细胞穿透肽（Arg）_9与力达霉素强化融合蛋白的构建及其抗肿瘤活性研究
|
摘要
力达霉素(Lidamycin, LDM, C-1027)是由球孢链霉菌(Streptomyces globisporus)产生的烯二炔类抗生素,是迄今报道过的对肿瘤细胞杀伤作用最强的大分子肽类抗肿瘤抗生素,具有很强的细胞毒性、抗血管生成活性及抑制实验肿瘤生长的作用。LDM的分子由烯二炔结构的发色团(AE)和辅基蛋白(LDP)构成,两者可进行拆分和重建,以往的研究表明发色团是力达霉素分子的活性部分,辅基蛋白具有稳定和保护发色团的功能。辅基蛋白是否具有抗肿瘤活性,目前还没有定论。
神经胶质瘤是最常见的原发性脑肿瘤,是高度血管化的血管生成依赖性生长的人类肿瘤。目前的报道表明神经胶质瘤的血管微环境在神经胶质瘤发生与发展、侵袭、临床诊断及治疗效果中起关健性作用。由于血脑屏障的存在,限制了大部分抗血管生成药物在神经胶质瘤治疗方面的应用。
细胞穿透肽(cell penetrating peptides, CPPs)是一类以非受体依赖方式、非经典内吞方式直接穿过细胞膜进入细胞的多肽,它们的长度一般不超过30个氨基酸且富含碱性氨基酸,能够介导与其相连的各种本不能通过细胞膜的生物活性物质进入细胞。迄今的研究提示CPPs能在体外或体内介导一系列生物活性分子如蛋白质、肽类、寡聚核苷酸、质粒及脂质体等进入各种不同组织和细胞,这些重要发现为将亲水性蛋白、寡肽及寡核苷酸应用于药学、基因治疗、细胞生物学等研究领域开辟了一条新的道路。
本研究利用本室构建表达的力达霉素重组辅基蛋白rLDP,研究其对多种肿瘤细胞的体外亲和活性及其体内外抗肿瘤活性,通过分子重建制备重组力达霉素(rLDM),研究其与正常力达霉素的结构和活性异同；利用基因工程方法构建表达辅基蛋白与细胞穿透肽(Arg)9的融合蛋白(Arg)9-LDP,对其穿膜能力和抗肿瘤活性进行研究,再通过分子强化技术组装发色团AE,得到强化融合蛋白(Arg)9-LDP-AE,研究其对神经胶质瘤细胞的体外杀伤作用以及体内抑瘤活性。1.重组力达霉素的制备及活性分析
利用本室保存的含有pET30-sldp质粒的E.coli BL21(DE3)starTM菌株,IPTG诱导带有组氨酸标签肽(His-tag)的重组辅基蛋白的表达。重组辅基蛋白rLDP分子量约为12 kDa,除存在于周质腔中外,还分泌到培养液上清中。Ni2+亲和层析对培养液上清的蛋白进行纯化,得到的目的蛋白经SDS-PAGE电泳检测纯度在98%以上,每升发酵液得到23 mg活性蛋白。细胞免疫荧光和基于流式细胞术的荧光实验结果显示rLDP蛋白对肿瘤细胞,如人肺癌A549细胞、人卵巢癌OVCAR3细胞、人肝癌Bel-7402细胞、人神经胶质瘤U87细胞、人非小细胞肺癌H460细胞均有很强的结合活性,而对正常肝组织来源的L02细胞则无亲和活性。细胞增殖实验证明rLDP蛋白可抑制Bel-7402细胞的增殖。流式细胞仪检测结果表明rLDP蛋白可引起Bel-7402细胞周期的G2／M期阻滞,且存在浓度依赖性。体内抑瘤实验表明rLDP蛋白能够显著抑制小鼠肝癌H22移植瘤的生长,且毒副作用较小。rLDP蛋白静脉注射15 mg/kg、30 mg/kg和60 mg/kg 3个剂量组11天的抑瘤率分别为44.6%、57.1%和50.1%,与对照组相比有显著差异；rLDP蛋白腹腔注射30mg/kg、60mg/kg和120 mg/kg 3个剂量组11天的抑瘤率分别为52.1%、49.3%和59.5%,与对照组相比有显著差异；rLDP蛋白灌胃给药30 mg/kg、60 mg/kg和120 mg/kg 3个剂量组11天的抑瘤率分别为34.3%、30.2%和60.3%,与对照组相比有显著差异。将AE分子与rLDP蛋白在体外进行分子组装后,经HPLC检测在350 nm处出现了特定的吸收峰,表明AE分子与rLDP蛋白进行了成功组装,得到了重组力达霉素rLDM。MTT试验结果显示重组力达霉素rLDM对不同的肿瘤细胞系均表现出了强烈的杀伤作用,其对U87细胞、MCF-7细胞、SKOV3细胞和Bel-7402细胞的IC50值与力达霉素LDM接近。流式细胞仪检测结果表明rLDM和LDM在浓度为1 nM时引起U87细胞和SKOV3细胞周期的S期阻滞和G2／M期阻滞,在浓度为0.001 nM时SKOV3细胞出现G2／M期阻滞,U87细胞出现G1期阻滞。
2.细胞穿透肽(Arg)9与力达霉素融合蛋白(Arg)9-LDP的构建及活性分析
利用基因重组技术扩增含有细胞穿透肽(Arg)9和力达霉素辅基蛋白LDP基因的(arg)9ldp片段,连接到质粒pET30a(+)上,构建重组质粒pET30-(arg)9ldp,转化到E.coli BL21(DE3)starTM中,IPTG诱导带有组氨酸标签肽(His-tag)的融合蛋白的表达。融合蛋白分子量约为15 kDa,主要以包涵体形式存在。Ni2+亲和柱纯化融合蛋白,得到的目的蛋白经SDS-PAGE电泳检测纯度在95%以上,每升发酵液得到15 mg活性蛋白。细胞荧光检测和基于流式细胞术的荧光实验结果显示带有穿透肽的融合蛋白可以穿透细胞膜进入细胞内部,与重组辅基蛋白和牛血清白蛋白相比有明显差异,其在37℃的穿透能力优于4℃,说明穿透过程与能量有关。细胞增殖实验证明融合蛋白可抑制肿瘤细胞的增殖,其对U87细胞的IC50值为5.25×10-5mol／L。流式细胞仪检测结果表明20μM的融合蛋白可引起U87细胞周期的G1期阻滞,浓度为2001μM时引起U87细胞周期的G2／M期阻滞和S期阻滞。
3.细胞穿透肽(Arg)9与力达霉素强化融合蛋白(Arg)9-LDP-AE的制备及活性分析
将含有细胞穿透肽和力达霉素辅基蛋白的融合蛋白(Arg)9-LDP与力达霉素活性发色团组装,经HPLC检测在350 nm处出现了特定的吸收峰,表明强化融合蛋白(Arg)9-LDP-AE组装成功。细胞增殖实验证明强化融合蛋白对肿瘤细胞具有强杀伤作用,强化融合蛋白和力达霉素对U87细胞和MCF-7细胞的IC50值相近,强化融合蛋白对OVCAR3细胞和Bel-7402细胞的IC50值分别是力达霉素的1／20和1／10。流式细胞仪检测结果表明强化融合蛋白可引起U87细胞周期的G2／M期阻滞和S期阻滞,Annexin V-FITC/PI双染结合流式细胞仪结果表明强化融合蛋白以剂量依赖方式诱导细胞凋亡。体内抑瘤实验表明强化融合蛋白(Arg)9-LDP-AE对肝癌H22小鼠移植瘤具有明显的抑制作用,且毒副作用较LDM小。强化融合蛋白0.2 mg/kg和0.3 mg/kg剂量组抑瘤率分别为85.3%和89.2%,与LDM0.05 mg/kg组相比有明显提高。(Arg)9-LDP-AE对人神经胶质瘤U87细胞形成的实体瘤有明显的抑制作用,28天时0.1 mg/kg和0.2mg/kg 2个剂量组抑瘤率分别为79.2%和88.8%,与LDM组存在显著差异(P<0.01)。
Lidamycin (LDM, C-1027), a macromolecular peptide antibiotic produced by Streptomyces globisporus, showed extremely potent cytotoxicity against tumor cell lines, anti-angiogenic activity and marked growth inhibition of transplantable tumors in mice. LDM consists of an active enedinye chomophore (AE) and an apoprotein (LDP), which can be separated and reconstituted without losing its activity. The primary role of LDP is as a packing carrier protein for the chemically unstable chromophore, however, the antitumor activity of LDP remains unidentified.
Glioma, the most common primary brain tumor, is highly vascularized and angiogenesis-dependent. Current reports show that the vascular microenvironment of glioma plays a central role in glioma progression, invasion, diagnosis and the effectiveness of clinic treatment. Because of blood-brain barrier, the application of antiangiogenesis drugs in glioma therapy is limited.
Cell-penetrating peptides (CPPs) are short peptides of less than 30 amino acids that are able to penetrate cell membranes and translocate different cargoes into cells. The mechanism of cell translocation is not known but it is apparently receptor and energy independent, although, in certain cases, translocation can be partially mediated by endocytosis. Cargoes that are successfully internalized by CPPs range from small molecules, oligonucleotide to proteins, bangosome and supramolecular particles. CPPs are novel vehicles for the translocation of cargo into cells, their properties make them potential drug delivery agents of interest for future use.
In this study, recombinant lidamycin apoprotein rLDP was prepared and recombinant lidamycin rLDM was reconstituted, the affinity activity of rLDP to tumor cells and the antitumor activity of rLDP and rLDM was observed. Fusion protein (Arg)9-LDP was prepared and energized fusion protein (Arg)9-LDP-AE was reconstituted. The membrane translocation ability of fusion protein and the potent cytotoxicity of fusion protein and its energized protein were observed.
1. Preparation of recombinant lidamycin and the study of its activity
E.coli BL21 (DE3) starTM cells which has the recombinant plasmid pET30-sldp were preserved in our laboratory. The recombinant lidamycin apoprotein rLDP with His-tag was successfully secreted into the culture medium and periplasmic space of E.coli after IPTG inducing. rLDP was purified by Ni2+ affinity chromatography and the purity of fusion proteins was all over 98%as determined by SDS-PAGE. Finally about 23 mg of purified protein was obtained from 1 L of culture medium. The immunofluorescent cytochemical staining and FACS-based fluorescence assay showed that rLDP has strong binding activity to cancer cell lines, such as human lung carcinoma A549 cells and H460 cells, human ovarian carcinoma OVCAR3 cells, human glioma U87 cells, and human hepatoma Bel-7402 cells. However, rLDP has no binding activity to L02 cells came from normal hepatic tissue. The results of MTT assay proved that rLDP displayed cytotoxicity to Bel-7402 cells with IC50 value of 7.05×10-5 mol/L. FACS analysis of cell cycle showed that the cells were arrested in G2/M phase after rLDP treatment, and the degree of arrest was concentration dependent. Transplantable hepatoma 22 (H22) in Kunming mice was used to investigate the inhibitory effects of rLDP. Evidently, rLDP suppressed H22 tumor growth. The inhibition rates of rLDP at dose of 15 mg/kg,30 mg/kg and 60 mg/kg were 44.6%,57.1%and 50.1%respectively for intravenous injection. The inhibition rates of rLDP at dose of 30 mg/kg,60 mg/kg and 120 mg/kg were 52.1%,49.3%and 59.5%respectively for intraperitoneal injection. The inhibition rates of rLDP at dose of 30 mg/kg,60 mg/kg and 120 mg/kg were 34.3%, 30.2%and 60.3%respectively for intragastric administration. The recombinant lidamycin rLDM was prepared by the reconstitution of rLDP and AE. The results of MTT assay showed that the IC50 values of rLDM for SKOV3 cells, U87 cells, MCF-7 cells and Bel-7402 cells were close to those of LDM. FACS analysis of cell cycle showed that rLDM and LDM induced similar cell cycle arrest in SKOV3 cells and U87cells. Cells were both arrested in G2/M phase and S phase after rLDM and LDM treatment at 1 nM, SKOV3 cells were arrested in G2/M phase and U87cells were arrested in G1 phase after treatment at 0.001 nM.
2. Construction of fusion protein (Arg)9LDP and the study of its activity
(arg)9ldp gene was ligated into the plasmid pET-30a(+), then the recombinant plasmid pET30-(arg)yldp was transformed into competent E.coli BL21 (DE3) starTM cells. Fusion protein (Arg)9-LDP with His-tag was produced in the form of inclusion after IPTG inducing. (Arg)9-LDP was purified by Ni2+ affinity chromatography and the purity of fusion proteins was all over 95%as determined by SDS-PAGE. Finally about 15 mg of purified protein was obtained from 1 L of culture medium. The result of fluorescent cytochemical staining showed that (Arg)9-LDP could be transferred into tumor cells and distributed in cytoplasm for 2 h incubation. The FACS-based fluorescence assay revealed that the translocation of (Arg)9-LDP was influenced by temperature. The results of MTT assay proved that (Arg)9-LDP had moderate cytotoxicity to U87 cells with IC50 value of 5.25×10-5 mol/L. FACS ananlysis of cell cycle showed that the cells were arrested in G2/M phase and S phase after (Arg)9-LDP treatment at 200μM and G1 phase at 20μM.
3. Preparation of energized fusion protein (Arg)9-LDP-AE and the study of its antitumor activity
The energized fusion protein (Arg)9-LDP-AE was prepared by the reconstitution of (Arg)9-LDP and AE. The results of MTT assay proved that (Arg)9-LDP-AE had stronger cytotoxicity than LDM. The IC50 values of (Arg)9-LDP-AE for U87 cells and MCF-7 cells were close to those of LDM, the IC50 values of (Arg)9-LDP-AE was only 1/20 of that of LDM for OVCAR3 cells and 1/10 for Bel-7402 cells respectively. FACS ananlysis of cell cycle showed that the U87 cells were arrested in G2/M phase and S phase after (Arg)9-LDP-AE treatment, and the degree of arrest was concentration dependent. The results of Annexin V-FITC/PI assay indicated the potent and dose-dependent proapoptotic effect of (Arg)9-LDP-AE on U87 cells. In vivo, the energized fusion protein (Arg)9-LDP-AE showed stronger antitumor activity than LDM. The inhibition rates of (Arg)9-LDP-AE against murine hepatoma H22 at dose of 0.2 mg/kg and 0.3 mg/kg were 85.3%and 89.2%respectively, while that of LDM at 0.05 mg/kg was 74.6%. (Arg)9-LDP-AE at the dose of 0.1 mg/kg and 0.2 mg/kg inhibited the growth of human glioma U87 xenografts by 79.2%and 88.8%, while that of LDM at 0.05 mg/kg was 62.9%.
神经胶质瘤是最常见的原发性脑肿瘤,是高度血管化的血管生成依赖性生长的人类肿瘤。目前的报道表明神经胶质瘤的血管微环境在神经胶质瘤发生与发展、侵袭、临床诊断及治疗效果中起关健性作用。由于血脑屏障的存在,限制了大部分抗血管生成药物在神经胶质瘤治疗方面的应用。
细胞穿透肽(cell penetrating peptides, CPPs)是一类以非受体依赖方式、非经典内吞方式直接穿过细胞膜进入细胞的多肽,它们的长度一般不超过30个氨基酸且富含碱性氨基酸,能够介导与其相连的各种本不能通过细胞膜的生物活性物质进入细胞。迄今的研究提示CPPs能在体外或体内介导一系列生物活性分子如蛋白质、肽类、寡聚核苷酸、质粒及脂质体等进入各种不同组织和细胞,这些重要发现为将亲水性蛋白、寡肽及寡核苷酸应用于药学、基因治疗、细胞生物学等研究领域开辟了一条新的道路。
本研究利用本室构建表达的力达霉素重组辅基蛋白rLDP,研究其对多种肿瘤细胞的体外亲和活性及其体内外抗肿瘤活性,通过分子重建制备重组力达霉素(rLDM),研究其与正常力达霉素的结构和活性异同；利用基因工程方法构建表达辅基蛋白与细胞穿透肽(Arg)9的融合蛋白(Arg)9-LDP,对其穿膜能力和抗肿瘤活性进行研究,再通过分子强化技术组装发色团AE,得到强化融合蛋白(Arg)9-LDP-AE,研究其对神经胶质瘤细胞的体外杀伤作用以及体内抑瘤活性。1.重组力达霉素的制备及活性分析
利用本室保存的含有pET30-sldp质粒的E.coli BL21(DE3)starTM菌株,IPTG诱导带有组氨酸标签肽(His-tag)的重组辅基蛋白的表达。重组辅基蛋白rLDP分子量约为12 kDa,除存在于周质腔中外,还分泌到培养液上清中。Ni2+亲和层析对培养液上清的蛋白进行纯化,得到的目的蛋白经SDS-PAGE电泳检测纯度在98%以上,每升发酵液得到23 mg活性蛋白。细胞免疫荧光和基于流式细胞术的荧光实验结果显示rLDP蛋白对肿瘤细胞,如人肺癌A549细胞、人卵巢癌OVCAR3细胞、人肝癌Bel-7402细胞、人神经胶质瘤U87细胞、人非小细胞肺癌H460细胞均有很强的结合活性,而对正常肝组织来源的L02细胞则无亲和活性。细胞增殖实验证明rLDP蛋白可抑制Bel-7402细胞的增殖。流式细胞仪检测结果表明rLDP蛋白可引起Bel-7402细胞周期的G2／M期阻滞,且存在浓度依赖性。体内抑瘤实验表明rLDP蛋白能够显著抑制小鼠肝癌H22移植瘤的生长,且毒副作用较小。rLDP蛋白静脉注射15 mg/kg、30 mg/kg和60 mg/kg 3个剂量组11天的抑瘤率分别为44.6%、57.1%和50.1%,与对照组相比有显著差异；rLDP蛋白腹腔注射30mg/kg、60mg/kg和120 mg/kg 3个剂量组11天的抑瘤率分别为52.1%、49.3%和59.5%,与对照组相比有显著差异；rLDP蛋白灌胃给药30 mg/kg、60 mg/kg和120 mg/kg 3个剂量组11天的抑瘤率分别为34.3%、30.2%和60.3%,与对照组相比有显著差异。将AE分子与rLDP蛋白在体外进行分子组装后,经HPLC检测在350 nm处出现了特定的吸收峰,表明AE分子与rLDP蛋白进行了成功组装,得到了重组力达霉素rLDM。MTT试验结果显示重组力达霉素rLDM对不同的肿瘤细胞系均表现出了强烈的杀伤作用,其对U87细胞、MCF-7细胞、SKOV3细胞和Bel-7402细胞的IC50值与力达霉素LDM接近。流式细胞仪检测结果表明rLDM和LDM在浓度为1 nM时引起U87细胞和SKOV3细胞周期的S期阻滞和G2／M期阻滞,在浓度为0.001 nM时SKOV3细胞出现G2／M期阻滞,U87细胞出现G1期阻滞。
2.细胞穿透肽(Arg)9与力达霉素融合蛋白(Arg)9-LDP的构建及活性分析
利用基因重组技术扩增含有细胞穿透肽(Arg)9和力达霉素辅基蛋白LDP基因的(arg)9ldp片段,连接到质粒pET30a(+)上,构建重组质粒pET30-(arg)9ldp,转化到E.coli BL21(DE3)starTM中,IPTG诱导带有组氨酸标签肽(His-tag)的融合蛋白的表达。融合蛋白分子量约为15 kDa,主要以包涵体形式存在。Ni2+亲和柱纯化融合蛋白,得到的目的蛋白经SDS-PAGE电泳检测纯度在95%以上,每升发酵液得到15 mg活性蛋白。细胞荧光检测和基于流式细胞术的荧光实验结果显示带有穿透肽的融合蛋白可以穿透细胞膜进入细胞内部,与重组辅基蛋白和牛血清白蛋白相比有明显差异,其在37℃的穿透能力优于4℃,说明穿透过程与能量有关。细胞增殖实验证明融合蛋白可抑制肿瘤细胞的增殖,其对U87细胞的IC50值为5.25×10-5mol／L。流式细胞仪检测结果表明20μM的融合蛋白可引起U87细胞周期的G1期阻滞,浓度为2001μM时引起U87细胞周期的G2／M期阻滞和S期阻滞。
3.细胞穿透肽(Arg)9与力达霉素强化融合蛋白(Arg)9-LDP-AE的制备及活性分析
将含有细胞穿透肽和力达霉素辅基蛋白的融合蛋白(Arg)9-LDP与力达霉素活性发色团组装,经HPLC检测在350 nm处出现了特定的吸收峰,表明强化融合蛋白(Arg)9-LDP-AE组装成功。细胞增殖实验证明强化融合蛋白对肿瘤细胞具有强杀伤作用,强化融合蛋白和力达霉素对U87细胞和MCF-7细胞的IC50值相近,强化融合蛋白对OVCAR3细胞和Bel-7402细胞的IC50值分别是力达霉素的1／20和1／10。流式细胞仪检测结果表明强化融合蛋白可引起U87细胞周期的G2／M期阻滞和S期阻滞,Annexin V-FITC/PI双染结合流式细胞仪结果表明强化融合蛋白以剂量依赖方式诱导细胞凋亡。体内抑瘤实验表明强化融合蛋白(Arg)9-LDP-AE对肝癌H22小鼠移植瘤具有明显的抑制作用,且毒副作用较LDM小。强化融合蛋白0.2 mg/kg和0.3 mg/kg剂量组抑瘤率分别为85.3%和89.2%,与LDM0.05 mg/kg组相比有明显提高。(Arg)9-LDP-AE对人神经胶质瘤U87细胞形成的实体瘤有明显的抑制作用,28天时0.1 mg/kg和0.2mg/kg 2个剂量组抑瘤率分别为79.2%和88.8%,与LDM组存在显著差异(P<0.01)。
Lidamycin (LDM, C-1027), a macromolecular peptide antibiotic produced by Streptomyces globisporus, showed extremely potent cytotoxicity against tumor cell lines, anti-angiogenic activity and marked growth inhibition of transplantable tumors in mice. LDM consists of an active enedinye chomophore (AE) and an apoprotein (LDP), which can be separated and reconstituted without losing its activity. The primary role of LDP is as a packing carrier protein for the chemically unstable chromophore, however, the antitumor activity of LDP remains unidentified.
Glioma, the most common primary brain tumor, is highly vascularized and angiogenesis-dependent. Current reports show that the vascular microenvironment of glioma plays a central role in glioma progression, invasion, diagnosis and the effectiveness of clinic treatment. Because of blood-brain barrier, the application of antiangiogenesis drugs in glioma therapy is limited.
Cell-penetrating peptides (CPPs) are short peptides of less than 30 amino acids that are able to penetrate cell membranes and translocate different cargoes into cells. The mechanism of cell translocation is not known but it is apparently receptor and energy independent, although, in certain cases, translocation can be partially mediated by endocytosis. Cargoes that are successfully internalized by CPPs range from small molecules, oligonucleotide to proteins, bangosome and supramolecular particles. CPPs are novel vehicles for the translocation of cargo into cells, their properties make them potential drug delivery agents of interest for future use.
In this study, recombinant lidamycin apoprotein rLDP was prepared and recombinant lidamycin rLDM was reconstituted, the affinity activity of rLDP to tumor cells and the antitumor activity of rLDP and rLDM was observed. Fusion protein (Arg)9-LDP was prepared and energized fusion protein (Arg)9-LDP-AE was reconstituted. The membrane translocation ability of fusion protein and the potent cytotoxicity of fusion protein and its energized protein were observed.
1. Preparation of recombinant lidamycin and the study of its activity
E.coli BL21 (DE3) starTM cells which has the recombinant plasmid pET30-sldp were preserved in our laboratory. The recombinant lidamycin apoprotein rLDP with His-tag was successfully secreted into the culture medium and periplasmic space of E.coli after IPTG inducing. rLDP was purified by Ni2+ affinity chromatography and the purity of fusion proteins was all over 98%as determined by SDS-PAGE. Finally about 23 mg of purified protein was obtained from 1 L of culture medium. The immunofluorescent cytochemical staining and FACS-based fluorescence assay showed that rLDP has strong binding activity to cancer cell lines, such as human lung carcinoma A549 cells and H460 cells, human ovarian carcinoma OVCAR3 cells, human glioma U87 cells, and human hepatoma Bel-7402 cells. However, rLDP has no binding activity to L02 cells came from normal hepatic tissue. The results of MTT assay proved that rLDP displayed cytotoxicity to Bel-7402 cells with IC50 value of 7.05×10-5 mol/L. FACS analysis of cell cycle showed that the cells were arrested in G2/M phase after rLDP treatment, and the degree of arrest was concentration dependent. Transplantable hepatoma 22 (H22) in Kunming mice was used to investigate the inhibitory effects of rLDP. Evidently, rLDP suppressed H22 tumor growth. The inhibition rates of rLDP at dose of 15 mg/kg,30 mg/kg and 60 mg/kg were 44.6%,57.1%and 50.1%respectively for intravenous injection. The inhibition rates of rLDP at dose of 30 mg/kg,60 mg/kg and 120 mg/kg were 52.1%,49.3%and 59.5%respectively for intraperitoneal injection. The inhibition rates of rLDP at dose of 30 mg/kg,60 mg/kg and 120 mg/kg were 34.3%, 30.2%and 60.3%respectively for intragastric administration. The recombinant lidamycin rLDM was prepared by the reconstitution of rLDP and AE. The results of MTT assay showed that the IC50 values of rLDM for SKOV3 cells, U87 cells, MCF-7 cells and Bel-7402 cells were close to those of LDM. FACS analysis of cell cycle showed that rLDM and LDM induced similar cell cycle arrest in SKOV3 cells and U87cells. Cells were both arrested in G2/M phase and S phase after rLDM and LDM treatment at 1 nM, SKOV3 cells were arrested in G2/M phase and U87cells were arrested in G1 phase after treatment at 0.001 nM.
2. Construction of fusion protein (Arg)9LDP and the study of its activity
(arg)9ldp gene was ligated into the plasmid pET-30a(+), then the recombinant plasmid pET30-(arg)yldp was transformed into competent E.coli BL21 (DE3) starTM cells. Fusion protein (Arg)9-LDP with His-tag was produced in the form of inclusion after IPTG inducing. (Arg)9-LDP was purified by Ni2+ affinity chromatography and the purity of fusion proteins was all over 95%as determined by SDS-PAGE. Finally about 15 mg of purified protein was obtained from 1 L of culture medium. The result of fluorescent cytochemical staining showed that (Arg)9-LDP could be transferred into tumor cells and distributed in cytoplasm for 2 h incubation. The FACS-based fluorescence assay revealed that the translocation of (Arg)9-LDP was influenced by temperature. The results of MTT assay proved that (Arg)9-LDP had moderate cytotoxicity to U87 cells with IC50 value of 5.25×10-5 mol/L. FACS ananlysis of cell cycle showed that the cells were arrested in G2/M phase and S phase after (Arg)9-LDP treatment at 200μM and G1 phase at 20μM.
3. Preparation of energized fusion protein (Arg)9-LDP-AE and the study of its antitumor activity
The energized fusion protein (Arg)9-LDP-AE was prepared by the reconstitution of (Arg)9-LDP and AE. The results of MTT assay proved that (Arg)9-LDP-AE had stronger cytotoxicity than LDM. The IC50 values of (Arg)9-LDP-AE for U87 cells and MCF-7 cells were close to those of LDM, the IC50 values of (Arg)9-LDP-AE was only 1/20 of that of LDM for OVCAR3 cells and 1/10 for Bel-7402 cells respectively. FACS ananlysis of cell cycle showed that the U87 cells were arrested in G2/M phase and S phase after (Arg)9-LDP-AE treatment, and the degree of arrest was concentration dependent. The results of Annexin V-FITC/PI assay indicated the potent and dose-dependent proapoptotic effect of (Arg)9-LDP-AE on U87 cells. In vivo, the energized fusion protein (Arg)9-LDP-AE showed stronger antitumor activity than LDM. The inhibition rates of (Arg)9-LDP-AE against murine hepatoma H22 at dose of 0.2 mg/kg and 0.3 mg/kg were 85.3%and 89.2%respectively, while that of LDM at 0.05 mg/kg was 74.6%. (Arg)9-LDP-AE at the dose of 0.1 mg/kg and 0.2 mg/kg inhibited the growth of human glioma U87 xenografts by 79.2%and 88.8%, while that of LDM at 0.05 mg/kg was 62.9%.
引文
[1]Fischer I, Gagner JP, Law M, Newcomb EW, Zagzag D. Angiogenesis in gliomas: biology and molecular pathophysiology. Brain Pathol 2005; 15:297-310.
[2]Lakka SS, Rao JS. Antiangiogenic therapy in brain tumors. Expert Rev Neurother 2008;8:1457-73.
[3]Folkman J, Merler E, Abernathy C, Williams G. Isolation of a tumor factor responsible for angiogenesis. J Exp Med 1971;133:275-88.
[4]Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249-57.
[5]Albini A, Sporn MB. The tumour microenvironment as a target for chemoprevention. Nat Rev Cancer 2007;7:139-47.
[6]Norton L, Massague J. Is cancer a disease of self-seeding? Nat Med 2006;12:875-8.
[7]Leung CT, Brugge JS. Tumor self-seeding:bidirectional flow of tumor cells. Cell 2009;139:1226-8.
[8]Penas-Prado M, Gilbert MR. Molecularly targeted therapies for malignant gliomas:advances and challenges. Expert Rev Anticancer Ther 2007;7:641-61.
[9]Aghi M, Cohen KS, Klein RJ, Scadden DT, Chiocca EA. Tumor stromal-derived factor-1 recruits vascular progenitors to mitotic neovasculature, where microenvironment influences their differentiated phenotypes. Cancer Res 2006;66:9054-64.
[10]Dietrich J, Norden AD, Wen PY. Emerging antiangiogenic treatments for gliomas-efficacy and safety issues. Curr Opin Neurol 2008;21:736-44.
[11]Jain RK, di Tomaso E, Duda DG, Loeffler JS, Sorensen AG, Batchelor TT. Angiogenesis in brain tumours. Nat Rev Neurosci 2007;8:610-22.
[12]Narayana A, Kelly P, Golfinos J, Parker E, Johnson G, Knopp E, et al. Antiangiogenic therapy using bevacizumab in recurrent high-grade glioma: impact on local control and patient survival. J Neurosurg 2009; 110:173-80.
[13]Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999;285:1569-72.
[14]Green M, Loewenstein PM. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 1988;55:1179-88.
[15]Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 1988;55:1189-93.
[16]Gupta B, Levchenko TS, Torchilin VP. Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv Drug Deliv Rev 2005;57:637-51.
[17]Console S, Marty C, Garcia-Echeverria C, Schwendener R, Ballmer-Hofer K. Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans. J Biol Chem 2003;278:35109-14.
[18]Wadia JS, Stan RV, Dowdy SF. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 2004; 10:310-5.
[19]Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, et al. Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem 2003;278:585-90.
[20]Ter-Avetisyan G, Tunnemann G, Nowak D, Nitschke M, Herrmann A, Drab M, et al. Cell entry of arginine-rich peptides is independent of endocytosis. J Biol Chem 2009;284:3370-8.
[21]Zaro JL, Shen WC. Cytosolic delivery of a p16-peptide oligoarginine conjugate for inhibiting proliferation of MCF7 cells. J Control Release 2005; 108:409-17.
[22]Jewell CM, Fuchs SM, Flessner RM, Raines RT, Lynn DM. Multilayered films fabricated from an oligoarginine-conjugated protein promote efficient surface-mediated protein transduction. Biomacromolecules 2007;8:857-63.
[23]Fuchs SM, Raines RT. Polyarginine as a multifunctional fusion tag. Protein Sci 2005;14:1538-44.
[24]Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake:peptoid molecular transporters. Proc Natl Acad Sci U S A 2000;97:13003-8.
[25]Tunnemann G, Ter-Avetisyan G, Martin RM, Stockl M, Herrmann A, Cardoso MC. Live-cell analysis of cell penetration ability and toxicity of oligo-arginines. J Pept Sci 2008;14:469-76.
[26]Wu RP, Youngblood DS, Hassinger JN, Lovejoy CE, Nelson MH, Iversen PL, et al. Cell-penetrating peptides as transporters for morpholino oligomers:effects of amino acid composition on intracellular delivery and cytotoxicity. Nucleic Acids Res 2007;35:5182-91.
[27]Furuhata M, Kawakami H, Toma K, Hattori Y, Maitani Y. Intracellular delivery of proteins in complexes with oligoarginine-modified liposomes and the effect of oligoarginine length. Bioconjug Chem 2006;17:935-42.
[28]Fuchs SM, Raines RT. Pathway for polyarginine entry into mammalian cells. Biochemistry 2004;43:2438-44.
[29]Goncalves E, Kitas E, Seelig J. Binding of oligoarginine to membrane lipids and heparan sulfate:structural and thermodynamic characterization of a cell-penetrating peptide. Biochemistry 2005;44:2692-702.
[30]Nicolaou KC, Smith AL, Yue EW. Chemistry and biology of natural and designed enediynes. Proc Natl Acad Sci U S A 1993;90:5881-8.
[31]Shao RG, Zhen YS. [Relationship between the molecular composition of C1027, a new macromolecular antibiotic with enediyne chromophore, and its antitumor activity]. Yao Xue Xue Bao 1995;30:336-42.
[32]Hu JL, Xue YC, Xie MY, Zhang R, Otani T, Minami Y, et al. A new macromolecular antitumor antibiotic, C-1027. I. Discovery, taxonomy of producing organism, fermentation and biological activity. J Antibiot (Tokyo) 1988;41:1575-9.
[33]Xu YJ, Zhen YS, Goldberg IH. C1027 chromophore, a potent new enediyne antitumor antibiotic, induces sequence-specific double-strand DNA cleavage. Biochemistry 1994;33:5947-54.
[34]Xu YJ, Xi Z, Zhen YS, Goldberg IH. A single binding mode of activated enediyne C1027 generates two types of double-strand DNA lesions:deuterium isotope-induced shuttling between adjacent nucleotide target sites. Biochemistry 1995;34:12451-60.
[35]Xu YJ, Xi Z, Zhen YS, Goldberg IH. Mechanism of formation of novel covalent drug.DNA interstrand cross-links and monoadducts by enediyne antitumor antibiotics. Biochemistry 1997;36:14975-84.
[36]Shao RG, Zhen YS. Enediyne anticancer antibiotic lidamycin:chemistry, biology and pharmacology. Anticancer Agents Med Chem 2008;8:123-31.
[37]Qiu Q, Wang Z, Jiang JM, Li DD. [Effect of lidamycin on mitochondria initiated apoptotic pathway in human cancer cells]. Yao Xue Xue Bao 2007;42:132-8.
[38]Chen L, Jiang J, Cheng C, Yang A, He Q, Li D, et al. P53 dependent and independent apoptosis induced by lidamycin in human colorectal cancer cells. Cancer Biol Ther 2007;6:965-73.
[39]Liu JS, Kuo SR, Yin X, Beerman TA, Melendy T. DNA damage by the enediyne C-1027 results in the inhibition of DNA replication by loss of replication protein A function and activation of DNA-dependent protein kinase. Biochemistry 2001;40:14661-8.
[40]He QY, Jiang B, Li DD, Zhen YS. [Effects of lidamycin on apoptotic gene expressions and cytoskeleton in human hepatoma bel-7402 cells]. Ai Zheng 2002;21:351-5.
[41]Inoue M, Usuki T, Lee N, Hirama M, Tanaka T, Hosoi F, et al. Antitumor enediyne chromoprotein C-1027:mechanistic investigation of the chromophore-mediated self-decomposition pathway. J Am Chem Soc 2006;128:7896-903.
[42]Zhen HY, Huang YH, Zhen YS. Lidamycin inhibits the cancer cell PKC activity induced by basic fibroblast growth factor. Yao Xue Xue Bao 2005;40:1110-5.
[43]Chen J, Ouyang ZG, Zhang SH, Zhen YS. Down-regulation of the nuclear factor-kappaB by lidamycin in association with inducing apoptosis in human pancreatic cancer cells and inhibiting xenograft growth. Oncol Rep 2007;17:1445-51.
[44]Zhen YZ, Lin YJ, Li Y, Zhen YS. Lidamycin shows highly potent cytotoxic to myeloma cells and inhibits tumor growth in mice. Acta Pharmacol Sin 2009;30:1025-32.
[45]Zhang SH, Chen J, Jiang M, Zhen YS. [Lidamycin induces apoptosis of human gastric carcinoma BGC823 cells and inhibits xenograft growth in nude mice]. Yao Xue Xue Bao 2008;43:601-4.
[46]Zhen H, Xue Y, Zhen Y. [Inhibition of angiogenesis by antitumor antibiotic C1027 and its effect on tumor metastasis]. Zhonghua Yi Xue Za Zhi 1997;77:657-60.
[47]Xu YJ, Li DD, Zhen YS. Mode of action of C-1027, a new macromolecular antitumor antibiotic with highly potent cytotoxicity, on human hepatoma BEL-7402 cells. Cancer Chemother Pharmacol 1990;27:41-6.
[48]Liu X, Bian C, Ren K, Jin H, Li B, Shao RG. Lidamycin induces marked G2 cell cycle arrest in human colon carcinoma HT-29 cells through activation of p38 MAPK pathway. Oncol Rep 2007;17:597-603.
[49]Huang YH, Shang BY, Zhen YS. Antitumor efficacy of lidamycin on hepatoma and active moiety of its molecule. World J Gastroenterol 2005; 11:3980-4.
[50]Liu YP, Li QS, Huang YR, Zhou MJ, Liu CX. Pharmacokinetics of C-1027 in mice as determined by TCA-RA method. World J Gastroenterol 2005; 11:717-20.
[51]Otani T, Minami Y, Marunaka T, Zhang R, Xie MY. A new macromolecular antitumor antibiotic, C-1027. Ⅱ. Isolation and physico-chemical properties. J Antibiot (Tokyo) 1988;41:1580-5.
[52]Otani T. Conformation studies on and assessment by spectral analysis of the protein-chromophore interaction of the macromolecular antitumor antibiotic C-1027. J Antibiot (Tokyo) 1993;46:791-802.
[53]Sakata N, Tsuchiya KS, Moriya Y, Hayashi H, Hori M, Otani T, et al. Aminopeptidase activity of an antitumor antibiotic, C-1027. J Antibiot (Tokyo) 1992;45:113-7.
[54]Cai L, Chen H, Miao Q, Wu S, Shang Y, Zhen Y, et al. Binding capability of the enediyne-associated apoprotein to human tumors and constitution of a ligand oligopeptide-integrated protein. J Biotechnol 2009; 144:142-50.
[55]Zein N, Reiss P, Bernatowicz M, Bolgar M. The proteolytic specificity of the natural enediyne-containing chromoproteins is unique to each chromoprotein. Chem Biol 1995;2:451-5.
[56]Matsumoto T, Okuno Y, Sugiura Y. Specific interaction between a novel enediyne chromophore and apoprotein in macromolecular antitumor antibiotic C-1027. Biochem Biophys Res Commun 1993; 195:659-66.
[57]Urbaniak MD, Muskett FW, Finucane MD, Caddick S, Woolfson DN. Solution structure of a novel chromoprotein derived from apo-neocarzinostatin and a synthetic chromophore. Biochemistry 2002;41:11731-9.
[58]Otani T, Yasuhara T, Minami Y, Shimazu T, Zhang R, Xie MY. Purification and primary structure of C-1027-AG, a selective antagonist of antitumor antibiotic C-1027, from Streptomyces globisporus. Agric Biol Chem 1991;55:407-17.
[59]Chen Y, Shao R, Bartlam M, Li J, Jin L, Gao Y, et al. Crystallization and preliminary X-ray crystallographic studies of a macromolecular antitumour antibiotic, C1027. Acta Crystallogr D Biol Crystallogr 2002;58:173-5.
[60]Tanaka T, Fukuda-Ishisaka S, Hirama M, Otani T. Solution structures of C-1027 apoprotein and its complex with the aromatized chromophore. J Mol Biol 2001;309:267-83.
[61]Allen TM, Cullis PR. Drug delivery systems:entering the mainstream. Science 2004;303:1818-22.
[62]de Wolf FA, Brett GM. Ligand-binding proteins:their potential for application in systems for controlled delivery and uptake of ligands. Pharmacol Rev 2000;52:207-36.
[63]Sudhahar CG, Chin DH. Aponeocarzinostatin--a superior drug carrier exhibiting unusually high endurance against denaturants. Bioorg Med Chem 2006;14:3543-52.
[64]Hariharan P, Liang W, Chou SH, Chin DH. A new model for ligand release. Role of side chain in gating the enediyne antibiotic. J Biol Chem 2006;281:16025-33.
[65]Nozaki S, Tomioka Y, Hishinuma T, Inoue M, Nagumo Y, Tsuruta LR, et al. Design, production, and characterization of recombinant neocarzinostatin apoprotein in Escherichia coli. J Biochem 2002;131:729-38.
[66]Tomioka Y, Kisara S, Yoshizawa S, Ozawa M, Suzuki N, Yamaguchi H, et al. Preparation of neocarzinostatin apoprotein mutants and the randomized library on the chromophore-binding cavity. Biol Pharm Bull 2006;29:1010-4.
[67]Mohanty S, Sieker LC, Drobny GP. Sequential 1H NMR assignment of the complex of aponeocarzinostatin with ethidium bromide and investigation of protein-drug interactions in the chromophore binding site. Biochemistry 1994;33:10579-90.
[68]Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ. Immunotoxin treatment of cancer. Annu Rev Med 2007;58:221-37.
[69]Zhen YS. [Bright prospects of antibody-based drug for targeted cancer therapy]. Zhonghua Yi Xue Za Zhi 2009;89:1729-31.
[70]Awada A, Cardoso F, Atalay G, Giuliani R, Mano M, Piccart MJ. The pipeline of new anticancer agents for breast cancer treatment in 2003. Crit Rev Oncol Hematol 2003;48:45-63.
[71]Zhong G, Zhang S, Li Y, Liu X, Gao R, Miao Q, et al. A tandem scFv-based fusion protein and its enediyne-energized analogue show intensified therapeutic efficacy against lung carcinoma xenograft in athymic mice. Cancer Lett.
[72]Liu X, He H, Feng Y, Zhang M, Ren K, Shao R. Difference of cell cycle arrests induced by lidamycin in human breast cancer cells. Anticancer Drugs 2006; 17:173-9.
[73]Trehin R, Merkle HP. Chances and pitfalls of cell penetrating peptides for cellular drug delivery. Eur J Pharm Biopharm 2004;58:209-23.
[74]Kim HK, Davaa E, Myung CS, Park JS. Enhanced siRNA delivery using cationic liposomes with new polyarginine-conjugated PEG-lipid. Int J Pharm.
[75]Choi Y, McCarthy JR, Weissleder R, Tung CH. Conjugation of a photosensitizer to an oligoarginine-based cell-penetrating peptide increases the efficacy of photodynamic therapy. ChemMedChem 2006; 1:458-63.
[76]Yang SR, Kim SB, Joe CO, Kim JD. Intracellular delivery enhancement of poly(amino acid) drug carriers by oligoarginine conjugation. J Biomed Mater Res A 2008;86:137-48.
[77]Khalil IA, Kogure K, Futaki S, Harashima H. High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression. J Biol Chem 2006;281:3544-51.
[78]Khalil IA, Kogure K, Futaki S, Hama S, Akita H, Ueno M, et al. Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery. Gene Ther 2007;14:682-9.
[79]Kim WJ, Christensen LV, Jo S, Yockman JW, Jeong JH, Kim YH, et al. Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adenocarcinoma. Mol Ther 2006;14:343-50.
[80]Abes R, Arzumanov A, Moulton H, Abes S, Ivanova G, Gait MJ, et al. Arginine-rich cell penetrating peptides:design, structure-activity, and applications to alter pre-mRNA splicing by steric-block oligonucleotides. J Pept Sci 2008;14:455-60.
[81]Mino T, Mori T, Aoyama Y, Sera T. Cell-permeable artificial zinc-finger proteins as potent antiviral drugs for human papillomaviruses. Arch Virol 2008;153:1291-8.
[82]Vincent B, Vincent JP, Checler F. Purification and characterization of human endopeptidase 3.4.24.16. Comparison with the porcine counterpart indicates a unique cleavage site on neurotensin. Brain Res 1996;709:51-8.
[83]Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB. Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 2000;56:318-25.
[84]Li M, Su ZG, Janson JC. In vitro protein refolding by chromatographic procedures. Protein Expr Purif 2004;33:1-10.
[85]Tsumoto K, Ejima D, Kumagai I, Arakawa T. Practical considerations in refolding proteins from inclusion bodies. Protein Expr Purif 2003;28:1-8.
[86]Guo XF, Zhu XF, Shang Y, Zhang SH, Zhen YS. A bispecific enediyne-energized fusion protein containing ligand-based and antibody-based oligopeptides against epidermal growth factor receptor and human epidermal growth factor receptor 2 shows potent antitumor activity. Clin Cancer Res 16:2085-94.
[87]Rhee M, Davis P. Mechanism of uptake of C105Y, a novel cell-penetrating peptide. J Biol Chem 2006;281:1233-40.
[88]Cheng G, Mi L, Cao Z, Xue H, Yu Q, Carr L, et al. Functionalizable and Ultrastable Zwitterionic Nanogels. Langmuir.
[89]Anandhakumar S, Nagaraja V, Raichur AM. Reversible polyelectrolyte capsules as carriers for protein delivery. Colloids Surf B Biointerfaces.
[90]Thoren PE, Persson D, Isakson P, Goksor M, Onfelt A, Norden B. Uptake of analogs of penetratin, Tat(48-60) and oligoarginine in live cells. Biochem Biophys Res Commun 2003;307:100-7.
[91]Nakase I, Niwa M, Takeuchi T, Sonomura K, Kawabata N, Koike Y, et al. Cellular uptake of arginine-rich peptides:roles for macropinocytosis and actin rearrangement. Mol Ther 2004; 10:1011-22.
[92]Zaro JL, Shen WC. Evidence that membrane transduction of oligoarginine does not require vesicle formation. Exp Cell Res 2005;307:164-73.
[93]Zaro JL, Rajapaksa TE, Okamoto CT, Shen WC. Membrane transduction of oligoarginine in HeLa cells is not mediated by macropinocytosis. Mol Pharm 2006;3:181-6.
[94]Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992;267:10931-4.
[95]Carmeliet P. Angiogenesis in health and disease. Nat Med 2003;9:653-60.
[96]Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669-76.
[97]Lee JE, Didier DN, Lockett MR, Scalf M, Greene AS, Olivier M, et al. Characterization of vascular endothelial growth factor receptors on the endothelial cell surface during hypoxia using whole cell binding arrays. Anal Biochem 2007;369:241-7.
[98]Vaisman N, Gospodarowicz D, Neufeld G. Characterization of the receptors for vascular endothelial growth factor. J Biol Chem 1990;265:19461-6.
[99]Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 1992;359:845-8.
[100]Stratmann A, Risau W, Plate KH. Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis. Am J Pathol 1998;153:1459-66.
[101]Weindel K, Moringlane JR, Marme D, Weich HA. Detection and quantification of vascular endothelial growth factor/vascular permeability factor in brain tumor tissue and cyst fluid:the key to angiogenesis? Neurosurgery 1994;35:439-48; discussion 48-9.
[102]Schmidt NO, Westphal M, Hagel C, Ergun S, Stavrou D, Rosen EM, et al. Levels of vascular endothelial growth factor, hepatocyte growth factor/scatter factor and basic fibroblast growth factor in human gliomas and their relation to angiogenesis. Int J Cancer 1999;84:10-8.
[103]Lamszus K, Lengler U, Schmidt NO, Stavrou D, Ergun S, Westphal M. Vascular endothelial growth factor, hepatocyte growth factor/scatter factor, basic fibroblast growth factor, and placenta growth factor in human meningiomas and their relation to angiogenesis and malignancy. Neurosurgery 2000;46:938-47; discussion 47-8.
[104]Miao QF, Liu XY, Shang BY, Ouyang ZG, Zhen YS. An enediyne-energized single-domain antibody-containing fusion protein shows potent antitumor activity. Anticancer Drugs 2007;18:127-37.
[105]Miao Q, Shang B, Ouyang Z, Liu X, Zhen Y. Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin. Sci China C Life Sci 2007;50:447-56.
[106]Li XQ, Zhang SH, Ouyang ZG, Zhen YS. [Inhibitory effect of lidamycin upon vasculogenic mimicry and its induction of apoptosis in glioma cells]. Zhonghua Yi Xue Za Zhi 2009;89:1736-40.
[107]Sheng WJ, Miao QF, Zhen YS. [Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv]. Yao Xue Xue Bao 2009;44:597-602.
[108]Guo XF, Zhong GS, Miao QF, Zhen YS. [Construction of energized fusion protein consisting of epidermal growth factor receptor oligopeptide ligand and lidamycin and its antitumor activity]. Ai Zheng 2009;28:561-8.
[109]Santra S, Yang H, Stanley JT, Holloway PH, Moudgil BM, Walter G, et al. Rapid and effective labeling of brain tissue using TAT-conjugated CdS:Mn/ZnS quantum dots. Chem Commun (Camb) 2005:3144-6.
[110]Mathupala SP. Delivery of small-interfering RNA (siRNA) to the brain. Expert Opin Ther Pat 2009; 19:137-40.
[111]Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994;84:1415-20.
[1]Schwarze SR, Ho A, Vocero Akbani A,et al. In vivo protein transduction:delivery of a biologically active protein into the mouse [J].Science,1999,285(5433):1569-1572.
[2]Green M, Loewenstein PM. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein [J]. Cell, 1988,55:1179-1188.
[3]Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immuno-deficiency virus [J]. Cell,1988,55:1189-1193.
[4]Gupta B, Levchenko TS, Torchilin VP. Intracellular delivery of large molecules and small particles by cell penetrating proteins and peptides [J]. Adv Drug Deliv Rev.2005,57(4):637-651.
[5]Console S, Marty C, Garci A-Echeverria C. Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans [J]. Biol Chem,2003,278 (37):35109-35114.
[6]Wadia J S, Stan R V, Dowdy S F, et al. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion p roteins after lipidraft macrop inocytosis [J]. NatMed,2004,10(3):310-315.
[7]Jean Philippe Richard, Kamran Melikov, Eric Vives, et al. Cell-penetrating Peptides:a reevaluation of the mechanism of cellular uptake [J]. The Journal Of Biological Chemistry,2003,278(1):585-590.
[8]Gohar Ter-Avetisyan, Gisela Tunnemann, Danny Nowak,et al. Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis [J]. The Journal Of Biological Chemistry 2009,284(6):3370-3380.
[9]Selivanova G, Iotsova V, Okan I, et al. Restoration of the growth suppression func-tion of mutant p53 by a synthetic peptide derived from the p53 C-terminal domain[J]. Nature Medicine,1997,3:632-638.
[10]Harbour J W, Worley L, MaD, et al. Transducible peptide therapy for uveal melanoma and retinoblastoma [J]. Arch Ophthalmol,2002,120:1341-1346.
[11]Snyder EL, Meade BR, Saenz CC, et al. Treatment of terminal peritoneal carcin -omatosis by a transducible P53-activating peptide. PLoS Biol,2004,2:186-193.
[12]Snyder E, Saenz C, Denicourt C, et al. Enhanced targeting and killing of tumor cells expressing the CXC chemokine receptor 4 by transducible anticancer peptides [J]. Cancer Res,2005,65:10646-10650.
[13]Mizukawa K, Kawamura A, Sasayama T. Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines [J]. J Neurooncol,2006,77(3):247-255.
[14]Arnt CR, Chiorean MV, Heldebrant MP, et al. Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAPI in Situ [J]. J Biol Chem,2002,277:44236-44243.
[15]Yan H, Thomas J, Liu T, et al. Induction of melanoma cell apoptosis and inhibiti-on of tumor growth using a cell-permeable Survivin antagonist [J].Oncogene,2006, 25:6968-6974.
[16]Holinger EP, Chittenden T, Lutz RJ, et al. Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases. J Biol Chem,1999,274:13298-13304.
[17]Naotaka Shibagakin, Udeym C. Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity [J]. J Immunol,2002, 168 (5):2393-2401.
[18]Nico Michel, Wolfram Osen, Lutz Gissmann, et al.Enhanced Immunogenicity of HPV 16 E7 Fusion Proteins in DNA Vaccination [J]. Virology,2002,294 (1):47-59.
[19]Michel N, Ohlschlager P, Osen W,et al. T cell response to human papillomavirus 16 E7 in mice:comparison of Cr release assay, intracellular IFN-gamma production, ELISPOT and tetramer staining [J]. Intervirology.2002,45(4-6):290-299.
[20]Peng S, Trimble C, Ji H, et al. Characterization of HPV-16 E6 DNA vaccines employing intracellular targeting and intercellular spreading strategies [J]. Biomed Sci.2005,12(5):689-700.
[21]Batchu RB, Moreno AM, Szmania SM, et al. Protein transduction of dendritic cells for NY-ESO-1-Based immunotherapy of myeloma [J]. Cancer Res,2005, 65:10041-10049.
[22]Jennica L, Zaro, Wei-Chiang Shen, Cytosolic delivery of a p16-peptide oligoarginine conjugate for inhibiting proliferation of MCF7 cells[J] Journal of Controlled Release,2005,108(2-3):409-417.
[23]Ezhevsky SA, Nagahara H, Vocero Akbani AM, et al. Hypo-phosphorylation of the retinoblastoma protein(PRb) cyclin D:Cdk4/6 complexes results in active pRb [J]. Proc Natl Acad Sci USA,1997.94:10699-10704.
[24]Fahraeus R, Paramio JM, Ball KL, et al. Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide derived from p16 CDKN2/INK4A [J]. Curr Biol,1996,6:94-91.
[25]Gius DR, Ezhevsky SA, Becker-Hapak M, et al. Transduced p16INK4a peptides inhibit hypophosphorylation of the retinoblastoma protein and cell cycle progression prior to activation of Cdk2 complexes in late G1 [J]. Cancer Res, 1999,59:2577-2580.
[26]el-Deiry WS, Harper JW, O'Connor JM, et al. WAFI/CIP1 is induced in p53-mediated G1 arrest and apoptosis [J]. Cancer Res,1994,54:1169-1174.
[27]Bonfanti M, Taverna S, Salmona M, et al. p21 WAFT-derived peptides linked to an internalization peptide inhibit human cancer cell growth [J]. Cancer Res,1997, 57:1442-1446.
[28]Nagahara H, Vocero-Akbani AM, Snyder EL, et al.Transduction of full-length TAT fusion proteins into mammalian cells:TAT-p27 Kipl induces cell migration [J]. Nat Med,1998,4:1449-1452.
[29]Parada Y, Banerji L, Glassford J, et al. BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27 Kipl expression. J Biol Chem,2001,276:23572-23580.
[30]Huang LE, Gu J, Schau M,et al. Regulation of hypoxia-inducible factor 1 alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway [J]. Proc Natl Acad Sci USA,1998,95(14):7987-7992.
[31]Harada H, Hiraoka M, Kizaka-Kondoh S. Antitumor effect of TAT-oxygen-dep-endent degradation-caspase 3 fusion protein specifically stabilized and activated in hypoxic tumor cells [J]. Cancer Res,2002,62:2013-2018.
[32]Bryan R. Meade, Steven F. Dowdy. Enhancing the cellular uptake of siRNA duplexes followingnoncovalent packaging with protein transduction domain Peptides [J]. Adv Drug Deliv Rev,2008,60(4-5):530-536.
[33]Nakase I, Niwa M, Takeuchit,et al. Cellular uptake of arginine-rich peptides:roles for macropinocytosis and actin rearrangement [J]. Mol. Ther.2004,10(6): 1011-1022.
[34]Magzoub M, Pramanik A, Graslund A,et al.Modeling the endosomal escape of cell-penetrating peptides:transmembrane pH gradient driven translocation across phospholipid bilayers [J]. Biochemistry,2005,44(45):14890-14897.
[35]Skehel JJ, Cross K, Steinhauer D,et al. Influenza fusion peptides[J]. Biochem Soc Trans,2001,29(4):623-626.
[36]Michiue H, Tomizawa K, Wei FY, et al. The NH2 terminus of influenza virus hemagglutinin-2 subunit peptides enhances the antitumor potency of polyarginine-mediated p53 protein transduction[J]. J Biol Chem,2005,280(9): 8285-8289.
[37]Nadine K, Patricia M, Thorsten N,et al. A Membrane-Destabilizing Peptide in Capsid Protein L2 Is Required for Egress of Papillomavirus Genomes from Endosomes [J]. Journal of Virology,2006,80(2):759-768.
[38]Maiolo JR 3rd, Ottinger EA, Ferrer M,et al. Specific redistribution of cell-penetrating peptides from endosomes to the cytoplasm and nucleus upon laser illumination [J]. J Am Chem Soc.2004,126(47):15376-15377
[39]Wadia JS, Stan RV, Dowdy SF. Ansducible TAT HA fusogenic peptide enhances escape of TAT fusion proteins after lipid raft macropinocytosis [J]. Nat Med,2004, 10(3):310-315.
[40]Shiraishi T, Pankratova S, Nielsen PE. Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides [J]. Chem. Biol,2005,12(8):923-929.
[41]Kim D,Jeon C,Kim JH, et al.Cytoplasmic transduction peptide (CTP):new approach for the delivery of biomolecules into cytoplasm in vitro and in vivo [J]. Exp Cell Res,2006,312(8):1277-1288.
[42]Zhao K, Luo G, Giannelli S,et al. Mitochondria-targeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines [J]. Biochem Pharmacol.2005,70(12):1796-806.
[43]Snyder EL, Saenz CC, Denicourt C,et al. Enhanced targeting and killing of tumor cells expressing the CXC chemokine receptor 4 by transducible anticancer peptides[J].Cancer Res.2005,65(23):10646-10650.
[44]Ruoslahti E, Duza T, Zhang L.Vascular homing peptides with cell-penetrating properties [J].Curr Pharm Des.2005, 11(28):3655-3660.
[45]Robinson P, Stuber D, Deryckere F,et al. Identification using phage display of peptides promoting targeting and internalization into HPV-transformed cell lines [J]. J Mol Recognit.2005,18(2):175-182.
[2]Lakka SS, Rao JS. Antiangiogenic therapy in brain tumors. Expert Rev Neurother 2008;8:1457-73.
[3]Folkman J, Merler E, Abernathy C, Williams G. Isolation of a tumor factor responsible for angiogenesis. J Exp Med 1971;133:275-88.
[4]Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249-57.
[5]Albini A, Sporn MB. The tumour microenvironment as a target for chemoprevention. Nat Rev Cancer 2007;7:139-47.
[6]Norton L, Massague J. Is cancer a disease of self-seeding? Nat Med 2006;12:875-8.
[7]Leung CT, Brugge JS. Tumor self-seeding:bidirectional flow of tumor cells. Cell 2009;139:1226-8.
[8]Penas-Prado M, Gilbert MR. Molecularly targeted therapies for malignant gliomas:advances and challenges. Expert Rev Anticancer Ther 2007;7:641-61.
[9]Aghi M, Cohen KS, Klein RJ, Scadden DT, Chiocca EA. Tumor stromal-derived factor-1 recruits vascular progenitors to mitotic neovasculature, where microenvironment influences their differentiated phenotypes. Cancer Res 2006;66:9054-64.
[10]Dietrich J, Norden AD, Wen PY. Emerging antiangiogenic treatments for gliomas-efficacy and safety issues. Curr Opin Neurol 2008;21:736-44.
[11]Jain RK, di Tomaso E, Duda DG, Loeffler JS, Sorensen AG, Batchelor TT. Angiogenesis in brain tumours. Nat Rev Neurosci 2007;8:610-22.
[12]Narayana A, Kelly P, Golfinos J, Parker E, Johnson G, Knopp E, et al. Antiangiogenic therapy using bevacizumab in recurrent high-grade glioma: impact on local control and patient survival. J Neurosurg 2009; 110:173-80.
[13]Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999;285:1569-72.
[14]Green M, Loewenstein PM. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 1988;55:1179-88.
[15]Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 1988;55:1189-93.
[16]Gupta B, Levchenko TS, Torchilin VP. Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv Drug Deliv Rev 2005;57:637-51.
[17]Console S, Marty C, Garcia-Echeverria C, Schwendener R, Ballmer-Hofer K. Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans. J Biol Chem 2003;278:35109-14.
[18]Wadia JS, Stan RV, Dowdy SF. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 2004; 10:310-5.
[19]Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, et al. Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem 2003;278:585-90.
[20]Ter-Avetisyan G, Tunnemann G, Nowak D, Nitschke M, Herrmann A, Drab M, et al. Cell entry of arginine-rich peptides is independent of endocytosis. J Biol Chem 2009;284:3370-8.
[21]Zaro JL, Shen WC. Cytosolic delivery of a p16-peptide oligoarginine conjugate for inhibiting proliferation of MCF7 cells. J Control Release 2005; 108:409-17.
[22]Jewell CM, Fuchs SM, Flessner RM, Raines RT, Lynn DM. Multilayered films fabricated from an oligoarginine-conjugated protein promote efficient surface-mediated protein transduction. Biomacromolecules 2007;8:857-63.
[23]Fuchs SM, Raines RT. Polyarginine as a multifunctional fusion tag. Protein Sci 2005;14:1538-44.
[24]Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake:peptoid molecular transporters. Proc Natl Acad Sci U S A 2000;97:13003-8.
[25]Tunnemann G, Ter-Avetisyan G, Martin RM, Stockl M, Herrmann A, Cardoso MC. Live-cell analysis of cell penetration ability and toxicity of oligo-arginines. J Pept Sci 2008;14:469-76.
[26]Wu RP, Youngblood DS, Hassinger JN, Lovejoy CE, Nelson MH, Iversen PL, et al. Cell-penetrating peptides as transporters for morpholino oligomers:effects of amino acid composition on intracellular delivery and cytotoxicity. Nucleic Acids Res 2007;35:5182-91.
[27]Furuhata M, Kawakami H, Toma K, Hattori Y, Maitani Y. Intracellular delivery of proteins in complexes with oligoarginine-modified liposomes and the effect of oligoarginine length. Bioconjug Chem 2006;17:935-42.
[28]Fuchs SM, Raines RT. Pathway for polyarginine entry into mammalian cells. Biochemistry 2004;43:2438-44.
[29]Goncalves E, Kitas E, Seelig J. Binding of oligoarginine to membrane lipids and heparan sulfate:structural and thermodynamic characterization of a cell-penetrating peptide. Biochemistry 2005;44:2692-702.
[30]Nicolaou KC, Smith AL, Yue EW. Chemistry and biology of natural and designed enediynes. Proc Natl Acad Sci U S A 1993;90:5881-8.
[31]Shao RG, Zhen YS. [Relationship between the molecular composition of C1027, a new macromolecular antibiotic with enediyne chromophore, and its antitumor activity]. Yao Xue Xue Bao 1995;30:336-42.
[32]Hu JL, Xue YC, Xie MY, Zhang R, Otani T, Minami Y, et al. A new macromolecular antitumor antibiotic, C-1027. I. Discovery, taxonomy of producing organism, fermentation and biological activity. J Antibiot (Tokyo) 1988;41:1575-9.
[33]Xu YJ, Zhen YS, Goldberg IH. C1027 chromophore, a potent new enediyne antitumor antibiotic, induces sequence-specific double-strand DNA cleavage. Biochemistry 1994;33:5947-54.
[34]Xu YJ, Xi Z, Zhen YS, Goldberg IH. A single binding mode of activated enediyne C1027 generates two types of double-strand DNA lesions:deuterium isotope-induced shuttling between adjacent nucleotide target sites. Biochemistry 1995;34:12451-60.
[35]Xu YJ, Xi Z, Zhen YS, Goldberg IH. Mechanism of formation of novel covalent drug.DNA interstrand cross-links and monoadducts by enediyne antitumor antibiotics. Biochemistry 1997;36:14975-84.
[36]Shao RG, Zhen YS. Enediyne anticancer antibiotic lidamycin:chemistry, biology and pharmacology. Anticancer Agents Med Chem 2008;8:123-31.
[37]Qiu Q, Wang Z, Jiang JM, Li DD. [Effect of lidamycin on mitochondria initiated apoptotic pathway in human cancer cells]. Yao Xue Xue Bao 2007;42:132-8.
[38]Chen L, Jiang J, Cheng C, Yang A, He Q, Li D, et al. P53 dependent and independent apoptosis induced by lidamycin in human colorectal cancer cells. Cancer Biol Ther 2007;6:965-73.
[39]Liu JS, Kuo SR, Yin X, Beerman TA, Melendy T. DNA damage by the enediyne C-1027 results in the inhibition of DNA replication by loss of replication protein A function and activation of DNA-dependent protein kinase. Biochemistry 2001;40:14661-8.
[40]He QY, Jiang B, Li DD, Zhen YS. [Effects of lidamycin on apoptotic gene expressions and cytoskeleton in human hepatoma bel-7402 cells]. Ai Zheng 2002;21:351-5.
[41]Inoue M, Usuki T, Lee N, Hirama M, Tanaka T, Hosoi F, et al. Antitumor enediyne chromoprotein C-1027:mechanistic investigation of the chromophore-mediated self-decomposition pathway. J Am Chem Soc 2006;128:7896-903.
[42]Zhen HY, Huang YH, Zhen YS. Lidamycin inhibits the cancer cell PKC activity induced by basic fibroblast growth factor. Yao Xue Xue Bao 2005;40:1110-5.
[43]Chen J, Ouyang ZG, Zhang SH, Zhen YS. Down-regulation of the nuclear factor-kappaB by lidamycin in association with inducing apoptosis in human pancreatic cancer cells and inhibiting xenograft growth. Oncol Rep 2007;17:1445-51.
[44]Zhen YZ, Lin YJ, Li Y, Zhen YS. Lidamycin shows highly potent cytotoxic to myeloma cells and inhibits tumor growth in mice. Acta Pharmacol Sin 2009;30:1025-32.
[45]Zhang SH, Chen J, Jiang M, Zhen YS. [Lidamycin induces apoptosis of human gastric carcinoma BGC823 cells and inhibits xenograft growth in nude mice]. Yao Xue Xue Bao 2008;43:601-4.
[46]Zhen H, Xue Y, Zhen Y. [Inhibition of angiogenesis by antitumor antibiotic C1027 and its effect on tumor metastasis]. Zhonghua Yi Xue Za Zhi 1997;77:657-60.
[47]Xu YJ, Li DD, Zhen YS. Mode of action of C-1027, a new macromolecular antitumor antibiotic with highly potent cytotoxicity, on human hepatoma BEL-7402 cells. Cancer Chemother Pharmacol 1990;27:41-6.
[48]Liu X, Bian C, Ren K, Jin H, Li B, Shao RG. Lidamycin induces marked G2 cell cycle arrest in human colon carcinoma HT-29 cells through activation of p38 MAPK pathway. Oncol Rep 2007;17:597-603.
[49]Huang YH, Shang BY, Zhen YS. Antitumor efficacy of lidamycin on hepatoma and active moiety of its molecule. World J Gastroenterol 2005; 11:3980-4.
[50]Liu YP, Li QS, Huang YR, Zhou MJ, Liu CX. Pharmacokinetics of C-1027 in mice as determined by TCA-RA method. World J Gastroenterol 2005; 11:717-20.
[51]Otani T, Minami Y, Marunaka T, Zhang R, Xie MY. A new macromolecular antitumor antibiotic, C-1027. Ⅱ. Isolation and physico-chemical properties. J Antibiot (Tokyo) 1988;41:1580-5.
[52]Otani T. Conformation studies on and assessment by spectral analysis of the protein-chromophore interaction of the macromolecular antitumor antibiotic C-1027. J Antibiot (Tokyo) 1993;46:791-802.
[53]Sakata N, Tsuchiya KS, Moriya Y, Hayashi H, Hori M, Otani T, et al. Aminopeptidase activity of an antitumor antibiotic, C-1027. J Antibiot (Tokyo) 1992;45:113-7.
[54]Cai L, Chen H, Miao Q, Wu S, Shang Y, Zhen Y, et al. Binding capability of the enediyne-associated apoprotein to human tumors and constitution of a ligand oligopeptide-integrated protein. J Biotechnol 2009; 144:142-50.
[55]Zein N, Reiss P, Bernatowicz M, Bolgar M. The proteolytic specificity of the natural enediyne-containing chromoproteins is unique to each chromoprotein. Chem Biol 1995;2:451-5.
[56]Matsumoto T, Okuno Y, Sugiura Y. Specific interaction between a novel enediyne chromophore and apoprotein in macromolecular antitumor antibiotic C-1027. Biochem Biophys Res Commun 1993; 195:659-66.
[57]Urbaniak MD, Muskett FW, Finucane MD, Caddick S, Woolfson DN. Solution structure of a novel chromoprotein derived from apo-neocarzinostatin and a synthetic chromophore. Biochemistry 2002;41:11731-9.
[58]Otani T, Yasuhara T, Minami Y, Shimazu T, Zhang R, Xie MY. Purification and primary structure of C-1027-AG, a selective antagonist of antitumor antibiotic C-1027, from Streptomyces globisporus. Agric Biol Chem 1991;55:407-17.
[59]Chen Y, Shao R, Bartlam M, Li J, Jin L, Gao Y, et al. Crystallization and preliminary X-ray crystallographic studies of a macromolecular antitumour antibiotic, C1027. Acta Crystallogr D Biol Crystallogr 2002;58:173-5.
[60]Tanaka T, Fukuda-Ishisaka S, Hirama M, Otani T. Solution structures of C-1027 apoprotein and its complex with the aromatized chromophore. J Mol Biol 2001;309:267-83.
[61]Allen TM, Cullis PR. Drug delivery systems:entering the mainstream. Science 2004;303:1818-22.
[62]de Wolf FA, Brett GM. Ligand-binding proteins:their potential for application in systems for controlled delivery and uptake of ligands. Pharmacol Rev 2000;52:207-36.
[63]Sudhahar CG, Chin DH. Aponeocarzinostatin--a superior drug carrier exhibiting unusually high endurance against denaturants. Bioorg Med Chem 2006;14:3543-52.
[64]Hariharan P, Liang W, Chou SH, Chin DH. A new model for ligand release. Role of side chain in gating the enediyne antibiotic. J Biol Chem 2006;281:16025-33.
[65]Nozaki S, Tomioka Y, Hishinuma T, Inoue M, Nagumo Y, Tsuruta LR, et al. Design, production, and characterization of recombinant neocarzinostatin apoprotein in Escherichia coli. J Biochem 2002;131:729-38.
[66]Tomioka Y, Kisara S, Yoshizawa S, Ozawa M, Suzuki N, Yamaguchi H, et al. Preparation of neocarzinostatin apoprotein mutants and the randomized library on the chromophore-binding cavity. Biol Pharm Bull 2006;29:1010-4.
[67]Mohanty S, Sieker LC, Drobny GP. Sequential 1H NMR assignment of the complex of aponeocarzinostatin with ethidium bromide and investigation of protein-drug interactions in the chromophore binding site. Biochemistry 1994;33:10579-90.
[68]Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ. Immunotoxin treatment of cancer. Annu Rev Med 2007;58:221-37.
[69]Zhen YS. [Bright prospects of antibody-based drug for targeted cancer therapy]. Zhonghua Yi Xue Za Zhi 2009;89:1729-31.
[70]Awada A, Cardoso F, Atalay G, Giuliani R, Mano M, Piccart MJ. The pipeline of new anticancer agents for breast cancer treatment in 2003. Crit Rev Oncol Hematol 2003;48:45-63.
[71]Zhong G, Zhang S, Li Y, Liu X, Gao R, Miao Q, et al. A tandem scFv-based fusion protein and its enediyne-energized analogue show intensified therapeutic efficacy against lung carcinoma xenograft in athymic mice. Cancer Lett.
[72]Liu X, He H, Feng Y, Zhang M, Ren K, Shao R. Difference of cell cycle arrests induced by lidamycin in human breast cancer cells. Anticancer Drugs 2006; 17:173-9.
[73]Trehin R, Merkle HP. Chances and pitfalls of cell penetrating peptides for cellular drug delivery. Eur J Pharm Biopharm 2004;58:209-23.
[74]Kim HK, Davaa E, Myung CS, Park JS. Enhanced siRNA delivery using cationic liposomes with new polyarginine-conjugated PEG-lipid. Int J Pharm.
[75]Choi Y, McCarthy JR, Weissleder R, Tung CH. Conjugation of a photosensitizer to an oligoarginine-based cell-penetrating peptide increases the efficacy of photodynamic therapy. ChemMedChem 2006; 1:458-63.
[76]Yang SR, Kim SB, Joe CO, Kim JD. Intracellular delivery enhancement of poly(amino acid) drug carriers by oligoarginine conjugation. J Biomed Mater Res A 2008;86:137-48.
[77]Khalil IA, Kogure K, Futaki S, Harashima H. High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression. J Biol Chem 2006;281:3544-51.
[78]Khalil IA, Kogure K, Futaki S, Hama S, Akita H, Ueno M, et al. Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery. Gene Ther 2007;14:682-9.
[79]Kim WJ, Christensen LV, Jo S, Yockman JW, Jeong JH, Kim YH, et al. Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adenocarcinoma. Mol Ther 2006;14:343-50.
[80]Abes R, Arzumanov A, Moulton H, Abes S, Ivanova G, Gait MJ, et al. Arginine-rich cell penetrating peptides:design, structure-activity, and applications to alter pre-mRNA splicing by steric-block oligonucleotides. J Pept Sci 2008;14:455-60.
[81]Mino T, Mori T, Aoyama Y, Sera T. Cell-permeable artificial zinc-finger proteins as potent antiviral drugs for human papillomaviruses. Arch Virol 2008;153:1291-8.
[82]Vincent B, Vincent JP, Checler F. Purification and characterization of human endopeptidase 3.4.24.16. Comparison with the porcine counterpart indicates a unique cleavage site on neurotensin. Brain Res 1996;709:51-8.
[83]Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB. Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 2000;56:318-25.
[84]Li M, Su ZG, Janson JC. In vitro protein refolding by chromatographic procedures. Protein Expr Purif 2004;33:1-10.
[85]Tsumoto K, Ejima D, Kumagai I, Arakawa T. Practical considerations in refolding proteins from inclusion bodies. Protein Expr Purif 2003;28:1-8.
[86]Guo XF, Zhu XF, Shang Y, Zhang SH, Zhen YS. A bispecific enediyne-energized fusion protein containing ligand-based and antibody-based oligopeptides against epidermal growth factor receptor and human epidermal growth factor receptor 2 shows potent antitumor activity. Clin Cancer Res 16:2085-94.
[87]Rhee M, Davis P. Mechanism of uptake of C105Y, a novel cell-penetrating peptide. J Biol Chem 2006;281:1233-40.
[88]Cheng G, Mi L, Cao Z, Xue H, Yu Q, Carr L, et al. Functionalizable and Ultrastable Zwitterionic Nanogels. Langmuir.
[89]Anandhakumar S, Nagaraja V, Raichur AM. Reversible polyelectrolyte capsules as carriers for protein delivery. Colloids Surf B Biointerfaces.
[90]Thoren PE, Persson D, Isakson P, Goksor M, Onfelt A, Norden B. Uptake of analogs of penetratin, Tat(48-60) and oligoarginine in live cells. Biochem Biophys Res Commun 2003;307:100-7.
[91]Nakase I, Niwa M, Takeuchi T, Sonomura K, Kawabata N, Koike Y, et al. Cellular uptake of arginine-rich peptides:roles for macropinocytosis and actin rearrangement. Mol Ther 2004; 10:1011-22.
[92]Zaro JL, Shen WC. Evidence that membrane transduction of oligoarginine does not require vesicle formation. Exp Cell Res 2005;307:164-73.
[93]Zaro JL, Rajapaksa TE, Okamoto CT, Shen WC. Membrane transduction of oligoarginine in HeLa cells is not mediated by macropinocytosis. Mol Pharm 2006;3:181-6.
[94]Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992;267:10931-4.
[95]Carmeliet P. Angiogenesis in health and disease. Nat Med 2003;9:653-60.
[96]Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669-76.
[97]Lee JE, Didier DN, Lockett MR, Scalf M, Greene AS, Olivier M, et al. Characterization of vascular endothelial growth factor receptors on the endothelial cell surface during hypoxia using whole cell binding arrays. Anal Biochem 2007;369:241-7.
[98]Vaisman N, Gospodarowicz D, Neufeld G. Characterization of the receptors for vascular endothelial growth factor. J Biol Chem 1990;265:19461-6.
[99]Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 1992;359:845-8.
[100]Stratmann A, Risau W, Plate KH. Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis. Am J Pathol 1998;153:1459-66.
[101]Weindel K, Moringlane JR, Marme D, Weich HA. Detection and quantification of vascular endothelial growth factor/vascular permeability factor in brain tumor tissue and cyst fluid:the key to angiogenesis? Neurosurgery 1994;35:439-48; discussion 48-9.
[102]Schmidt NO, Westphal M, Hagel C, Ergun S, Stavrou D, Rosen EM, et al. Levels of vascular endothelial growth factor, hepatocyte growth factor/scatter factor and basic fibroblast growth factor in human gliomas and their relation to angiogenesis. Int J Cancer 1999;84:10-8.
[103]Lamszus K, Lengler U, Schmidt NO, Stavrou D, Ergun S, Westphal M. Vascular endothelial growth factor, hepatocyte growth factor/scatter factor, basic fibroblast growth factor, and placenta growth factor in human meningiomas and their relation to angiogenesis and malignancy. Neurosurgery 2000;46:938-47; discussion 47-8.
[104]Miao QF, Liu XY, Shang BY, Ouyang ZG, Zhen YS. An enediyne-energized single-domain antibody-containing fusion protein shows potent antitumor activity. Anticancer Drugs 2007;18:127-37.
[105]Miao Q, Shang B, Ouyang Z, Liu X, Zhen Y. Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin. Sci China C Life Sci 2007;50:447-56.
[106]Li XQ, Zhang SH, Ouyang ZG, Zhen YS. [Inhibitory effect of lidamycin upon vasculogenic mimicry and its induction of apoptosis in glioma cells]. Zhonghua Yi Xue Za Zhi 2009;89:1736-40.
[107]Sheng WJ, Miao QF, Zhen YS. [Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv]. Yao Xue Xue Bao 2009;44:597-602.
[108]Guo XF, Zhong GS, Miao QF, Zhen YS. [Construction of energized fusion protein consisting of epidermal growth factor receptor oligopeptide ligand and lidamycin and its antitumor activity]. Ai Zheng 2009;28:561-8.
[109]Santra S, Yang H, Stanley JT, Holloway PH, Moudgil BM, Walter G, et al. Rapid and effective labeling of brain tissue using TAT-conjugated CdS:Mn/ZnS quantum dots. Chem Commun (Camb) 2005:3144-6.
[110]Mathupala SP. Delivery of small-interfering RNA (siRNA) to the brain. Expert Opin Ther Pat 2009; 19:137-40.
[111]Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994;84:1415-20.
[1]Schwarze SR, Ho A, Vocero Akbani A,et al. In vivo protein transduction:delivery of a biologically active protein into the mouse [J].Science,1999,285(5433):1569-1572.
[2]Green M, Loewenstein PM. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein [J]. Cell, 1988,55:1179-1188.
[3]Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immuno-deficiency virus [J]. Cell,1988,55:1189-1193.
[4]Gupta B, Levchenko TS, Torchilin VP. Intracellular delivery of large molecules and small particles by cell penetrating proteins and peptides [J]. Adv Drug Deliv Rev.2005,57(4):637-651.
[5]Console S, Marty C, Garci A-Echeverria C. Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans [J]. Biol Chem,2003,278 (37):35109-35114.
[6]Wadia J S, Stan R V, Dowdy S F, et al. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion p roteins after lipidraft macrop inocytosis [J]. NatMed,2004,10(3):310-315.
[7]Jean Philippe Richard, Kamran Melikov, Eric Vives, et al. Cell-penetrating Peptides:a reevaluation of the mechanism of cellular uptake [J]. The Journal Of Biological Chemistry,2003,278(1):585-590.
[8]Gohar Ter-Avetisyan, Gisela Tunnemann, Danny Nowak,et al. Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis [J]. The Journal Of Biological Chemistry 2009,284(6):3370-3380.
[9]Selivanova G, Iotsova V, Okan I, et al. Restoration of the growth suppression func-tion of mutant p53 by a synthetic peptide derived from the p53 C-terminal domain[J]. Nature Medicine,1997,3:632-638.
[10]Harbour J W, Worley L, MaD, et al. Transducible peptide therapy for uveal melanoma and retinoblastoma [J]. Arch Ophthalmol,2002,120:1341-1346.
[11]Snyder EL, Meade BR, Saenz CC, et al. Treatment of terminal peritoneal carcin -omatosis by a transducible P53-activating peptide. PLoS Biol,2004,2:186-193.
[12]Snyder E, Saenz C, Denicourt C, et al. Enhanced targeting and killing of tumor cells expressing the CXC chemokine receptor 4 by transducible anticancer peptides [J]. Cancer Res,2005,65:10646-10650.
[13]Mizukawa K, Kawamura A, Sasayama T. Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines [J]. J Neurooncol,2006,77(3):247-255.
[14]Arnt CR, Chiorean MV, Heldebrant MP, et al. Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAPI in Situ [J]. J Biol Chem,2002,277:44236-44243.
[15]Yan H, Thomas J, Liu T, et al. Induction of melanoma cell apoptosis and inhibiti-on of tumor growth using a cell-permeable Survivin antagonist [J].Oncogene,2006, 25:6968-6974.
[16]Holinger EP, Chittenden T, Lutz RJ, et al. Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases. J Biol Chem,1999,274:13298-13304.
[17]Naotaka Shibagakin, Udeym C. Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity [J]. J Immunol,2002, 168 (5):2393-2401.
[18]Nico Michel, Wolfram Osen, Lutz Gissmann, et al.Enhanced Immunogenicity of HPV 16 E7 Fusion Proteins in DNA Vaccination [J]. Virology,2002,294 (1):47-59.
[19]Michel N, Ohlschlager P, Osen W,et al. T cell response to human papillomavirus 16 E7 in mice:comparison of Cr release assay, intracellular IFN-gamma production, ELISPOT and tetramer staining [J]. Intervirology.2002,45(4-6):290-299.
[20]Peng S, Trimble C, Ji H, et al. Characterization of HPV-16 E6 DNA vaccines employing intracellular targeting and intercellular spreading strategies [J]. Biomed Sci.2005,12(5):689-700.
[21]Batchu RB, Moreno AM, Szmania SM, et al. Protein transduction of dendritic cells for NY-ESO-1-Based immunotherapy of myeloma [J]. Cancer Res,2005, 65:10041-10049.
[22]Jennica L, Zaro, Wei-Chiang Shen, Cytosolic delivery of a p16-peptide oligoarginine conjugate for inhibiting proliferation of MCF7 cells[J] Journal of Controlled Release,2005,108(2-3):409-417.
[23]Ezhevsky SA, Nagahara H, Vocero Akbani AM, et al. Hypo-phosphorylation of the retinoblastoma protein(PRb) cyclin D:Cdk4/6 complexes results in active pRb [J]. Proc Natl Acad Sci USA,1997.94:10699-10704.
[24]Fahraeus R, Paramio JM, Ball KL, et al. Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide derived from p16 CDKN2/INK4A [J]. Curr Biol,1996,6:94-91.
[25]Gius DR, Ezhevsky SA, Becker-Hapak M, et al. Transduced p16INK4a peptides inhibit hypophosphorylation of the retinoblastoma protein and cell cycle progression prior to activation of Cdk2 complexes in late G1 [J]. Cancer Res, 1999,59:2577-2580.
[26]el-Deiry WS, Harper JW, O'Connor JM, et al. WAFI/CIP1 is induced in p53-mediated G1 arrest and apoptosis [J]. Cancer Res,1994,54:1169-1174.
[27]Bonfanti M, Taverna S, Salmona M, et al. p21 WAFT-derived peptides linked to an internalization peptide inhibit human cancer cell growth [J]. Cancer Res,1997, 57:1442-1446.
[28]Nagahara H, Vocero-Akbani AM, Snyder EL, et al.Transduction of full-length TAT fusion proteins into mammalian cells:TAT-p27 Kipl induces cell migration [J]. Nat Med,1998,4:1449-1452.
[29]Parada Y, Banerji L, Glassford J, et al. BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27 Kipl expression. J Biol Chem,2001,276:23572-23580.
[30]Huang LE, Gu J, Schau M,et al. Regulation of hypoxia-inducible factor 1 alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway [J]. Proc Natl Acad Sci USA,1998,95(14):7987-7992.
[31]Harada H, Hiraoka M, Kizaka-Kondoh S. Antitumor effect of TAT-oxygen-dep-endent degradation-caspase 3 fusion protein specifically stabilized and activated in hypoxic tumor cells [J]. Cancer Res,2002,62:2013-2018.
[32]Bryan R. Meade, Steven F. Dowdy. Enhancing the cellular uptake of siRNA duplexes followingnoncovalent packaging with protein transduction domain Peptides [J]. Adv Drug Deliv Rev,2008,60(4-5):530-536.
[33]Nakase I, Niwa M, Takeuchit,et al. Cellular uptake of arginine-rich peptides:roles for macropinocytosis and actin rearrangement [J]. Mol. Ther.2004,10(6): 1011-1022.
[34]Magzoub M, Pramanik A, Graslund A,et al.Modeling the endosomal escape of cell-penetrating peptides:transmembrane pH gradient driven translocation across phospholipid bilayers [J]. Biochemistry,2005,44(45):14890-14897.
[35]Skehel JJ, Cross K, Steinhauer D,et al. Influenza fusion peptides[J]. Biochem Soc Trans,2001,29(4):623-626.
[36]Michiue H, Tomizawa K, Wei FY, et al. The NH2 terminus of influenza virus hemagglutinin-2 subunit peptides enhances the antitumor potency of polyarginine-mediated p53 protein transduction[J]. J Biol Chem,2005,280(9): 8285-8289.
[37]Nadine K, Patricia M, Thorsten N,et al. A Membrane-Destabilizing Peptide in Capsid Protein L2 Is Required for Egress of Papillomavirus Genomes from Endosomes [J]. Journal of Virology,2006,80(2):759-768.
[38]Maiolo JR 3rd, Ottinger EA, Ferrer M,et al. Specific redistribution of cell-penetrating peptides from endosomes to the cytoplasm and nucleus upon laser illumination [J]. J Am Chem Soc.2004,126(47):15376-15377
[39]Wadia JS, Stan RV, Dowdy SF. Ansducible TAT HA fusogenic peptide enhances escape of TAT fusion proteins after lipid raft macropinocytosis [J]. Nat Med,2004, 10(3):310-315.
[40]Shiraishi T, Pankratova S, Nielsen PE. Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides [J]. Chem. Biol,2005,12(8):923-929.
[41]Kim D,Jeon C,Kim JH, et al.Cytoplasmic transduction peptide (CTP):new approach for the delivery of biomolecules into cytoplasm in vitro and in vivo [J]. Exp Cell Res,2006,312(8):1277-1288.
[42]Zhao K, Luo G, Giannelli S,et al. Mitochondria-targeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines [J]. Biochem Pharmacol.2005,70(12):1796-806.
[43]Snyder EL, Saenz CC, Denicourt C,et al. Enhanced targeting and killing of tumor cells expressing the CXC chemokine receptor 4 by transducible anticancer peptides[J].Cancer Res.2005,65(23):10646-10650.
[44]Ruoslahti E, Duza T, Zhang L.Vascular homing peptides with cell-penetrating properties [J].Curr Pharm Des.2005, 11(28):3655-3660.
[45]Robinson P, Stuber D, Deryckere F,et al. Identification using phage display of peptides promoting targeting and internalization into HPV-transformed cell lines [J]. J Mol Recognit.2005,18(2):175-182.