Development of a novel method for the purification of C-phycocyanin pigment from a local cyanobacterial strain Limnothrix sp. NS01 and evaluation of its anticancer properties

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.

Impact of incubation conditions on protein and C-Phycocyanin recovery from Arthrospira platensis post- pulsed electric field treatment

“…Generally severe pH alterations result in protein denaturation as the pH affects the charges of the ionisable groups of the proteins and eventually the protein stability The integrity of C-Phycocyanin with an isoelectric point of about 4.5 to 5.0 (Safaei et al, 2019) can be altered in a pH far from its isoelectric point.…”

“…This cyanobacteria has been receiving enormous attention during the last decades due to its high content of proteins (up to 70% of dry weight) that can be used for food or pharmaceutical applications (Ali and Saleh, 2012). Among various proteins, phycobiliproteins like C-Phycocyanins are supplementary light-absorbing complexes that are present in high concentration in A. platensis (up to 20% of dry weight) (Safaei et al, 2019). Not only can phycobiliproteins like C-Phycocyanin be used as pharmaceuticals, they can also be utilized as natural blue colorants for food applications (Chaiklahan et al, 2012).…”

“…In fact, C-Phycocyanin are oligomeric proteins that are composed of α and β subunits (monomers) (Berns and MacColl, 1989). In general, they can be found as trimers (αβ)3 or hexamers (αβ)6 (Safaei et al, 2019).…”

“…Since C-Phycocyanin are water-soluble proteins located on the outer membrane of thylakoids (Safaei et al, 2019), PEF treatment can greatly enhance their release into the suspension. Indeed, (Scherer et al, 2019) have also shown that the extraction of membrane-bound proteins by PEF treatment are often limited, whereas water-soluble proteins can be extracted very efficiently.…”

A highly purified C-phycocyanin from thermophilic cyanobacterium Thermosynechococcus elongatus and its cytotoxic activity assessment using an in vitro cell-based approach

“…Previous assessment of the anti-tumour activity of C-PC have been focused on the C-PC isolated mainly from Spirulina platensis [5], in addition to other rich sources including Arthronema africanum [43], Limnothrix sp. [44,45], Porphyra yezoensis [46], and Porphyra haitanensis [47]. To the best of our knowledge, the potential antitumour effect of C-PC isolated from T. elongatus remains unexplored.…”