重组海蛇碱性磷脂酶A_2的抗肿瘤活性与相对酶活性的关系  

Correlation of Antitumor Effect of Recombinant Sea Snake Basic Phospholipase A_2 to Its Enzymatic Activity

作　　者：梁永钜[1,2] 杨小平[1,2] 卫剑文[3] 符立梧[1,2] 姜孝玉[3] 陈尚武[3] 杨文利[3] 

机构地区：[1]华南肿瘤学国家重点实验室 [2]中山大学肿瘤防治中心肿瘤研究所,广东广州510060 [3]中山大学生命科学学院生化系海洋生物功能基因组开放实验室

出　　处：《癌症》2005年第12期1474-1478,共5页Chinese Journal of Cancer

基　　金：国家自然科学基金(No.30200049)~~

摘　　要：背景与目的:蛇毒磷脂酶A2(snakevenomphospholipaseA2,SVPLA2)是一类分子量为14ku左右的蛋白家族,除了酶活性,该蛋白还具有多种药理活性。迄今,陆地蛇PLA2的结构与功能关系特别是其酶活性与药理活性的关系已被广泛研究,但对海蛇PLA2的研究则较少。本文旨在研究重组平颏海蛇碱性磷脂酶A2(recombinantseasnakebasicphospholipaseA2,rSSBPLA2)及其突变体(rN48和rK49)体内外抗肿瘤作用,并探讨酶活性相关位点对抑瘤作用的影响。方法:对酶活性相关位点48、49位氨基酸进行His→Asn和Asp→Lys的定点突变,用MTT法检测rSSBPLA2及其突变体rN48和rK49对人原髓白血病细胞HL-60、人神经母细胞瘤细胞SK-N-SH、人胃癌细胞MGC-803和人肝癌细胞HepG2的生长抑制作用;用小鼠S180移植瘤和艾氏腹水癌(EAC)模型对rSSBPLA2进行抗肿瘤作用观察;并检测突变体对小鼠移植瘤的抗瘤活性。结果:与rSSBPLA2相比较,突变体rN48和rK49的相对酶活力分别为0和5%。rSSBPLA2对HL-60细胞、SK-N-SH细胞和MGC-803细胞的IC50值分别为(45.28±0.09)μg/ml、(57.07±0.12)μg/ml和(69.34±0.35)μg/ml,对HepG2细胞生长没有抑制作用;而rN48和rK49对以上细胞均无抑制作用。rSSBPLA2对小鼠S180移植瘤具有明显的抑制作用,按2mg/kg(qd×10)、2mg/kg(q2d×5)、4mg/kg(qd×1)、4mg/kg(q5d×2)实验给药,其抑瘤率分别为50.8%、43.2%、38.2%、55.5%;rSSBPLA2按4mg/kg(q5d×2)剂量给药时,对EAC抑瘤率为33.5%,与对照组比较均有显著性差异(P<0.01)。而突变体rN48和rK49分别按4mg/kg(q2d×5)和7mg/kg(qd×10)方案进行实验,无明显抗肿瘤作用。结论:rSSBPLA2的抑瘤活性可能与其酶活性密切相关。BACKGROUND ＆ OBJECTIVE：Snake venom phospolipase A2 （PLA2）, a large family of homologous （14 ku） soluble proteins, exerts diverse pharmacologic activities as well as enzymatic activities. So far, the structure and function of terrestrial snake PLA2, especially the relationship of its enzymatic and pharmacologic activities have been studied extensively, but the investigation of sea snake PLA2 are limited. This study was to investigate the in vitro and in vivo antitumor effects of recombinant sea snake basic PLA2 （rSSBPLA2） and its mutants rN48 and rK4 from sea snake Lapemis hardwickii venom, and to explore the influence of 2 residues related with the enzymatic activity on the antitumor effects. METHODS： Site-directed mutagenesis of the 2 conserved residues related with enzymatic activity （His48 mutated to Asn and Asp49 mutated to Lys） was performed. The inhibitory effects of rSSBPLA2, rN48 and rK49 on proliferation of human myeloid leukemia cell line HL-60, human neuroblastoma cell line SK-N-SH, human gastric cancer cell line MGC-803, and human liver cancer cell line HepG2 were assessed by MTT assay. Their antitumor effects on sarcoma cell line S180 xenograft and EAC ascites cancer model in mice were detected. RESULTS： The relative enzymatic activities of rN48 and rK49 were 0 and 5% of that of rSSBPLA2. The 50% inhibitory concentration （IC50） of rSSBPLA2 for HL60, SK-N-SH, and MGC-803 cells were （45.28±0.09） μg/ml, （57.07±0.12） μg/ml, and （69.34± 0.35） μg/ml, respectively, but it had no inhibitory effect on proliferation of HepG2 cells, rSSBPLA2 obviously inhibited growth of S180 xenograft in mice; the inhibitory rates were 50.8%, 43.2%, 38.2%, and 55.5%, respectively, under the dose of 2 mg/kg （qd×10）, 2 mg/kg （q2d×5）, 4 mg/kg （qd×1） and 4 mg/kg （q5d×2）. The inhibitory rate of EAC model was 33.5% under the dose of 4 mg/kg （q5d×2）. The inhibitory rates were significantly higher in test groups than in control groups （P〈0.01）. rN48 and rK49

关 键 词：重组海蛇碱性磷脂酶A2 抗肿瘤活性 酶活性 S180肉瘤 定点突变 

分 类 号：R73-36[医药卫生—肿瘤]