商陆皂苷甲的联用可显著提高tApoptin凋亡蛋白的抗肿瘤活性  

The Combination of Esculentoside A Can Significantly Improve the Anti-tumor Activity of tApoptin

作　　者：李柳美 曹雪玮 王晓旦 王富军 赵健[1] LI Liu-Mei;CAO Xue-Wei;WANG Xiao-Dan;WANG Fu-Jun;ZHAO Jian(State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China;Zhejiang FONOW Medicine Co.Ltd,Dongyang 322100,Zhejiang,China;)Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]华东理工大学生物反应器工程重点实验室,上海200237 [2]浙江孚诺医药股份有限公司,浙江东阳322100 [3]上海中医药大学中药研究所,上海201203

出　　处：《中国生物化学与分子生物学报》2019年第10期1098-1107,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No 81571795)资助~~

摘　　要：凋亡蛋白(apoptin)因可特异性诱导肿瘤细胞和转化细胞凋亡而备受广大研究人员的关注。但由于凋亡蛋白质本身结构特性等方面的原因,导致通过原核表达的蛋白质不稳定,很容易聚集沉淀,并且凋亡蛋白质本身也不能自主跨膜进入胞内发挥其生物学作用。本文考察了一种野生型凋亡蛋白N-末端缺失突变体tApoptin(1~43氨基酸残基缺失)在大肠杆菌中以可溶性形式表达,并且通过荧光显微镜和共聚焦显微镜观察到,这种N-端缺失突变体tApoptin与绿色荧光蛋白(green fluorescent protein,EGFP)的融合蛋白质具有自主进入A549、HeLa、H460和SK-OV-3等多种肿瘤细胞的能力,而且这种转运效率具有细胞特异性。与肝素钠共孵育可抑制tApoptin进入细胞的能力,推测tApoptin可能是通过与细胞表面硫酸乙酰肝素多糖结合后被内吞进入细胞。MTT的结果显示,突变体tApoptin依然保持了全长凋亡蛋白质的抗肿瘤特性,对所测试的多种肿瘤细胞生长都具有不同程度的抑制作用。与tApoptin给药组相比,tApoptin与传统中药材来源的商陆皂苷甲(esculentoside,EsA)联用的给药组,对测试的4种肿瘤细胞的药效提升约100倍以上。其中,对SK-OV-3细胞的提升效果最显著,提升463倍,半抑制浓度(half maximal inhibitory concentration,IC50)从27.37±2.25μmol/L降低到0.059±0.003μmol/L。流式细胞术分析表明,与tApoptin给药组相比,tApoptin与EsA联合的给药组细胞凋亡率显著增加(6.46±0.34%vs.41.9±0.47%,P<0.001)。与溶酶体共定位的共聚焦结果表明,EsA可能通过破坏细胞中的溶酶体促使tApoptin释放到胞质内有效发挥其药理作用。Apoptin has attracted much more interests due to its ability to selectively induce apoptosis transformed and tumor cells but not in normal cells.However,due to the structural tendency of apoptin expressed in prokaryotic systems are unstable and easy to aggregate and precipitate,and the cell-impermeable property of apoptin restrict its therapeutic application.Here,we described the construction of a truncated variant of apoptin,named as tApoptin(lack of residues 1 to 43),which can be expressed in E.coli in a soluble form.In this study,we first observed that green fluorescent protein(EGFP)fused to tApoptin(named as EGFP-tApoptin)could autonomously and selectively enter A549,HeLa,H460 and SK-OV-3 cells according to the results obtained by confocal microscopy.Heparin sodium significantly inhibited the transmembrane ability of EGFP-tApoptin,demonstrating that heparin sulfate on the cell membrane is involved in the intracellular delivery of EGFP-tApoptin.Furthermore,MTT assay results showed that the mutant tApoptin retained partial anti-tumor activity of full-length Apoptin,but the inhibition of the growth of various tumor cells tested was not particularly significant.According to the results of confocal microscopy,most of tApoptin transported into the cell subsequently entered lysosomes,which may hinder the cytotoxicity of tApoptin.Therefore,esculentoside A(EsA),originated from a kind of Chinese traditional herb,was co-administrated with tApoptin to improve the cytotoxicity of tApoptin.Co-administration with EsA enhanced the inhibition of tApoptin on four test tumor cells by more than 100-fold.Especially,the enhancement was about 463-fold in SK-OV-3 cells,decreasing the 50%inhibitory concentration(IC50)from 27.37±2.25μmol/L to 0.059±0.003μmol/L.Additionally,flow cytometry analysis showed that EsA could significantly enhance the ability of tApoptin to induce cell apoptosis(tApoptin alone:6.46±0.34%vs.tApoptin with EsA:41.9±0.47%,P<0.001).Consequently,this study constructed a novel mutant tApoptin which can be expr

关 键 词：凋亡蛋白突变体 商陆皂苷甲 抗肿瘤 药物运输 

分 类 号：Q71[生物学—分子生物学]