Phycocyanin: A Potential Drug for Cancer Treatment

Abstract

Phycocyanin isolated from marine organisms has the characteristics of high efficiency and low toxicity, and it can be used as a functional food. It has been reported that phycocyanin has anti-oxidative function, anti-inflammatory activity, anti-cancer function, immune enhancement function, liver and kidney protection pharmacological effects. Thus, phycocyanin has an important development and utilization as a potential drug, and phycocyanin has become a new hot spot in the field of drug research. So far, there are more and more studies have shown that phycocyanin has the anti-cancer effect, which can block the proliferation of cancer cells and kill cancer cells. Phycocyanin exerts anti-cancer activity by blocking tumor cell cell cycle, inducing tumor cell apoptosis and autophagy, thereby phycocyanin can serve as a promising anti-cancer agent. This review discusses the therapeutic use of phycocyanin and focuses on the latest advances of phycocyanin as a promising anti-cancer drug.

Introduction: Phycocyanin

In the past few decades, natural products have become increasingly important for the application of chemical prevention and treatment for diseases ¹. Natural products derived from food or food supplements could be used as drugs in the treatment for diseases, and their chemical prevention and chemotherapy effects have been fully studied ^{2, 3}. Recently, marine natural products with pharmacological activity have been shown to have potent anti-cancer activity, and have less or no toxic side effects ⁴. Thus, marine natural products have an important development and utilization in recent years. In addition, marine natural products have become one of the most important resources of novel lead compounds for critical diseases. Phycocyanin (PC), a marine natural extract, has been studied for its anti-cancer effect on malignant solid tumors ⁵. In addition, phycocyanin is a toxin on cancer cells while it is non-toxic to normal cells ⁶.

Phycocyanin is a biologically active nutrient compound which is isolated and purified from a variety of seaweeds ⁷. Phycocyanin obtained from different species, such as Aphanizomenon sp. ⁸, Spirulina sp. ⁹, Phormidium sp. ¹⁰, Lyngbya sp. ¹¹, Synechocystis sp. ¹² and Synechococcus sp. ¹³, has been separated and studied.

Phycocyanin belongs to the phycobiliprotein (PBP) family ¹⁴, which is characterized by a deep and intense blue color. According to the colored molecules, phycobiliproteins can be divided into three categories: phycoerythrin (PE, PE is red), phycocyanin (PC, PC is blue), and allophycocyanin (AP, AP is bluish green) ^{15, 16}.

Phycocyanin is a kind of photosynthetic assistant protein which can efficiently capture light energy ¹⁷. Phycobiliprotein is one of the components of phycobilisome ¹⁵, which is a supramolecular protein complex that auxiliarily collects light energy. Phycobilisome plays an important role in photosynthesis energy absorption and transmission ¹⁸. Phycobiliprotein acts as an antenna molecule in algae photosynthesis, which can absorb light energy and can be capable of efficiently delivering light energy to a reaction center containing chlorophyll by a non-radioactive process ¹⁹.

Phycocyanin: General properties

Phycocyanin has a deep and intense blue color and consists of α and β subunits ²⁰. In general, the α and β subunits of the phycocyanin form a stable heterodimeric monomer (αβ) and then polymerize it into a multimer (αβ)n (n=1~6) ²¹. Most phycocyanins are present as a trimer (αβ)₃. The α and β subunits of C-phycocyanin have similar 3D structures; however, their sequences are different ²². The α and β subunits contain about 160 to 180 amino acid residues, respectively. The molecular weight of α and β subunits ranges 10~19kD and14~21kD, and the ratio of α and β subunits is usually 1:1. Each subunit is linked to l~4 chromophores, so that phycobiliproteins have a specific absorption spectrum ^23-25. The amino acid sequences of α and β subunits from Spirulina platensisis were just as follows (table 1):

Table 1.

The amino acid sequences of α and β subunits of C-PC from Spirulina platensisis

α chain	β chain
MKTPLTEAVSIADSQGRFLS	MFDAFTKVVSQADTRGEMLS
STEIQVAFGRFRQAKAGLEA	TAQIDALSQMVAESNKRLDA
AKALTSKADSLISGAAQAVY	VNRITSNASTIVSNAARSLF
NKFPYTTQMQGPNYAADQRG	AEQPQLIAPGGNAYTSRRMA
KDKCARDIGYYLRMVTYCLI	ACLRDMEIILRYVTYAVFAG
AGGTGPMDEYLIAGIDEINR	DASVLEDRCLNGLRETYLAL
TFELSPSWYIEALKYIKANH	GTPGSSVAVGVGKMKEAALA
GLSGDAATEANSYLDYAINA	IVNDPAGITPGDCSALASEI
LS	ASYFDRACAAVS

The amino acid sequence of C-phycocyanin is gotten from the Protein Database of the United States Research Collaboratory for Structural Bioinformatics (RCSB PDB, http://www.rcsb.org).

Each polypeptide chain of phycocyanin consists of apoprotein and chromophore with a ring-opening tetrapyrrole structure. The chromophore in phycobiliprotein is called phycobiliin. Phycocyanin contains for phycocyanobilin (PCB) chromophores, and the PCB chromophores span an absorption range from 590 to 670 nm ²¹.

At present, there are a variety of phycocyanins to obtain high purity crystals, and the high-resolution X-ray crystallography was used to determine the high-level structure. The Protein Database of the United States Research Collaboratory for Structural Bioinformatics (RCSB PDB, http://www.rcsb.org) has included more than 20 advanced structures of phycocyanins (Table 2).

Table 2.

Three-dimensional structures of phycocyanins in RCSB PDB

PDB ID	Type	Source	Resolution	Ref.
5TOU	C-Phycocyanin	Pseudanabaena sp. lw0831	2.04 Å	26
4ZIZ	C-Phycocyanin	Thermosynechococcus elongatus	1.75Å	27
4Z8K	C-Phycocyanin	Thermosynechococcus elongatus	2.5Å	28
4L1E	C-Phycocyanin	Leptolyngbya sp. N62DM	2.61Å	29
4N6S	C-Phycocyanin	Thermosynechococcus vulcanus	2.4Å	30
4F0T	C-Phycocyanin	Synechocystis sp. PCC 6803	2.61Å	31
4GXE	C-Phycocyanin	Thermosynechococcus vulcanus	3.0Å	31
4GY3	C-Phycocyanin	Thermosynechococcus vulcanus	2.5Å	31
4H0M	C-Phycocyanin	Synechococcus elongatus	2.2Å	31
3O18	C-Phycocyanin	Thermosynechococcus vulcanus	1.35Å	32
3O2C	C-Phycocyanin	Thermosynechococcus vulcanus	1.5Å	32
2BV8	C-Phycocyanin	Gracilaria chilensis	2.01Å	33
1ON7	C-Phycocyanin	Thermosynechococcus vulcanus	2.7Å	20
1JBO	C-Phycocyanin	Synechococcus elongatus	1.45Å	34
1HA7	C-Phycocyanin	Arthrospira platensis	2.2Å	35
1KTP	C-Phycocyanin	Thermosynechococcus vulcanus	1.6Å	36
1F99	R-Phycocyanin	Polysiphonia urceolata	2.4Å	37
1I7Y	C-Phycocyanin	Thermosynechococcus vulcanus	2.5Å	38
1PHN	C-Phycocyanin	Cyanidium caldarium	1.65Å	39
1CPC	C-Phycocyanin	Microchaete diplosiphon	1.66Å	40
1GHO	C-Phycocyanin	Arthrospira platensis	2.2Å	41

The isoelectric point of phycocyanin is between 4.1 and 6.4, depending on the source of extraction and the method of extraction and purification ^{42, 43}. Recent research suggests that phycocyanin is light sensitive, so phycocyanin must be preserved in the dark ⁴³. Since phycocyanin is sensitive to heat ^{44, 45}, phycocyanin must be purified at 4 ~ 5 ° C ^{46, 47}.

Isolation and Purification

So far, phycocyanins are isolated from algae in all studies. Purification involves multiple steps of crude extraction, including extraction with ammonium sulfate and separation on an ion exchange column ⁴⁸, hydroxyapatite column ⁸ or gel filtration chromatography ⁴⁹.

The determination of phycocyanin purity is based on the absorbance ratio A₆₂₀ / A₂₈₀, and the absorbance at 620 and 280 nm corresponds to phycocyanin and total protein, respectively ⁵⁰. When A₆₂₀/A₂₈₀≤0.7, phycocyanin is considered to be food grade, when 0.7≤A₆₂₀/A₂₈₀≤3.9, phycocyanin is considered to be reagent grade, when A₆₂₀/A₂₈₀≥4.0, phycocyanin is considered to be analysis level ^{51, 52}. Similarly, the phycocyanin concentration can be calculated based on the absorbance values of A₆₂₀ and A₆₅₂ which corresponds to the allophycocyanin ^{42, 48}. The formula is as follows:

C-PC(mg/ml) = (A_620nm-0.474A_652nm)/5.34

Purity=A₆₂₀/A₂₈₀

Clinical uses of phycocyanin

Phycocyanin can have a potent potential as a drug in a wide range of clinical applications. Phycocyanin shows a wide range of pharmacological effects, with anti-oxidation ⁵³, anti-cancer ⁵⁴, anti-inflammatory activity ⁵⁵, photo-induced cytotoxicity ⁵⁶ and stimulating the immune system ⁵⁷.

Phycocyanin plays an antioxidant role in inhibiting hepatic lipid peroxidation and being helpful to liver protection ^{58, 59}. Phycocyanin also scavenges free radicals from damaged nerve cells, which could avoid DNA oxidative damage cause from free radicals and prevent neuronal cell apoptosis ^{60, 61}. There are more and more researches have shown that phycocyanin plays an effective anti-cancer role in various cancer cell types (such as breast cancer ^{57, 62}, liver cancer ⁶³, lung cancer^{64, 65}, colon cancer ⁶⁶, Leukemia ⁶⁷ and bone marrow cancer ⁶⁸ and so on) in vitro and in vivo. Morcos first discovered the laser-induced cytotoxicity of phycocyanin, which could kill tumor cells, while the damage to normal tissue is minimal ⁶⁹. When in combination with He-Ne light, C-phycocyanin can serve as a photosensitize agent in the photodynamic therapy, which could provide a possible tumor therapy ⁵⁷. C-phycocyanin shows specific affinity to the scavenger receptor-A (SR-A) of tumor-associated macrophages (TAMs), which is highly expressed on TAM. Thus, C-phycocyanin can act as a new class of TAM-targeted photosensitiser, which exhibits an efficient in vitro photodynamic activity, and selectively accumulates in tumour sites probably due to the affinity to TAM, which provides a novel strategy to enhance the efficacy for cancer therapy ⁷⁰. Moreover, C-phycocyanin displays an anti-inflammatory potential, so C-phycocyanin is a potential natural anti-inflammatory agent ^{71, 72}. Phycocyanin can promote animal blood cell regeneration, improve lymphocyte activity and lymphatic system to improve immune function, and comprehensively enhance the disease resistance of the body ⁷³. In addition, C-phycocyanin could inhibit TGF-β1-induced EMT and C-PC might be a potential anti-fibrosis drug ⁷⁴.

Molecular targets of phycocyanin

Phycocyanin can affect cancer cell cycle progression, leading to cell cycle arrest. Cell cycle arrest in G0/G1 phase was described in breast cancer MDA-MB-231 ⁶, colon cancer HT29 and adenocarcinoma A549 cells ⁵⁴. Phycocyanin could increase the expression of p21, meanwhile, Phycocyanin could down-regulate the expression of Cyclin E and CDK2 in the MDA-MB-231 cells ⁶.

Phycocyanin is able to activate the apoptotic pathway (the mitochondrial/ cytochrome C pathway), activate caspase and induce poly (ADP-ribose) polymerase-1 (PARP-1) cleavage, alter the Bcl-2/Bax ratio (Bcl-2/Bax ratio represents apoptosis degree) ⁶⁷. In addition, phycocyanin can alter the mitochondrial membrane potential (MMP) ⁶², which can stimulate the release of cytochrome c and promote the formation of reactive oxygen species (ROS), ultimately lead to cancer cell apoptosis ⁷⁵.

Phycocyanin can promote the expression of CD59 protein and Fas protein in Hela cells, While Phycocyanin had no significant effect on CD59 and Fas protein expression in normal CHO cells ⁷⁶. C-phycocyanin can down-regulate the expression of pro-inflammatory cytokines (IL-1β, IL-2, interferon-γ and tumor necrosis factor-α), transcription factors (Janus kinase 3 (Jak3), signal transducers and activators of transcription 3(stat3)), and enhance the expression of anti-inflammatory cytokines IL-4 ⁷⁷. Phycocyanin can inhibit the expression of COX-2 and prostaglandin E2 (PGE2), and down-regulate the MMP-9 expression by mitogen-activated protein kinase (MAPK) signaling pathway ⁶. C-phycocyanin from Spirulina platensis could inhibit MMP-2 and MMP-9 expression and TIMP-2 at a mRNA expression level; however, C-phycocyanin could not have any effect on MMP-1 expressed in MDA-MB231 and TIMP-1 expressed in HepG2 cells ⁷⁸. In addition, C-phycocyanin could bind to VEGFR1 alone, and down-regulated levels of VEGF-A, MMP-2 and MMP-9 ⁷⁹.

The anti- proliferation effect of phycocyanin is mediated by BCR-ABL signaling and inactivation of the downstream PI3K/Akt pathway ⁸⁰. In addition, MAPK, PI3K/Akt/mTOR and NF-κB pathways involved phycocyanin-induced cell death ⁸¹.

C-phycocyanin could down-regulate the expression of vimentin, type 1 collagen and fibronectin, and up-regulating the expression of E-cadherinin in TGF-β1-treated cells. Thus, C-phycocyanin C could inhibit Epithelial-Mesenchymal Transition (EMT) ⁷⁴.

In addition, C-phycocyanin shows specific affinity to the scavenger receptor-A (SR-A) of tumour-associated macrophages (TAMs) ⁷⁰. C-PC inhibits the expression of MDR1 in a reactive oxygen species and COX-2 dependent manner in HepG2 cells. Further, C-PC inhibits NF-κB and AP-1 mediated signal transduction pathways which involved in the regulation of MDR1 expression ⁸².

Phycocyanin and cancer

Cancer is a generic term for malignancy diseases, their basic characteristics are cell proliferation and apoptosis out of control, and hyperplasia to form a new organism (neoplasm). The pharmacological effects of anti-cancer drugs generally include inhibition of tumor cell proliferation, induction of tumor cell apoptosis, cell cycle arrest and inhibition of tumor cell metastasis and so on. In fact, most drugs with anti-cancer properties are derived from natural compounds ⁸³. Among them, phycocyanin, a marine drug, plays an anti-proliferation and pro-apoptotic effects on different cancer cell lines in vitro, while phycocyanin have no side effects on normal tissue cells ^{84, 85}. More and more evidences have proved that phycocyanin has an effective anti-cancer effect on various cancer cell types (such as breast cancer ^{57, 62}, liver cancer ⁶³, lung cancer ^{64, 65}, colon cancer ⁶⁶, Leukemia ⁶⁷ and bone marrow cancer ⁶⁸) in vitro and in vivo. High-dose phycocyanin does not induce significant toxic symptoms or mortality in animal experiments ^{81, 86}. These studies demonstrated the therapeutic potential of phycocyanin in cancer therapy.

Cell cycle arrest

Cell cycle regulation plays an important role in cell proliferation, differentiation and apoptosis. In recent years, it has been reported that cell cycle regulation dysfunction is closely related to the development of tumor. Thus, pharmacological interventions for cell cycle are becoming a potential pathway for tumor therapy. Normal cell cycle is controllable while tumor cell cycle is out of control, thus tumor cells can infinitely proliferate. So it is particularly important that how to regulate cell cycle in tumor therapy.

The cell cycle is divided into two phases: interphase and division phase. The interphase includes G1 phase, S phase and G2 phase. The cell cycle includes three major cell cycle checkpoints: the G1 / S detection point, the S phase detection point, and the G2 / M detection point, which are the critical steps for the cell cycle. Cell cycle checkpoints are the most significant targets for many anticancer drugs, which further induce tumor cell apoptosis by blocking these checkpoints.

When C-PC treated tumor cells HT-29 and A549, it was reported that cell cycle was blocked in the cell cycle G0 / G1 phase, DNA synthesis was blocked, thus, tumor cell proliferation was inhibited ⁵⁴. Similarly, when C-PC treated human breast tumor cell MDA-MB-231 and human squamous carcinoma cell 686LN-M4C1, these tumor cells were found to have different degrees of cell cycle arrest in G0 / G1 phase ^{6, 66}. Phycocyanin could increase the expression of p21, meanwhile, Phycocyanin could down-regulate the expression of Cyclin E and CDK2 in the MDA-MB-231 cell ⁶.Moreover, phycocyanin could prevent K562 cells into S phase and the cells were arrested in G1 phase ⁸⁷.

In addition, Gaoyong Liao found that phycocyanin blocked G2 / M cell cycle progression and induced apoptosis of PANC-1 cells ⁸¹. Jun Ying proved that phycocyanin led to G2 / M phase arrest of SKOV-3 cells ⁸⁸. Chunyan Wang also confirmed that phycocyanin caused cell cycle G2 / M arrest and induced apoptosis in human hepatoma cell line HepG2 ⁵⁶. It was interesting to note that several groups reported the mechanism of PC-mediated cell cycle arrest (Table 3).

Table 3.

Mechanism of PC-mediated cell cycle arrest

Checkpoints	Cell line	Ref.
G0 / G1	HT-29	54
	A549	54
	MDA-MB-231	6
	686LN-M4C1	66
	K562	87
G2/ M	Panc-1	81
	SKOV-3	88
	HepG2	56

Pro-apoptotic effects

Apoptosis, also known as programmed cell death, is a complementary mechanism with cell proliferation in cell life. When the cells undergo apoptosis, the cells have the following morphological characteristics: cell membrane bubbling, cell shrinkage and cytoplasmic coagulation. When cell apoptosis occurs, activation of endonuclease / specific protease leads to genomic cleavage, chromosome DNA fragmentation and ultimately formation of apoptotic bodies ⁸⁹. Two major apoptotic pathways have been demonstrated: one is the mitochondrial / cytochrome C (endogenous) pathway, which activates Caspase-9 and Caspase-3; the second pathway is the cell membrane surface death receptor (exogenous) pathway, in which the exogenous signal activates Caspase-8 and Caspase-3 ⁹⁰. Caspase-3 is activated during most of the apoptosis ⁹¹. Caspase-3 will eventually induce apoptosis, including DNA fragmentation and cell shrinkage ⁹². Therefore, tumor cell apoptosis is the key to tumor therapy. It is extremely important to study drugs that can induce tumor cell apoptosis without affecting other normal cellular functions during tumor therapy.

It is a crucial to look for new anti-cancer therapies which could promote cancer cell apoptosis. In this case, several researches reported the pro-apoptotic effect of phycocyanin on various human cancer cells and some markers were found changeable after treatment (Table 4).

Table 4.

Pro-apoptotic effect of phycocyanin on different cancer cell lines

Cell line	Origin	Markers	Ref.
MDA-MB-231	Breast cancer	Caspase-9 activation Production of ROS and singlet oxygen radicals Cytochrome c release Decrease the levels of Bcl-2, p-AKT, cyclin E and CDK2 Increase the levels of p21, γ-H2AX and Bax Decrease the level of Bcl-2/Bax	6, 93
MCF-7	Breast cancer	Caspase-9 activation Cytochrome c release Decrease the level of Bcl-2 Up-regulation of Fas Disruption of MMP Down-regulation of NF-κB, P53 and CD44	57, 62
HT-29	Colorectal cancer	DNA fragmentation	66
HepG2	Hepatoma	Caspase-3 activation PARP-1 cleavage DNA fragmentation Disruption of MMP Cytochrome c release Decrease the level of Bcl-2 Increase the level of Bax Decrease the level of Bcl-2/Bax	56, 63, 94
K562	Leukemia	DNA fragmentation PARP-1 cleavage Cytochrome c release Decrease the level of Bcl-2 Decrease the level of Bcl-2/Bax	67
A549	Lung cancer	DNA fragmentation Down-regulation of NF-κB Increase the levels of p38 MAPK	64
A375	Melanoma	DNA fragmentation Disruption of MMP	62
686LN-M4C1	squamous cell carcinoma	Caspase-8 and -3 activation	66
AK-5	Rat histiocytic tumor	DNA fragmentation Production of ROS Decrease the levels of Bcl-2, COX-2	95
RAW 264.7	mouse macrophages cell	PARP-1 cleavage Cytochrome c release DNA fragmentation Production of ROS Decrease the levels of Bcl-2, COX-2, PEG2	75
HeLa	human cervical cancer	Caspase-2, 3, 4, 6, 8, 9 and 10 activation DNA fragmentation Down-regulation of Fas, ICAM-1 and Bcl-2 Cytochrome c release Decrease the levels of CD59, cyclin D1 and CDK4	76, 96, 97
LNCaP	Prostate carcinoma	Caspase-9 and -3 activation ROS production	98
PANC-1	pancreatic cancer	Caspase-3 activation PARP-1 cleavage Stimulation of the NF-κB pathway Inhibition of the Akt/mTOR/p70S6K pathway Increase the levels of p-JNK and p-p38 Decrease the level of p-ERK	81
HEP-2	Laryngeal cancer	Caspase-3, 8 and 9 activation ROS production Increase the levels of Bax, Fas and p53 Decrease the level of Bcl-2 Decrease the level of Bcl-2/Bax	99
SKOV-3	Human ovarian cancer	Apoptosis bodies Improve the intracellular content of ROS Caspase-3, 8 and 9 activation Increase the level of mtSSB Decrease the levels of PSME3, HSP60, nucleolin, PPase and prdx-4	100
COLO 205	Human colon carcinoma	Chromatin condensation Nuclear fragmentation DNA strand break Caspase-3 and 9 activation Mitochondrial membrane depolarization Increase the level of Bax Cytochrome c release	101

MMP: mitochondrial membrane potential;

PARP-1: poly (ADP-ribose) polymerase-1;

ROS: reactive oxygen species.

As shown in Table 4, phycocyanin is capable of activating the mitochondrial / cytochrome C (endogenous) pathway, altering the Bcl-2/Bax ratio (Bcl-2, anti-apoptotic protein; Bax, pro-apoptotic protein; Bcl-2/Bax ratio represents apoptosis degree) and activating caspases and inducing poly (ADP-ribose) polymerase-1 (PARP-1) cleavage ⁶⁷. In addition, phycocyanin can alter the mitochondrial membrane potential (MMP), which leads to a decrease of Bcl-2 expression ⁶². Mitochondrial membrane changes stimulate the release of cytochrome c, promote the formation of reactive oxygen species (ROS) that has cytotoxic effects on cancer cells, and ultimately trigger apoptosis ⁷⁵. Under confocal microscopy, the phycocyanin is located within the mitochondria, which can explain its apoptotic effect ⁵⁶. Similarly, phycocyanin can activate the cell membrane surface death receptor (exogenous) pathway, activate the FAS protein, and promote apoptosis ^{57, 96}.

When phycocyanin treated breast cancer MBA-MD-231 cells, phycocyanin triggered the apoptosis of MBA-MD-231 cells by forming ROS, overexpressing p21 protein and lowering Bcl-2 / Bax levels ^{6, 93}. Apoptosis of MCF7 cells induced by phycocyanin was related to FAS over-expression, MMP changes, down-regulation of NF-κB, P53 and Bcl-2 expression ^{57, 62}. Apoptosis of leukemia cell line K562 induced by phycocyanin involved down-regulation of Bcl-2 / Bax and activation of PARP-1 ⁶⁷. HepG2 cells showed that phycocyanin stimulated the apoptotic process by activating PARP-1, altering MMP and decreasing Bcl-2 / Bax levels ⁶³. In addition, when AK5 was treated with C-phycocyanin, ROS was activated but Bcl-2 and COX-2 expression were inhibited ⁹⁵. C-phycocyanin-induced Hela cell apoptosis was caspase-dependent. Activation of caspase-2, -3, -4, -6, -8, -9 and -10 were involved in C-phycocyanin therapy ⁹⁶.In colon cancer HT-29 cells and lung adenocarcinoma A549 cells, phycocyanin decreased cell viability by inducing apoptosis in a dose-dependent manner. Phycocyanin treatment increased the cell nucleus cohesion and DNA fragments, and triggered HT-29 and A549 cells to stay in the G0 / G1 phase in a dose-dependent manner ⁵⁴.

Some researchers suggested that MAPK, PI3K/Akt/ mTOR and NF-κB signaling pathways were directly involved in phycocyanin-induced PANC-1 cell death ⁸¹. Gaoyong Liao found that phycocyanin increased the levels of p-JNK and p-p38 and reduced p-Erk levels in a time-dependent manner. MAPK signaling played an important role in phycocyanin-induced apoptosis of cancer cells. Similarly, phycocyanin inhibited p-Akt (Ser473) and p-mTOR (Ser2448) in a time-dependent manner, while phycocyanin treatment increased the nuclear fraction of NF-κB in a time-dependent manner without affecting cytoplasmic NF-κB.

Mathangi Ravi found that phycocyanin-induced apoptosis of MDA-MB-231 cells through down-regulation ERK1/2. Phycocyanin decreased phosphorylation of ERK1/2, down-regulated of Mcl-1 expression. In general, phycocyanin-induced apoptosis of MDA-MB-231 cells can be attributed to phycocyanin inhibition of MAPK signaling pathway, at the same time, phycocyanin is non-toxic to nonmalignant cells, platelets and RBCs. The study by Mathangi Ravi also demonstrated that the phycocyanin reduces the anti-angiogenic effect by inhibiting COX-2 levels, while C-phycocyanin reduces the expression of MMP-9 and blocks the invasion process ⁶.

In K562 cells, the levels of phosphorylated BCR-ABL and phosphorylated PI3K were decreased after treated with phycocyanin, whereas BCR-ABL and PI3K levels did not change significantly. AKT and p-AKT were reduced after phycocyanin treatment for 1 hour. Anti-proliferative effects of C-PC are mediated by BCR-ABL signaling and inactivation of downstream pathway PI3K / AKT ⁸⁰.

Combined use with drugs and radiation

Many studies have shown that two or more drug combinations can be used to treat the disease in order to improve the therapeutic effect ¹⁰². In the course of tumor treatment, different drug combinations can effectively improve the safety and efficacy of a single drug in cancer treatment regimen. When phycocyanin combined with these chemotherapy drugs, chemotherapy drugs dose can be reduced and thus minimize side effects. Phycocyanin can potentially improve the efficacy of currently available anti-cancer drugs. It is reported that diverse drugs and radiation may combine with phycocyanin to kill human cancer cells (Table 5).

Table 5.

Synergic effects of phycocyain with chemotherapy drugs or radiation

Drug	Cell lines/ animal model	origin	concentration	Markers	Ref.
Topotecan (TPT) + Phycocyanin (PC)	LNCaP	Limnothrix sp. 37-2-1	TPT (10% of typical dose)+ PC (500μg/L)	Reduce the effective dose of TPT Increase level of DNA fragmentation Increase level of ROS Increase activities of caspase-9 and caspase-3	98
Piroxicam+ C-phycocyanin (C-PC)	DMH-induced rat colon carcinogenesis	Spirulina platensis	Piroxicam (4mg/kg)+ C-PC (200mg/kg)	Reduce the toxicity and side effects of Piroxicam Significant decrease levels of COX-2 and PGE2 Significant increase the number of apoptotic cells Increase level of DNA fragmentation Reduce the number and size of tumors Increase the tumor inhibition rate Stimulate cytochrome c release Significant down-regulate the Bcl-2 expression Increase activities of caspase-9, caspase-3 and Apaf-1 Augment intracellular Ca(+2) Elevate calpain-9 expression Up-regulate Wnt/β-catenin pathway Down-regulation PPARγ expression	103-105
all-trans-retinoic acid (ATRA) + C-phycocyanin (C-PC)	HeLa	Spirulina platensis	ATRA (0.079mM)+ C-PC (96μg/L)	Reduce the effective dose and the toxicity of ATRA Enhance the inhibit effect of HeLa cells growth Inhibit the progress of the cell cycle Induce more G0/G1 cell cycle arrest Decrease cyclin D1 and CDK4 expressions Induce cell apoptosis Upregulate caspase-3 expression Downregulate the Bcl-2 expression Promote complement-mediated cytolysis. Downregulate the CD59 expression	65, 97, 106
Betaine + C-phycocyanin (C-PC)	A549	Spirulina platensis	Betaine (4%)+ C-PC (20μg/L)	Enhance betaine anti-cancer effects. Significant decrease A549 viability Inhibit the progress of the cell cycle Significant decrease NF-κB activation Significant increase total p38 MAPK expression Significant decrease in tumour weight	64
Doxorubicin (DOX)+ C-phycocyanin (C-PC)	Adult rat ventricular cardiomyocytes	Spirulina platensis	DOX (1μM)+ C-PC (10 μM) or Spirulina (50 μg/mL)	Significant attenuate the DOX-induced ROS increase Decrease the number of DOX-induced apoptotic cells Prevent the DOX-induced DNA fragmentation Inhibit DOX-induced Bax expression Inhibit the DOX-induced caspase-3 activation Inhibit the DOX-induced cytochrome c release Increase in the Bcl-2 /Bax ratio	107
Doxorubicin (DOX) + C-phycocyanin (C-PC)	HepG2	Spirulina platensis	DOX (50μM)+ C-PC (5, 25 and 50 μM)	Reduce effective dose of doxorubicin Synergistic effects on the anti-proliferation of HepG2 cells Increase the accumulation of doxorubicin in HepG2 cells	63
625-nm laser + C-phycocyanin (C-PC)	MDA-MB-231 HEK-293	Spirulina platensis	625-nm laser (80 Mw/cm2, 30 min)+ C-PC (200 μg/mL and 300 μg/mL)	Produce enough ROS and singlet oxygen radicals Disturb Cellular Morphology Nuclei Cleavage Cell Death Disruption of MMP	93
He-Ne laser + C-phycocyanin (C-PC)	HepG2	Microcystis aeruginosa	He-Ne laser (632.8 nm, 45mW/cm2, 26 J/cm2)+C-PC (200 μg/mL)	Induce high level of ROS accumulation Cause mitochondrial damage Increase cytochrome c release Activate caspase-3 G2/M cell cycle arrest Inhibit cell growth Induce HepG2 cell death by apoptosis Disruption of MMP C-PC is localized in mitochondria	56
He-Ne laser + C-phycocyanin (C-PC)	MCF-7	Spirulina platensis	He-Ne laser (632.8 nm, 24mW/cm2 76.43J/cm2,)+ C-PC (320mg/ml)	Strikingly inhibit tumor formation. Especially enhance immune-enhancing activity Promote the Fas expression of tumor tissue Restrain the CD44, NF-κB and P53 expressions Enhance inhibitory effect against the proliferation of MCF-7 cells in vitro Induce MCF-7 cells death by apoptosis. Increase caspase-9 activities and cytochrome c release Down-regulate the Bcl-2 protein	57

Miroslav ⁹⁸ treated LNCaP (prostate cancer) with 10% conventional doses of topotecan and C-phycocyanin (C-PC), the effect was better than single topotecan or C-PC treatment. The combination of C-phycocyanin and topotecan has activated a large number of caspase-9 and caspase-3, increased free radical oxygen (ROS) levels, induced apoptosis of tumor cells, and reduced side effects of topotecan.

Saini ¹⁰³ also demonstrated that when the combination of piroxicam (the traditional non-steroidal anti-inflammatory drug) and phycocyanin treated DMH-induced rat colon carcinogenesis, the effect was more than 70% higher than single-use drugs. DNA fragmentation, cyclooxygenase 2 (COX-2) expression and prostaglandin E2 (PGE-2) levels were significantly reduced. In addition, the number and size of tumors were also reduced.

Bing Li ⁹⁷ found that ATRA and C-phycocyanin combination treatment of HeLa cells could significantly reduce the dose and side effects of ATRA. The combination therapy can significantly down-regulate anti-apoptotic protein Bcl-2, up-regulate the expression of pro-apoptotic Caspase-3 protein, inhibit cell cycle related CDK-4 and Cyclin D1 protein expression, inhibit complement regulatory protein CD59 expression and induce the HeLa cell apoptosis.

When lung cancer A549 cells were treated with the combination therapy of betaine and C-phycocyanin (C-PC), A549 cell viability decreased up to 60%, and the effect was better than the conventional dose of betaine or C-phycocyanin. Betaine and C-phycocyanin (C-PC) combination therapy reduced NF-κB expression, increased the amount of pro-apoptotic protein p38 MAPK and induced cell G2/M cell cycle arrest. In additon, C-PC and betaine combination therapy might effectively inhibit tumor growth in rats ⁶⁴.

The formation of reactive oxygen species (ROS) and DNA fragmentation induced by Doxorubicin (DOX) was significantly attenuated when adult rats were pretreated with C-phycocyanin. Cell apoptosis rate induced by doxorubicin was significantly reduced. Thus, C-phycocyanin reduced doxorubicin-induced oxidative stress and cardiomyocyte apoptosis ¹⁰⁷.

Phycocyanin could be combined with He-Ne laser for the treatment of tumors ^{56, 57}. When HepG2 cells were pretreated with phycocyanin and laser irradiation (He-Ne), phycocyanin, as a photosensitizer, induced mitochondrial membrane potential loss, increased ROS, released cytochrome c, activated caspase-3, led to G2/M cell cycle arrest, then promoted HepG2 cells activate endogenous pathways to initiate apoptosis ⁵⁶. While phycocyanin could not play a role in normal human hepatocyte line HL7702.

Bing Li found that C-PC-mediated photodynamic therapy (PDT) could promote MCF-7 cell apoptosis in mouse tumor models. When C-PC combined with He-Ne laser irradiation, C-PC treatment effect was further improved, the immune function of mouse tumor model increased, the proliferation of immune cells increased, meanwhile, the expression of Fas protein increased, while tumor weight and anti-apoptosis protein (NF-kB) and CD44 mRNA expression decreased. In addition, caspase-9 expression was activated, cytochrome c released, Bcl-2 expression was down-regulated, apoptotic signal transduction was activated, eventually C-PC led to MCF-7 cell apoptosis ⁵⁷.

C-phycocyanin as a photosensitizer was used for low-level laser therapy under laser irradiation. In the 625nm laser irradiation, c-phycocyanin induced ROS to produce cytotoxic reaction, induced the apoptosis of MDA-MB-231 ⁹³.

Targeted therapy and nanoparticle therapy

The novel nano-drug may lead to the development of a new treatment strategy for cancer cells. Nano-drugs have opened up a new hot spot in the field of medicine research, which have the potential to overcome the shortcomings of conventional drugs with serious side effects in vivo ¹⁰⁸. The combination of medicine and nanotechnology provides a promising way to solve these problems, and many reports have proven the anticancer efficacy of nano-drugs for various cancer types in vivo and in vitro. The specifically expressed and overexpressed proteins in cancer cells are considered to be a useful molecular marker, which is constructed as tumor-targeted nano-drugs that specifically target nano-drugs to tumors. Thus, tumor-targeted nano-drugs are selective killing of cancer cells, while tumor-targeted nano-drugs have the least effect on normal cells. At present, tumor targeting Nano-drugs have become a hot topic.

Xiaoxia Liao successfully constructed a novel biomedical material, MWNT-CS-PC, with a combination of chitosan (CS), multiwalled carbon nanotubes (MWNTs) and phycocyanin (PC). Water-soluble chitosan served as a skeleton, which can bridge between two photo-responsive materials (phycocyanin and multiwalled carbon nanotubes). MWNT-CS-PC utilized different advantages of each component, such as the easy modification, solubility and low toxicity of chitosan, the photothermal effect of multiwalled carbon nanotubes in the near-infrared light region, and the low toxicity and photodynamic response of phycocyanin in the visible light region. It is reported that the average length of the MWNT-CS-PC was 233 nm and the zeta potential of the MWNT-CS-PC was 62.2 mV. It is noted that MWNT-CS-PC could inhibit the proliferation of L-o2, MCF-7 and HepG2 cells, and enhance the photoinduced cytotoxicity of MCF-7 and HepG2 cells with irradiation of both near-infrared light (808 nm) and visible light (532 nm). Phycocyanin could improve the phototoxicity of MWNT-CS-PC and also reduce the cytotoxicity of the carbon nanotube complex on normal cells L-o2. However, it is essential that MWNT-CS-PC conjugate tumor-associated antigen to enhance the targeting ability, so MWNT-CS-PC will possess high potential for photodynamic therapy of cancer ¹⁰⁹.

Peng Yang successfully constructed a novel nano-drug, C-PC/CMC-CD59sp nanoparticles, with a combination of C-phycocyanin (C-PC), Carboxymethyl chitosan (CMC) and CD59-specific ligand peptide (CD59sp). Carboxymethyl chitosan served as a vehicle, which can bridge between C-phycocyanin and CD59sp. It was reported that the C-PC/CMC-CD59sp nanoparticles were spherical, the mean length of C-PC/CMC-CD59sp nanoparticles was 200 nm, and the C-phycocyanin loading of C-PC/CMC-CD59sp nanoparticles was about 20%. The C-PC/CMC-CD59sp nanoparticles loaded with Water-soluble C-PC improved stability of C-PC and had sustained anticancer activity. Guided by the CD59-specific ligand peptide, the C-PC/CMC-CD59sp nanoparticles efficiently targeted the surface of HeLa cells and could be more effective in inhibiting HeLa cell proliferation. The C-PC/CMC-CD59sp nanoparticles induced HeLa cells apoptosis by up-regulation of cleaved caspase-3 and cleaved poly ADP-ribose polymerase proteins, and down-regulation of Bcl-2 proteins ¹¹⁰.

Dong-Hua Wan used C-phycocyanin (C-PC) as a vehicle of zinc phthalocyanine (ZnPc) to fabricate a ZnPc-CPC conjugate. It was reported that the zeta potential of the ZnPc-CPC conjugate is -44.2mV. The combination of ZnPc and C-PC could effectively enhance the solubility of ZnPc in aqueous media. C-phycocyanin have the specific affinity to the scavenger receptor-A (SR-A), similarly, zinc phthalocyanine could selectively bind the scavenger receptor-A (SR-A), which is highly expressed on tumor-associated macrophages (TAM). Thus, the non-covalent ZnPc-CPC conjugate could also target the tumour-associated macrophages. The tumour-associated macrophages have been shown to play a crucial role in the development and progression of cancer. So tumour-associated macrophages have been proposed to be a “target for cancer therapy”. The ZnPc-CPC conjugate selectively accumulated in tumor sites probably through the TAM-mediated mechanism. And the ZnPc-CPC conjugate exhibited an efficient in vitro photodynamic activity. Thus, C-phycocyanin can act as a new class of TAM-targeted photosensitiser as well as a desirable vehicle for photosensitisers. This provides a novel strategy to enhance the efficacy for cancer therapy ⁷⁰.

Effect on tumor progression and metastasis

Another basic feature of phycocyanin is the impact of tumor progression and metastasis potential, C-phycocyanin from Spirulina was used to chemically prevent DMH-induced colon cancer in rats. After C-phycocyanin treated DMH-induced rats, the number and size of tumors / lesions were reduced. At the same time, phycocyanin could bind to VEGFR1 alone. In addition, C-phycocyanin down-regulated levels of VEGF-A, MMP-2 and MMP-9, which are required for tumor metastasis and invasion to surrounding tissues. C-phycocyanin simultaneously down-regulated HIF-1 (associated with increased oxygen demand and angiogenesis) and MCP-1 expression (positively correlated with metastatic and poor prognosis in the tumor microenvironment), while C-phycocyanin promoted MIP-1 expression which plays a role in reducing angiogenesis ⁷⁹. Similarly, C-phycocyanin from Spirulina increased calpain-9 (calpain-9 could increase intracellular Ca²⁺ concentration, which contributes to drug-mediated apoptosis) and PPARγ (related to tumor progression), while C-phycocyanin inhibited colon cancer Wnt/β-catenin signal and played its anti-tumor effect, then down-regulates PPARα and PPARδ expression. So C-phycocyanin has the effects of promoting apoptosis and reducing carcinogenicity ¹¹¹.

C-phycocyanin inhibited the progression of cancer by inhibiting pro-inflammatory cytokines and Jak3 / Stat3 signaling ⁷⁷. C-phycocyanin from cyanobacteria down-regulated the expression of pro-inflammatory cytokines (IL-1β, IL-2, interferon-γ and tumor necrosis factor-α), transcription factors (Janus kinase 3 (Jak3) and signal transducers and activators of transcription 3(stat3)) and enhanced the expression of anti-inflammatory cytokines IL-4 in DMH-induced colon cancer.

Phycocyanin has a therapeutic effect on the invasion of tumor cells. After treatment with phycocyanin, actin filaments were destroyed in MDA-MB-231 cell, the cell polymerization capacity increased while the migration potential decreased ⁶. Phycocyanin is an inhibitor of COX-2 which biological function is to convert arachidonic acid to prostaglandins and plays a key role in tumor progression and chemical resistance ^{112, 113}. PGE-2 is a tightly regulated product of COX-2, which is characterized by promoting angiogenesis ¹¹⁴. It has been shown that COX-2 inhibitors up-regulated E-cadherin expression in colon cancer cell lines ¹¹⁵. The expression of COX-2 was positively correlated with tumor invasion, metastasis and poor prognosis in non-small cell lung cancer (NSCLC). Overexpression of COX-2 resulted in a decrease in E-cadherin expression and a decrease in cell aggregation capacity in NSCLC cells. Exogenous prostaglandin E2 (PGE2) significantly reduced E-cadherin in the treatment of NSCLC cells. ZEB1 and Snail (transcriptional inhibitors of E-cadherin) were up-regulated in COX-2 overexpressed cells or PGE2-treated NSCLC cells, and PGE2 enhanced the binding of ZEB1 and Snail to E-cadherin at the chromatin level. Therefore, PGE2 regulated the transcriptional inhibitory factor of E-cadherin to regulate the expression of COX-2-dependent E-cadherin in NSCLC ¹¹⁶.

C-phycocyanin inhibited EMT by reducing the expression of vimentin, type 1 collagen and fibronectin, and increasing the expression of E-cadherinin in TGF-β1-treated cells. Thus, C-phycocyanin inhibited TGF-β1-induced EMT, so C-phycocyanin might be a potential anti-fibrosis drug ⁷⁴.

Induction of autophagy

It was reported that autophagy is a double-edged sword, in addition to contributing to cell damage, as well as well-known to promote survival ^{117, 118}. Autophagy also plays a role in cancer cells, such as protecting cancer cells from death ¹¹⁹. But specific cancer cells can be killed by autophagy, in which case autophagy acts as a programmed cell death type II ^{120, 121}, sometimes, autophagy plays a role in killing tumor cells when autophagy interacted with other cell death mechanisms ¹²². In particular, autophagy and apoptosis are usually induced by the same stimulus and share similar effectors and regulators ^{123, 124}. These studies have shown that it is highly probable to develop anti-cancer treatment strategies by synergistically regulating autophagy and apoptosis. In view of these factors, many studies were actively looking for drugs that not only induce apoptosis but also induce autophagy. In this regard, some studies had shown that phycocyanin acted as a potential inducer for autophagy.

Gaoyong Liao ⁸¹ found that phycocyanin was able to induce autophagy in PANC-1 cells. When phycocyanin treated PANC-1 cells, the expression of Beclin 1 increased with the increase of phycocyanin dose and treatment time. As a mammalian homologous protein of yeast Atg6, Beclin 1 plays a crucial role in the process of autophagy induction and the formation of characteristic autophagosomes. Gaoyong Liao had shown that phycocyanin inhibited Akt/mTOR/p70S6K signaling pathways, which may contribute to phycocyanin-induced autophagy. Several studies have also shown that Akt/mTOR/p70S6K pathway plays an important role in the development of autophagy in various cancer cells, including liver cancer ¹²⁵, gastric cancer ¹²⁶, pancreatic cancer ¹²⁷ and malignant glioma ¹²⁸. Protein kinase Akt could inhibit tuberous sclerosis complex 2 (TSC2) by direct phosphorylation, to further up-regulate mTOR ¹²⁹, which has been considered a key regulator of autophagy ¹³⁰. Otherwise, cancer cells could be induced to trigger autophagy by inhibition of mTOR pathway ^{131, 132}. At the same time, phycocyanin activated JNK and p38 pathway and inhibited ERK signaling pathway. The study showed that MAPK signal pathway played an important role in phycocyanin-induced apoptosis of cancer cells. Autophagy and apoptosis usually coexisted and remained in balance with each other ¹³³.

Although there is a significant difference between apoptosis and autophagy, sometimes the same regulators can regulate both apoptosis and autophagy ¹³⁴. One such modulator is the NF-κB signaling pathway. When NF-κB signaling pathway is activated, it is well known that apoptosis would be inhibited ^{135, 136}, autophagy would be regulated in a positive or negative regulatory manner ^137-140.

Phycocyanin can induce NF-κB nuclear translocation, since NF-κB is one of the transcription factors of Beclin 1 which is a key promoter of autophagy in cells ¹⁴¹, so the expression of Beclin 1 was highly correlated with the amount of NF-κB transferred into the nucleus. In other words, phycocyanin-activated NF-κB nuclear translocation increased autophagy by increasing the transcription of the Beclin 1. Thus, phycocyanin-mediated complex interactions between autophagy and apoptosis may be due to NF-κB activation. In conclusion, phycocyanin induced apoptosis and autophagy by complex regulation of MAPK, Akt/mTOR and NF-κB signaling pathways.

In conclusion, the present study suggested that the NF-κB, MAPK, PI3K-Akt-mTOR pathways were involved in both phycocyanin-induced tumor cells apoptosis and autophagy (Figure 1). These findings therefore strongly suggested that the anticancer activities of phycocyanin.

Figure 1.

Schematic form of the proposed pathway mechanisms for phycocyanin-induced apoptosis and autophagy in tumor cells.

Concluding remarks

It is shown that phycocyanin has a clear potential as a drug in a wide range of studies. More and more literatures supported that phycocyanin might be a promising anticancer drug or supplementary drug of anticancer drug. On one hand, the constructions of targeted-tumor nano-drug or nanoparticles have become a hot spot for phycocyanin treatment of tumors. Targeted therapy and nanoparticle therapy have now become a hot topic in cancer treatment. Targeted therapy and nanoparticle therapy could increase anticancer effects toward cancer cells. In future, with the discovery of more and more tumor-specific antigens and nanomaterials, the construction of targeted nanoparticles is becoming more and more mature, the target to tumors is getting better, and the side effects of normal tissue cells are getting smaller. On the other hand, combination with other anticancer drugs and radiation for cancer therapy has become another research hotspot. Traditional anti-cancer drugs are often accompanied by serious side effects and short effective half-lives in vivo. When combined with other anticancer drugs and radiation, phycocyanin could reduce the effective dose of anticancer drugs, minimize side effects and improve the therapeutic effect. In addition, phycocyanin can effectively improve the safety and efficacy of a single anticancer drug in cancer treatment.

However, phycocyanin is currently not used as an anticancer drug for clinical use. Despite the phycocyanin target and mechanism were studied several times, it still has to investigate the molecular mechanism of phycocyanin on the death of cancer cells. Further clarifying the effect of phycocyanin on cancer and normal cells will help to develop effective strategies and instruct the clinical application.

Abbreviations

PC

Phycocyanin

C-PC

C-Phycocyanin

PBP

Phycobiliprotein

PE

Phycoerythrin

AP

Allophycocyanin

PCB

Phycocyanobilin

PEB

Phycoerythrobilin

PUB

Phycourobilin

PVB

Phycoviolobilin

Jak3

Janus kinase 3

STAT3

Signal transducers and activators of transcription 3

PGE2

Prostaglandin E2

MAPK

Mitogen-activated protein kinase

PARP-1

Poly (ADP-ribose) polymerase-1

EMT

Epithelial-Mesenchymal Transition

MMP

Mitochondrial membrane potential

ROS

Reactive oxygen species

TPT

Topotecan

ATRA

All-trans-retinoic acid

DOX

Doxorubicin

RCSB PDB

the Protein Database of the United States Research Collaboratory for Structural Bioinformatics

Authors' contributions

All authors have contributed to data preparation, drafting and revising the manuscripts. All authors have read and approved the final manuscript.