毛蚶抗癌蛋白对人乳腺癌细胞MCF-7的生长抑制作用

Inhibition effects of Arca subcrenata Lischke anticancer protein on human breast carcinoma MCF-7 cells

目的:从毛蚶软体部位中分离出具有一定抗癌活性的毛蚶蛋白组分,研究其对人乳腺癌细胞MCF-7的生长抑制作用。方法:以新鲜毛蚶软体部分为原材料,分离获得毛蚶抗癌蛋白组分(命名为NS);倒置相差显微镜观察不同浓度(50、100、200、400μg/mL)NS组分对MCF-7癌细胞生长的影响,将200μg/mL NS组分作用MCF-7细胞不同时间(12、24、48 h)后,Giemsa染色观察癌细胞及核形态的变化,流式细胞术检测NS组分对MCF-7细胞周期及细胞凋亡的影响,Western blot检测细胞周期及细胞凋亡相关蛋白的表达。结果:与阴性对照组比较,各浓度NS组分对人乳腺癌MCF-7细胞生长均具有明显的抑制作用(P<0.05或P<0.01);倒置相差显微镜下可见200μg/mL NS组分作用MCF-7细胞12 h后,部分细胞收缩变圆、脱落,24、48 h后细胞脱落现象更加明显;Giemsa染色发现,NS组分处理12 h后细胞出现有丝分裂相增加,继而出现多核或核碎裂等现象;流式细胞术检测发现NS组分主要引起细胞G2-M期阻滞;NS处理组凋亡率均较对照组明显升高(P均<0.01),且随着药物作用时间的延长,细胞凋亡比例逐渐增加。Western blot检测发现NS组分作用MCF-7细胞后p53及procaspase-3蛋白表达量均上调。结论:毛蚶抗癌蛋白NS组分体外对人乳腺癌MCF-7细胞的细胞毒作用主要通过引起细胞发生G2-M期阻滞、诱导细胞凋亡实现的,期间伴随p53及procaspase-3蛋白表达上调。

OBJECTIVE：To separate and extract Arca subcrenata Lischke protein component and investigate its anticancer mechanisms on human breast carcinoma MCF-7 cells in vitro.METHODS：Fresh Arca Subcrenata Lischke was used as the raw material to extract its anticancer protein component which was named as NS.The growing changes of cultured MCF-7 cells treated with different concentrations of NS（50,100,200,400 μg/mL） were observed under the inverted phase-contrast microscope.The cell and nucleus changes of MCF-7 cells treated with NS 200 μg/mL were analyzed by Giemsa staining.Flow cytometry was used to evaluate apoptosis and cell cycle of MCF-7 cells treated with NS.The expressions of apoptosis- and cell cycle-related proteins were analyzed by Western blot.RESULTS：Comparing with negative control group,obvious growth inhibition effects were found in MCF-7 cells treated with different concentrations of NS（50,100,200,400 μg/mL）（P〈0.05 or P〈0.01）.The appearance of cultured MCF-7 cells treated with NS 200 μg/ml,for 12,24 and 48 h were shrinking and falling off from the tissue culture flask in a time-dependent manner after treated with NS for 12 h.The percentage of MCF-7 cells in mitosis phase and the multinuclear or nuclear fragmentation cells were obviously increased.Flow cytometry analysis indicated that apoptotic cell increased and G2-M phase cells accumulated significantly with NS（P〈0.01）.The apoptosis rate of MCF-7 cells increased in a time- dependent manner.The expression of apoptotic protein procaspase-3 and p53 protein were up-regulated after NS treatment for 6 h.CONCLUSION：Inducing apoptosis and cell cycle arrest in G2-M phase were main mechanisms of Arca subcrenata Lischke anticancer protein component NS cytotoxic activity,and procaspase-3 and p53 signal pathways were upregulated in MCF-7 cells.