School of Food Science and Pharmacy of Zhejiang Ocean University, Zhejiang Provincial Key Engineering Technology Research Center of Biomedical Products, Zhoushan 316022, China.
Zhejiang Fisheries Research Laboratory, Zhoushan 316021, China.
Mar Drugs. 2018 Apr 12;16(4):125. doi: 10.3390/md16040125.
anti-tumor peptide (AAP-H) is a pentapeptide from the sea anemone with an amino acid sequence of Tyr-Val-Pro-Gly-Pro that is obtained by alkaline protease enzymatic hydrolysis extraction. In this study, we investigated the inhibitory effects of AAP-H on prostate cancer DU-145 cell proliferation using a methylthiazolyldiphenyl-tetrazolium bromide assay. Cell morphology was analyzed by hematoxylin-eosin staining, acridine orange/ethidium bromide fluorescence staining, Hoechst 33258 fluorescence staining, and scanning electron microscopy. The mitochondrial membrane potential was determined by flow cytometry following JC-1 staining. The cell apoptosis rate was measured by Annexin V-fluorescein isothiocyanate and propidium iodide staining followed by flow cytometric analysis, and the expression of apoptosis-associated proteins was assayed by Western blotting. The results demonstrated that AAP-H induced significant reductions in the number of viable cells and increased cell death in both a dose-dependent and time-dependent manner, with an IC of approximately 9.605 mM, 7.910 mM, and 2.298 mM at 24 h, 48 h, and 72 h, respectively. The morphologic characteristics of apoptotic cells were observed after treatment with AAP-H. The mitochondrial membrane potential was markedly decreased, and apoptosis increased after AAP-H treatment. Pro-apoptotic proteins, such as Bax, cytochrome-C, caspase-3, and caspase-9 were increased, but Bcl-2 was decreased. These findings suggest that AAP-H has moderate inhibitory effects on prostate cancer DU-145 cells, and the mechanism might involve the mitochondria-mediated apoptotic pathway. Therefore, AAP-H is a candidate anti-prostate cancer drug or health-care food.
抗肿瘤肽（AAP-H）是一种来源于海葵的五肽，其氨基酸序列为 Tyr-Val-Pro-Gly-Pro，通过碱性蛋白酶酶解提取获得。在本研究中，我们使用噻唑蓝比色法研究了 AAP-H 对前列腺癌细胞 DU-145 增殖的抑制作用。通过苏木精-伊红染色、吖啶橙/溴化乙锭荧光染色、Hoechst 33258 荧光染色和扫描电子显微镜观察细胞形态。通过 JC-1 染色后流式细胞术测定线粒体膜电位。通过 Annexin V-异硫氰酸荧光素和碘化丙啶染色后流式细胞术分析测定细胞凋亡率，并通过 Western blot 测定凋亡相关蛋白的表达。结果表明，AAP-H 以剂量和时间依赖的方式显著降低活细胞数量并增加细胞死亡，在 24 h、48 h 和 72 h 时，IC 分别约为 9.605 mM、7.910 mM 和 2.298 mM。用 AAP-H 处理后观察到凋亡细胞的形态特征。线粒体膜电位明显降低，AAP-H 处理后凋亡增加。促凋亡蛋白如 Bax、细胞色素 C、caspase-3 和 caspase-9 增加，而 Bcl-2 减少。这些发现表明 AAP-H 对前列腺癌细胞 DU-145 具有中等抑制作用，其机制可能涉及线粒体介导的凋亡途径。因此，AAP-H 是一种候选的抗前列腺癌药物或保健品。
