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1 生物工程学报 Chin J Biotech 2009, November 25; 25(11): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 医学与免疫生物技术 Surfactin MCF-7 曹小红, 王爱华, 王春玲, 鲁梅芳, 焦润芝, 朱慧, 赵思思, : 以体外培养的人乳腺癌细胞株 MCF-7 为研究对象, 探讨 surfactin 对肿瘤细胞增殖 凋亡及细胞骨架的影响 MTT 实验表明, surfactin 能抑制 MCF-7 的增殖, 并且呈现出浓度和时间的依赖关系, 作用 48 h 时的 IC 50 是 27.3 mol/l AO/EB 荧光染色法及流式细胞术检测发现, surfactin 可诱导 MCF-7 发生典型的凋亡形态学改变和 G 2 /M 期阻滞 免疫荧光和免疫印迹结果表明, surfactin 显著抑制了细胞内 vimentin 的表达, 诱导了 -tubulin 的解聚和重排, 使细胞的骨架系统发生了剧烈的变化 可见, surfactin 具有抑制 MCF-7 细胞增殖, 诱导细胞凋亡的作用, 其机制可能与细胞骨架蛋白的表达水平有关 : surfactin, 增殖, 凋亡, 细胞骨架 Effects of surfactin on proliferation, apoptosis and cytoskeleton in human breast cancer MCF-7 cells Xiaohong Cao, Aihua Wang, Chunling Wang, Meifang Lu, Runzhi Jiao, Hui Zhu, and Sisi Zhao College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin , China Abstract: We studied the effect of surfactin on cell proliferation, apoptosis and the cytoskeleton in human breast cancer cell line MCF-7 in vitro. The result of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) showed that the surfactin inhibited proliferation of MCF-7 cells in a dose- and time-dependent manner, with IC 50 at 48 h of 27.3 μmol/l. Surfactin-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes by AO/EB staining. Flow cytometric analysis also demonstrated that surfactin caused time-dependent apoptosis of MCF-7 cells through cell arrest at G 2 /M phase. Immunofluorescence and Western blotting showed that surfactin significantly suppressed the expression of vimentin, induced the α-tubulin depolymerization and rearrangement and then the skeleton system of the cells changed dramatically. Based on our findings, surfactin can significantly inhibit the growth of MCF-7 cells and induce apoptosis. Keywords: surfactin, proliferation, apoptosis, cytoskeleton, Received: July 21, 2009; Accepted: September 18, 2009 Supported by: National High Technology Research and Development Program of China (863 Program) (No. 2007AA10Z319). Corresponding author: Xiaohong Cao. Tel: ; Fax: ; wahwawayu@126.com 国家高技术研究发展计划 (863 计划 )(No. 2007AA10Z319) 资助

2 1706 ISSN CN /Q Chin J Biotech November 25, 2009 Vol.25 No.11 [1], ; : [2-3] ;, surfactin, surfactin, surfactin 1 材料与方法 1.1 材料 Bacillus natto TK-1( TK-1), MCF-7, MTT( -4, , 5- ) (PI) (AO) (EB) Sigma vimentin -tubulin FITC IgG Santa cruz 1.2 方法 surfactin HPLC [4] Bacillus natto TK D 15 surfactin MTT, /ml 96, 0.1 ml, 5 24 h, surfactin( 10 μmol/l 20 μmol/l 40 μmol/l 60 μmol/l 80 μmol/l 100 μmol/l), h, 4 h MTT, 4 h, DMSO 150 μl, 570 nm, % = OD / OD 100% (AO/EB ), / 50 ml, 24 h 27.3 μmol/l surfactin 24 h 48 h, PBS 2,, AO/EB 1,, 200, : (VN), ; (VA), ; (NVN), ; (NVA), 3, : (%) (VA+NVA)/(VN+NVN+VA+NVA) 100% (FCM), /ml, 24 h, MCF μmol/l, h,, 1000 r/min 5 min,, PBS, 1000 r/min 5 min, 1, 70% 4 o C 18 h,, PBS 2, 1.0 g/l A, min, 250 μl 75 μmol/l (PI), 4 o C 30 min,, 488 nm 630 nm Vimentin α-tubulin MCF-7, CO 2 24 h, 27.3 μmol/l surfactin : PBS 2, 3.7% 15 min, 0.1% Triton X min, 1% BSA-PBS 30 min, 4 o C ( anti-vimentin, anti- -tubulin) (1:100; 1% BSA-PBS ) PBS 3, 37 o C, FITC 1 h(1:100; 1% BSA-PBS ),

3 : Surfactin MCF Vimentin Kavallaris [5], 0.25% surfactin, PBS 2, 200 μl, 37 o C 10 min; r/min 10 min, 100 μl, 30 min, 1000 r/min 4 o C 5 min, Vimentin : SDS-PAGE,, 5% 30 min, 0.05% Tween20 PBS 3, ( anti-vimentin, anti- -tubulin), PBS 3, 1 h DAB -actin 2.2 荧光染色检测细胞凋亡 AO/EB ( 2a), surfactin 24 h ( 2b), AO,,, EB 48 h( 2c),, EB,, ( 1),, MCF-7,, P< 结果 2.1 Surfactin 对 MCF-7 人乳腺癌细胞增殖抑制作用 Surfactin MCF-7 1, Surfactin MCF-7,,, Surfactin 48 h IC μmol/l 图 1 Surfactin 对人乳腺癌细胞 MCF-7 的生长抑制作用 (MTT) Fig. 1 Growth inhibitory effect of Surfactin in MCF-7 human breast cancer cells (MTT). 图 2 AO/EB 染色法观察 MCF-7 细胞形态改变 Fig. 2 Morphological observation of MCF-7 cells using AO/EB staining. (a) Control. (b) 24 h. (c) 48 h. 表 1 MCF-7 细胞经 27.3 μmol/l surfactin 作用后细胞凋亡计数 Table 1 Effect of surfactin on the average apoptotic rate of MCF-7 cells Time (h) Cell numbers VA NVA VN NVN Amount Apoptotic cells (%) % % ** % ** Note: ** P< FCM 检测细胞周期阻断与凋亡结果 PI ( 3), 27.3 μmol/l surfactin MCF h, G 2 /M (9.2±2.4)% (42.7±1.9)%, (2.24±0.96)% (13.2±2.3)%,, surfactin MCF-7 G 2 /M, surfactin

4 1708 ISSN CN /Q Chin J Biotech November 25, 2009 Vol.25 No.11 图 3 Fig. 3 Surfactin 对 MCF-7 细胞周期分布的影响 Effect of cell cycle change of MCF-7 cell lines by surfactin. 2.4 Surfactin 对 MCF-7 细胞 vimentin 表达的影响 27.3 μmol/l surfactin MCF h, vimentin, Image Pro-Plus 6.0 (OD) ( 4, 5): surfactin vimentin( 4a), surfactin 24 h( 4b), 84.50% 73.25%, P<0.05, 48 h( 4c) 72 h( 4d), vimentin 40.00% 15.50%, P<0.01 ( 6), vimentin, surfactin h, vimentin 71.7% 66.2% 33.7% surfactin vimentin,, 2.5 免疫荧光观察 surfactin 作用后 MCF-7 细胞微管的变化, MCF-7, ( 7a) 27.3 μmol/l surfactin 12 h, ( 7b),, ( 7c) 48 h ( 7d),,,,,, tubulin

5 曹小红等: Surfactin 对人乳腺癌 MCF-7 细胞增殖 凋亡及细胞骨架的影响 图4 Surfactin 对 MCF-7 乳腺癌细胞 vimentin 表达的影 图 Surfactin 对 MCF-7 乳腺癌细胞 α-tubulin 表达的 响 影响 Fig. 4 Effect of surfactin on expression of vimentin protein in MCF-7 human breast cancer cells. (a) Control. (b) 24 h. (c) 48 h; (d) 72 h. Fig. 7 Effect of surfactin on expression of α-tubulin protein in MCF-7 human breast cancer cells. (a) Control. (b) 12 h. (c) 24 h. (d) 48 h. 图 8 蛋白免疫印迹分析 surfactin 对聚合微管蛋白表达 的影响 Fig. 8 Effect of surfactin on expression of polymeric tubulin protein by Western blotting analysis. 24.3% 结果表明, surfactin 抑制了细胞内微管蛋白 图5 Surfactin 作用不同时间后 vimentin 蛋白平均光密 度值变化 Fig. 5 Changes in average optical density of vimentin at different time after surfactin treatment (*P<0.05, **P<0.01). 的聚合, 并且随着作用时间的延长, 其对细胞内微 管蛋白聚合的抑制作用呈增加趋势 3 讨论 寻找化学结构新颖 作用机制独特的新药是新 抗肿瘤药研究的焦点 由于通过化学合成途径生产 抗癌药物步骤多 收率低 污染大, 人们已把注意 图6 蛋白免疫印迹分析 surfactin 对 vimentin 表达的影 响 Fig. 6 Effect of surfactin on expression of vimentin by Western blotting analysis. 力转移到微生物上, 现已发现许多微生物的提取 物 次级代谢产物具有抗肿瘤作用 本实验通过 MTT 发现 surfactin 对 MCF-7 细胞表现出较强的抑制作用, 其 中 细 胞 被 surfactin 处 理 48 h 时 的 IC50 是 μmol/l 诱导细胞凋亡是许多抗肿瘤药物发挥 聚合微管免疫印迹分析 免疫印迹结果显示(图 8), 随着 surfactin 作用时 作用的重要手段, 在本实验中通过 AO/EB 荧光双染 间的延长, 聚合的微管蛋白比例逐渐下降, 这与免 法观察到了凋亡的特征性改变, 流式细胞仪分析观 疫荧光观察到细胞内的微管网络稀疏结果一致 与 察到经 surfactin 处理的 MCF-7 细胞出现了亚二倍体 对照组相比, surfactin 作用 h 后, 聚合微 峰和 G2/M 期阻滞, 这都说明 surfactin 能够诱导 管蛋白的相对光密度值分别降至 71.4% 38.9%和 MCF-7 细胞产生凋亡

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