中国皖南尖吻蝮蛇毒中细胞毒素的分离纯化和抗癌活性研究

中国皖南尖吻蝮蛇毒中细胞毒素的分离纯化和抗癌活性研究

章良, 李虹, 吴梧桐^*

中国药科大学生物制药学院, 南京 210009

收稿日期:2003-12-25 修回日期:2004-05-10 出版日期:2004-06-15 发布日期:2004-06-15
通讯作者: 吴梧桐*

Purification and Characterization of Cytotoxins from Agkistrodon acutus Venom and Their Anticancer Activity

ZHANG Liang, LI Hong, WU Wu-tong^*

School of Biopharmaceutics, China Pharmaceutical University, Nanjing 210009, China

Received:2003-12-25 Revised:2004-05-10 Online:2004-06-15 Published:2004-06-15
Contact: WU Wu-tong*

摘要/Abstract

摘要： 目的　寻找皖南尖吻蝮蛇毒中的抗癌组分并初步研究其性质。方法　以DEAE-SepharoseFF, SOURCE-30S柱层析纯化粗毒; 以MTT法检测细胞毒活性; 以SDS-PAGE(银染)测定纯度; 以还原性及非还原SDS PAGE测定分子量; 又测定了它们的热稳定性和pH稳定性。结果　得到两个电泳纯的蛇毒类抗癌组分, 分别命名为ACTX-6和ACTX-8, 分子量为98 kDa和27 kDa, 都不是糖蛋白, 其中ACTX-6由两个分子量相近的亚单位以二硫键连接而成; 它们对肺癌细胞A549最敏感; ACTX-6对热稳定而ACTX-8对热不稳定; ACTX-6的稳定pH范围为7- 9, 而ACTX-8在6 - 9的范围内活性都比较稳定。

关键词: 尖吻蝮蛇, 尖吻蝮蛇, 尖吻蝮蛇, 蛇毒, 蛇毒, 蛇毒, 抗癌活性, 抗癌活性, 抗癌活性, 细胞毒素, 细胞毒素, 细胞毒素, 柱层析, 柱层析, 柱层析

Abstract: Aim To investigate the anticancer activity of two new cytotoxins from the venom of Agkistrodon acutus. Methods The venom was isolated by FPLC column chromatography consisting of DEAE Sepharose FF and Source 30S. The cytotoxic activity on tumor cells was detected by MTT method. Purity and molecular weight were determined by SDS-PAGE (silver staining). Their stabilities to temperature and pH were also detected. Results Two pure cytotoxins named ACTX-6 and ACTX-8 were obtained. Their molecular weights are 98 kDa and 27 kDa, respectively. ACTX-6 consists of two subunits bonded together by disulfide bonds. Conclusion ACTX-6 and ATCX-8 have highest inhibitory activity on lung cancer cell A549. ACTX-6 is stable to heat while ACTX-8 not. ACTX-6 is stable between pH 7-9 and ACTX-8 between pH 6-9.

Key words: Agkistrodon acutus, Agkistrodon acutus, snake venom, snake venom, anticancer activity, anticancer activity, cytotoxin, cytotoxin, column chromatography, column chromatography

中图分类号:

R963

R996.4

Supporting: Foundation item: National Natural Science Fundation of China (30171103).
^*Corresponding author. Tel.: 025-3220372; fax: 025-3220372

章良, 李虹, 吴梧桐^*. 中国皖南尖吻蝮蛇毒中细胞毒素的分离纯化和抗癌活性研究[J]. .

ZHANG Liang, LI Hong, WU Wu-tong^*. Purification and Characterization of Cytotoxins from Agkistrodon acutus Venom and Their Anticancer Activity[J]. .

参考文献

[1] Salvini TF, Belluzzo SS, Selistre de Araujo HS, et al. Regeneration and change of muscle fiber types after injury induced by a hemorrhagic fraction isolated from Agkistrodon contortrix laticinctus venom [J]. Toxicon, 2001, 39 (5): 641-649.
[2] Tani A, Ogawa T, Nose T, et al. Characterization, primary structure and molecular evolution of anticoagulant protein from Agkistrodon actus venom [J]. Toxicon, 2002, 40 (6): 803-813.
[3] Chaim-Matyas A, Ovadian M. Cytotoxic activity of various snake venoms on melanoma, B16F10 and chondrosarcoma [J]. Life Sci, 1987, 40 (16): 1601-1607.
[4] Scudiero DA, Shoemaker RH, Paull KD, et al. Evaluation of a soluble tetrazolium formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines [J]. Cancer Res, 1988, 48: 4827-4833.
[5] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 [J]. Nature, 1970, 227: 680-685.
[6] Swain M, Ross NW. A silver stain protocol for proteins yielding high resolution and transparent background in sodium dodecyl sulfate-polyacrylamide gels [J]. Electrophoresis, 1995, (16): 948-951.
[7] Andrews P. The gel-filtration behaviors of proteins related to their molecular weights over a wide range [J]. Biochem J, 1965, 96: 595-606.
[8] Young RL, Lowry OH. Quantitative methods for measuring the histochemical distribution of alanine, glutamate and glutamine in brain [J]. J Neurochem, 1966, 13 (9): 785-793.
[9] Higerd TB, Spizizen J. Isolation of two acetyl esterases from extracts of Bacillus subtilis [J]. J Bacteriol, 1973, 114(3): 1184-1192.
[10] Guo YJ. The experimental technology of protein electrophoresis [M]. Beijing: Science Publishing House, 2001, 151-153.
[11] Hou Q, Sherwin RP, Parrish C, et al. Contortrostatin, a dimeric disintegrin from Agkistrodon contortrix contortrix, inhibits breast cancer progression [J]. Breast Cancer Res Treat, 2000, 61 (3): 249-260.
[12] Wang LL, Tian L, Ma X, et al. Comparison of the hemorrhagic toxicity experiment methods using snake venom enzyme preparation [J]. J Snake, 1995, 7 (1): 4-6.
[13] Zaheer A, Braganca BM. Comparative study of three basic polypeptides from snake venoms in relation to their effects on the cell membrane of normal and tumor cells [J]. Cancer Biochem Biophys, 1980, 5 (1): 41-46.
[14] Zhang Y, Qing N, Gao R, et al. The study of biological effects of membrane Toxin-I from the venom of cobra (Naja naja atra) [J]. J Wuhan Univ (Nat Sci Ed), 1999, 45 (2): 195-199.