C-phycocyanin (C-PC) pigment, as a natural blue dye, has particular applications in various fields. It is a water-soluble protein which has anticancer, antioxidant and anti-inflammatory properties. Here, we introduce an efficient procedure for the purification of C-PC pigment, followed by conducting a comprehensive investigation of its cytotoxic effects on human breast cancer (MCF-7) cells and the underlying mechanisms. A novel four-step purification procedure including the adsorption of impurities with chitosan, activated charcoal, ammonium sulfate precipitation, and ion exchange chromatography was employed, achieving a high purity form of C-PC with purity index (PI) of 5.26. SDS-PAGE analysis showed the purified C-PC with two discrete bands, subunit α (17 kD) and β (20 kD), as confirmed its identity by Native-PAGE. A highly purified C-PC was employed to evaluate its anticancer activity and underlying molecular mechanisms of action. The inhibitory effects of highly purified C-PC on the proliferation of human breast cancer cells (MCF-7) have detected by MTT assay. The IC50 values for 24, 48, and 72 hours of exposure to C-PC were determined to be 5.92, 5.66, and 4.52 μg/μl, respectively. Flow cytometric analysis of cells treated with C-PC, by Annexin V/PI double staining, demonstrated to induce MCF-7 cells apoptosis. Also, the results obtained from propidium iodide (PI) staining showed that MCF-7 cells treated with 5.92 μg/μl C-PC for 24 h would arrest at the G2 phase and 5.66 and 4.52 μg/μl C-PC for 48 and 72 h could induce cell cycle arrest at both G2 and S phases. The oxidative damage and mitochondrial dysfunction were evaluated to determine the possible pathways involved in C-PC-induced apoptosis in MCF-7 cells. Our findings clearly indicated that the treatment of MCF-7 cells with C-PC (IC50 for 24 h) increased the production of reactive oxygen species (ROS). Consequently, an increase in the lipid peroxidation (LPO) level and a reduction in the ATP level, mitochondrial membrane potential (MMP), glutathione (GSH) and its oxidized form (GSSG), occurred over time. The reduced expression levels of anti-apoptotic proteins, Bcl2 and Stat3, plus cell cycle regulator protein, Cyclin D1, using Real-Time PCR confirm that the C-PC-induced death of MCF-7 human breast cancer cells occurred through the mitochondrial pathway of apoptosis. Collectively, the analyses presented here suggest that C-PC has the potential so that to develop it as a chemotherapeutic anticancer drug.

作为天然蓝色染料的C-藻蓝蛋白（C-PC）颜料在各个领域具有特殊的应用。它是一种具有抗癌，抗氧化和抗炎特性的水溶性蛋白质。在这里，我们介绍了一种纯化C-PC色素的有效程序，然后对其对人乳腺癌（MCF-7）细胞的细胞毒性作用及其潜在机制进行了全面研究。采用一种新颖的四步纯化程序，包括用壳聚糖吸附杂质，活性炭，硫酸铵沉淀和离子交换色谱，从而获得了纯度为5.26的高纯度C-PC形式。SDS-PAGE分析显示纯化的C-PC具有两个离散的带，亚基α（17 kD）和β（20 kD），已通过Native-PAGE确认了其身份。使用高度纯化的C-PC评估其抗癌活性和潜在的分子作用机理。通过MTT分析已经检测到高纯度的C-PC对人乳腺癌细胞（MCF-7）增殖的抑制作用。确定C，PC暴露24、48和72小时的IC50值分别为5.92、5.66和4.52μg/μl。通过膜联蛋白V / PI双重染色，对经C-PC处理的细胞进行流式细胞术分析，证明其可诱导MCF-7细胞凋亡。此外，从碘化丙啶（PI）染色获得的结果表明，用5.92μg/μlC-PC处理24 h的MCF-7细胞将在G2期停滞，而在5.66和4.52μg/μlC-PC的作用下将停滞于48和72 h可以诱导G2和S期的细胞周期停滞。评估氧化损伤和线粒体功能障碍，以确定可能的途径参与C-PC诱导MCF-7细胞凋亡。我们的发现清楚地表明，用C-PC处理MCF-7细胞（IC50 24小时）可增加活性氧（ROS）的产生。因此，随着时间的流逝，脂质过氧化（LPO）水平升高，ATP水平，线粒体膜电位（MMP），谷胱甘肽（GSH）及其氧化形式（GSSG）降低。抗凋亡蛋白的表达水平降低，随着时间的推移，脂质过氧化（LPO）水平增加，ATP水平，线粒体膜电位（MMP），谷胱甘肽（GSH）及其氧化形式（GSSG）降低。抗凋亡蛋白的表达水平降低，随着时间的推移，脂质过氧化（LPO）水平增加，ATP水平，线粒体膜电位（MMP），谷胱甘肽（GSH）及其氧化形式（GSSG）降低。抗凋亡蛋白的表达水平降低，Bcl2和Stat3，加上细胞周期调节蛋白Cyclin D1，使用实时PCR证实了C-PC诱导的MCF-7人乳腺癌细胞死亡是通过线粒体凋亡途径发生的。总体而言，此处介绍的分析表明C-PC具有潜力，因此有可能将其开发为化疗抗癌药。