青蛤多肽的酶法制备及对前列腺癌DU-145细胞的抑制活性

Enzymatic Preparation of Peptide from Cyclina sinensis Proteins and Its Inhibitory Activity toward Prostate Cancer DU-145 Cells

探讨青蛤多肽的制备工艺及体外抗前列腺癌DU-145细胞的活性。以氨基氮含量为检测指标,筛选最优酶种类并进行正交试验以获取最佳酶解青蛤内脏酶解工艺。经超滤截留、琼脂糖凝胶层析、高效液相色谱分离纯化及噻唑蓝(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT)活性筛选得到含有5个氨基酸的多肽(Ile-Leu-Tyr-Met-Pro)。对所得多肽采用MTT法测定DU-145细胞增殖抑制率;采用倒置显微镜、Hoechst 33258染色及透射电子显微镜技术观察细胞形态学变化;采用荧光法检测细胞内活性氧(reactive oxygen species,ROS)水平;采用线粒体膜电位检测DU-145细胞凋亡情况;采用免疫组织化学法检测非转移性克隆23型(nm23H1)蛋白的表达。结果表明:制备青蛤多肽最佳酶是木瓜蛋白酶,最佳酶解条件是料液比1:4、pH 7.0、加酶量1 500 U/g、温度45.0℃、酶解时间4 h;所得多肽对DU-145细胞的增殖具有明显的抑制作用,且呈时间与剂量依赖性;处理后的DU-145细胞出现凋亡的形态学特征;其质量浓度和细胞内ROS的表达量呈正相关关系;线粒体膜电位降低率从4.22%提高到25.07%;免疫组织化学法结果显示nm23H1蛋白的表达量增加。青蛤多肽能明显抑制DU-145细胞的增殖,并通过诱导其发生凋亡而发挥作用。

The purpose of this study was to investigate the enzymatic preparation of peptide from Cyclina sinensis proteins and to evaluate its activity against prostate cancer DU-145 cells. Different proteases were screened to increase the amino nitrogen content of hydrolysates and the hydrolysis conditions were optimized using orthogonal array experiments. An active peptide was purified from the hydrolysates through ultrafiltration, gel chromatography and preparative high performance liquid chromatography. The amino acid sequence of the purified peptide was identified as Ile-Leu-Tyr-Met-Pro. The antiproliferative activity of this peptide against DU-145 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and the morphologic change of DU-145 cells was observed by using an inverted microscope, Hoechst 33258 staining and transmission electron microscopy. A fluorescence method was used to detect the generation of intracellular reactive oxygen species(ROS). Cell apoptosis was evaluated by determination of mitochondrial membrane potential using flow cytometry. Furthermore, the expression of nm23H1 was detected by an immunohistochemical method. Our results showed that papain was the optimal protease for hydrolyzing Cyclina sinensis proteins and the optimum hydrolysis conditions were determined as follows: solid-to-solvent ratio 1:4, pH 7.0, enzyme dosage 1 500 U/g, temperature 45 ℃ and hydrolysis time 4 h. The purified peptide had a significant inhibitory effect on DU-145 cells in a time-and dosedependent manner. The treated cells exhibited apoptotic morphological characteristics. The concentration of the peptide was positively correlated with the generation of ROS in cells. Flow cytometry revealed that the percentage of cells with decreased mitochondrial membrane potential was increased from 4.22% to 25.07%. Immunohistochemical results indicated that the expression of nm23H1 was increased. Therefore, the enzymatic peptide from Cyclina sinensis could significantly inhibit the proliferation of DU-145 cells by inducing apoptosis.