate the purification process.(2) constructing cycling 7 peptides-E4orf4-His protein to reduce the number of disulfide bonds. The two fusion proteins were purified by His-tag affinity chromatography and gel exclusion chromatography.
Secondly,important part was to investigate the possible biological functions of the fusion protein EGF-E4orf4 using MDA-MB-231 and BGC823 cells that were characteristics for EGFR expression.The fusion protein stimulated EGFR phosphorylation in a dose- and time-dependent manner by western blotting analysis.Likewise,the results obtained from confocal microscopy confirmed the internalization and showed the differences between EGF-E4orf4 and EGF afterwards,EGF-E4orf4 significantly inhibited the ability of colony formation of BGC823.In vivo,EGF-E4orf4 suppressed tumor growth in a dose-dependent fashion with an inhibition ratio of 79%in MDA-MB-231 and 49%in BGC 823,respectively(P＜0.05).No adverse effects were observed in the nude mice even at the dose of 10mg/kg of EGF-E4orf4.
Final part was to create stable cell lines expressing E4orf4 and by MTT colorimetric assays,it was found that inducible expressing E4orf4 could inhibit cell growth. The first batch of microarray was carried out to identify changes in RNA levels resulting from E4orf4 expressing and the further independent experiments are still needed.
Our experiment results demonstrate that EGF-E4orf4 could be successfully introduced into the tumor cells by interaction between EGF and EGFR,and the antitumor efficacy of EGF-E4orf4 was evaluated involving clonogenic-cell-growth inhibition and the EGF-E4orf4-treated xenografts in nude mice.The fusion protein,EGF-E4orf4,with its high killing cancer cell potency and special selectivity,could be a promising drug for cancer therapy.
引文
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[5]Maoz T,Koren R,Ben-Ari I,Kleinberger T.YND1 interacts with CDC55 and is a novel mediator of E4orf4-induced toxicity.J Biol Chem.,2005;280(50):41270-7.
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[1]T(a|¨)uber B,Dobner T.Molecular regulation and biological function ofadenovirus early genes:the E40RFs.Gene,2001,278(1-2):1-23.
[2]Lechward K,Awotunde OS,Swiatek W,et al.Phosphatase 2A:variety of forms and diversity of functions.Acta Biochim Pol.,2001,48(4):921-933.
[3]Shtrichman R,Sharf R,Kleinberger T.Adenovirus E4orf4 protein interacts with both Balpha and B' subunits of protein phosphatase 2A,but E4orf4-induced apoptosis is mediated only by the interaction with Balpha..Oncogene,2000,19(33):3757-3765.
[4]Shtrichman R,Sharf R,Barr H,et al.Induction of apoptosis by adenovirus E4orf4protein is specific to transformed cells and requires an interaction with protein phosphatase 2A.Proc Natl Acad Sci U S A,1999,96(18):10080-10085.
[5]Maoz T,Koren R,Ben-Ari I,Kleinberger T.YND1 interacts with CDC55 and is a novel mediator of E4orf4-induced toxicity.J Biol Chem.,2005;280(50):41270-7.
[6]Kornitzer D,Sharf R,Kleinberger T.Adenovirus E4orf4 protein induces PP2A-dependent growth arrest in Saccharomyces cerevisiae and interacts with the anaphase-promoting complex/cyclosome.J Cell Biol,2001,154(2):3.31-344.
[7]Zhou BB,Li H,Yuan J,et al.Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells.Proc Natl Acad Sci U S A,1998,95(12):6785-6790.
[8]Miron MJ,Gallouzi IE,Lavoie JN.Nuclear localization of the adenovirus E4orf4 protein is mediated through an arginine-rich motif and correlates with cell death.Oncogene,2004,23(45):7458-7468.
[9]Gingras MC,Champagne C,Roy M,et al.Cytoplasmic death signal triggered by SRC-mediated phosphorylation of the adenovirus E4orf4 protein.Mol Cell Biol,2002,22(1):41-56.
[10]Robert A,Smadja-Lamere N,Landry MC,Champagne C,Petrie R,Lamarche-Vane N,Hosoya H,Lavoie JN.Adenovirus E4orf4 hijacks rho GTPase-dependent actin dynamics to kill cells:a role for endosome-associated actin assembly.Mol Biol Cell.2006;17(7):3329-44.
[11]Rohn JL,Noteborn MHThe viral death effector Apoptin reveals tumor-specific processes.Apoptosis.2004 May;9(3):315-22.
[12]Branton PE,Roopchand DE.The role of adenovirus E4orf4 protein in viral replication and cell killing.Oncogene,2001,20(54):7855-7865.
[13]Livne A,Shtrichman R,Kleinberger T.Caspase activation by adenovirus e4orf4 protein is cell line specific and Is mediated by the death receptor pathway.J Virol.2001 Jan;75(2):789-98.
[14]Mitrus I,Missol-Kolka E,Plucienniczak A,Szala S.Tumour therapy with genes encoding apoptin and E4orf4.Anticancer Res.2005 Mar-Apr;25(2A):1087-90.
[15]Wang DM,Zhou Y,Xie HJ,Ma XL,Wang X,Chen H,Huang BR..Cytotoxicity of a recombinant fusion protein of adenovirus early region 4 open reading frame 4 (E4orf4)and human epidermal growth factor on p53-deficient tumor cells.Anticancer Drugs.2006 Jun;17(5):527-37.