Abstract
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.
Highlights
Cyanobacteria formerly known as blue-green algae constitute a phylum of the bacterial domain which has the ability to acquire their energy by oxygenic photosynthesis
Since the increase in reactive oxygen species (ROS) production can lead to cell membrane damage through the chain reaction mechanism of oxidative degradation of lipids, we examined the level of lipid peroxidation (LPO) in MCF-7 cells after incubation with C-PC, at particular IC50 concentrations, in corresponding exposure time of 24, 48 and 72 h
Since mitochondria play a crucial role in activating apoptosis in mammalian cells, we examined the fundamental parameters involved in mitochondrial impairment including ROS production, the collapse of mitochondrial membrane potential (MMP), lipid peroxidation (LPO), ATP level and the GSH and GSSG contents to divulge the possible chain reactions responsible for mitochondrial apoptosis pathway
Summary
Cyanobacteria formerly known as blue-green algae constitute a phylum of the bacterial domain which has the ability to acquire their energy by oxygenic photosynthesis They use special pigments to capture the light energy which differs from those of the higher plants and are called phycobiliproteins[1,2]. C-PC is a water-soluble, non-toxic and blue colored photosynthetic pigment with an isoelectric point of about 4.5 to 5 This pigment is an oligomeric protein constructed from equal numbers of α and β subunits with the molecular weight of about 18 and 21 kDa, respectively[6]. Recent studies on the anticancer properties of C-PC exhibited a significant inhibitory effect on the growth of cancer cells in a time- and dose-dependent manner through multiple mechanisms including the induction of apoptosis, cell cycle arrest, inhibition of DNA replication and generation of reactive oxygen species (ROS)[22,23]. This study was aimed to introduce and establish, for the first time, an efficient procedure for the purification of high purity C-PC pigment from cyanobacterial strains, followed by conducting more detailed investigations on its cytotoxic effects on human breast cancer (MCF-7) cells and the underlying mechanisms
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