CAI Chuner, WANG Yuan, LI Chunxia, GUO Ziye JIA Rui, WU Weining HU Yan and HE Peimin,

1) Aquaculture and Life College, Shanghai Ocean University, Shanghai 201306, P. R. China

2) Institute of Marine Science, Shanghai Ocean University, Shanghai 201306, P. R. China

3) Laboratory of Pharmacology and Pharmacodynamics, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P. R. China

4) Shanghai CP Guojian Pharmaceutical Co., Ltd., Shanghai 201203, P. R. China

Purification and Photodynamic Bioactivity of Phycoerythrin and Phycocyanin from Porphyra yezoensis Ueda

CAI Chuner1),2), WANG Yuan3), LI Chunxia4), GUO Ziye1), JIA Rui1),2), WU Weining1), HU Yan1), and HE Peimin1),2),*

1) Aquaculture and Life College, Shanghai Ocean University, Shanghai 201306, P. R. China

2) Institute of Marine Science, Shanghai Ocean University, Shanghai 201306, P. R. China

3) Laboratory of Pharmacology and Pharmacodynamics, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P. R. China

4) Shanghai CP Guojian Pharmaceutical Co., Ltd., Shanghai 201203, P. R. China

Phycoerythrin and phycocyanin were purified fromPorphyra yezoensisUeda with their bioactivity determined in this study. Continuous precipitation with ammonium sulfate at different concentrations (10%, 20%, 40% and 50%) increased the purity (A564:A280) of phycoerythrin to 1.49, 3.92 fold of the raw extract (0.38) and the purity (A615:A280) of phycocyanin to 0.70, 3.33 fold of the raw extract (0.21). Two more times of chromatography with hydroxylapatites finally made the purity of phycoerythrin and phycocyanin reach 5.50, 14.47 fold of the raw extract, and 5.10, 24.29 fold of the raw extract, respectviely. The yield of high purity phycoerythrin and phycocyanin were 0.21% and 0.09% of driedP. yezoensisblade, respectively. The photodynamic cytotoxic experiment showed that both phycoerythrin and phycocyanin inhibited the growth of liver tumor cells significantly. It was found that 250 mg L-1purified phycoerythrin and phycocyanin inhibited the growth of hepatocellular carcinoma cells 24 h after laser-irradiation by 80% and 59%, respectively, and 100 mg L-1purified phycoerythrin and phycocyanin induced the apoptosis of 31.54% and 32.54% of the cells, respectively, 8 h after photodynamic therapy. Oue findings demonstrated thatP. yezoensiscan serve as photosensitizer (phycoerythrin and phycocyanin) producer.

Porphyra yezoensis; phycoerythrin; phycocyanin; purification; photosensitivity; apoptosis; hepatocellular carcinoma cell

1 Introduction

The statistics of World Health Organization showed that about thirteen million people are newly cancered and about seven million people died from diverse cancers each year worldwide (Lingwoodet al., 2008). There is an urgent need of finding active antitumor substances with new structures and functioning mechanisms (Chakrabortyet al., 2008). Photodynamic therapy (PDT) of cancer is a novel strategy which has been developing in last two decades. Since the approval of the first photosensitive drug Photofrin (trade name Porfimer Sodium) for the treatment of advanced bladder and esophageal cancer (Castanoet al., 2005) by Canadian government in 1993, PDT has been widely used to cancer diagnosis and treatment (Averyet al., 2010). PDT enriches photosensitive drug in cancer cells and eliminate them locally by light-activation. Its therapeutic effect depends largely on the efficiency of the photosensitizer. The first generation of photosensitizers with hematoporphyrine as the representative are complex in components and side effective (Sharmanet al., 1999), which are prepared from blood; however, the second generation of photosensitizers with delta-aminolevulinic acid (ALA), a heme precursor, as the representative, are synthesizablein vivo(Bunkeet al., 2000). Phycobiliproteins (PBPs) including phycoerythrin (PE) and phycocyanin (PC) are multi-subunit protein pigment complexes and sensitive to light. Their successful use to PDT of mouse myeloma (Morcoset al., 1988) has raised the interest in their advantages,e.g., high quantum yield of fluorescence and strong ability of killing tumor cells (Guoet al., 2008; Liet al., 2010). The price of purified PE and PC is continuously increasing, reaching 247 $ mg-1and 256 $ mg-1, respectively in 2013 (Sigma Co.). Many studies were carried out in order to improve the efficiency of PBPs purification from diverse materials. The quality and production varied among raw materials and procedures (Ranjitha and Kaushik, 2005). Some simple and convenient techniques of producing small quantity of PBPs have been developed (?t'astnáet al.,2000; Viskari and Colyer, 2002; Viskaria and Colyer, 2003). The routine preparing method of PBPs usually includes three steps: protein releasing, crude extraction and purification with chromatography (Moraes and Kalil, 2009). Crude extraction is important for improving the quality and quantity of final products; unfortunately, such a crucial step was less studied previously.

PBPs are extracted traditionally from PC- and allo PC-richSpirulina(Castanoet al., 2004).Porphyra, one of the three major economic macroalgae in China, has the highest productivity of PBPs. Extraction of PE and PC fromPorphyrawill greatly reduce the cost of PBPs production (Heet al., 2008). The methods available for the purification of PE and PC can be categorized into the expensive (mainly chromatography) and the simple (convenient for small quantity). In this study, we improved the efficiency of crude isolation step of the routine method and purified PE and PC fromP.yezoensis. We also investigated their photodynamic effect on the growth and apoptosis under irradiation of human hepatocellular carcinoma SMMC-7721 cells.

2 Materials and Methods

2.1 Experimental Materials

P. yezoensiswas collected in Jan 2011 from Rudong, Jiangsu Province, China. After washing with sea water, they were dried under dim light. Human hepatocellular carcinoma cell line SMMC-7721 was from the Cell Bank in Chinese Academy of Sciences.

2.2 PE and PC Extraction with Ammonium Sulfate Precipitation

Fifty grams of driedP. yezoensiswas immersed and homogenized in 250 mL 50 mmol L-1phosphate buffered saline in 1 mmol L-1EDTA (PBSE, pH 6.8), then centrifuged at 8500×g and 4℃ for 15 min. The supernatant was precipitated with ammonium sulfate at a concentration of 20% with the pellet discarded. The derived supernatant was precipitated again with 50% ammonium sulfate, with the pellet dissolved in PBSE. The solution was precipitated with 10% ammonium sulfate with the supernatant collected, which was then precipitated with ammonium sulfate again at a concentration of 40% with the supernatant discarded. The final precipitant was dissolved in 50 mM PBSE before an additional centrifugation at 19000×g and 4℃ for 30 min. It was then desalted with Sephadex G-25 and further purified with hydroxyapatite. By gradient eluting with a series of PBSE at different concentrations, the eluent containing PE and PC was collected, respectively. The eluted PE and PC were purified again using regenerated hydroxyapatite.

2.3 PE and PC Purity Examination

PE and PC collected in PBSE elution at different concentrations each were examined by protein absorption, SDS PAGE (14% separating and 5% stacking gel) and native PAGE (5% separating and 5% stacking gel). After electrophoresis, the protein bands were stained with Coomassie Bright Blue R-250 with the images analyzed on a PC enclosed with Gel Doc. Imaging system (Bio-Rad, USA). The spectrum of either solution or supernatant was read on Ultrospec 2000 Spectrophotometer to calculate the purity and content of PE and PC (Gao, 1993).

The purity of PE and PC was assessed according to the ratio of A565/A280and A615/A280, respectively (Liuet al., 2005). The average purity criterion for PBPs was 3.2. The concentration of PE and PC (in mg mL-1) was calculated from the absorbance at 565, 615 and 650 nm, respectively, using the formula

respectively (Gao, 1993). The yield of PE and PC was calculated with the following formula, respectively:

2.4 Determination of Photodynamic Cytotoxicity of PBPs

Human hepatocellular carcinoma SMMC-7721 cells were cultured in RPMI-1640. At the vigorous growing stage, the cells were digested with EDTA-trypsin. The digested cells were dispersed, then diluted to 109cell L-1with media and inoculated into 96 well plates, 1×104cells each (Liet al., 2001). Eighteen hours after inoculation, the cell media were replaced with the media containing different concentrations of PE or PC (30, 60, 120, 250 mg L-1). Four hours later, cells in those wells containing PE were irradiated with INNOVR 400 argon ion laser (Coherent Inc., Santa Clara, CA) at 514 nm (wavelength), 100 mW (output power) and 100 J cm-2(total energy density) for 15 min; while cells in those wells containing PC were irradiated with QHP He-Ne laser (Shanghai Glass Instrument Factory, China) at 632.8 nm, 30 mW and 35 J cm-2for 20 min. After irradiation, cells were further grown for 24 h with their number determined with MTT method using 630 Model Microplates Reader. The native cells (without any treatment) were used as blank controls and the cells which were treated with either PBPs or irradiation were used as negative controls. When cells were treated with PBPs, the concentration of PBPs was 30, 60, 120 and 250 mg L-1, respectively, and culturing time was 24, 48 and 72 h, respectively.

At 4, 8, or 12 h after inoculation, SMMC-7721 cells which were treated with 100 mg L-1PE and irradiated with argon ion laser at 100 J cm-2or with PC and irradi-ated with He-Ne laser at 35 J cm-2, were collected by trypsinization. The cells were washed with cold PBPs and spin-down at 2000×g for 5 min for three times, fixed with 2 mL Carnoy fixative (methanol: acetic acid = 3:l) and dispersed. In total, 1×106cells were used to cell apoptosis analysis each samples as described previously (Bonifacinoet al., 2007). Cells treated with 100 mg L-1PBPs or laser irradiation alone were used as negative control.

3 Results

3.1 PE and PC Purification

Fig.1 Examination of PE/PC during extraction and purification. (A), change of purity and yield. (B), change of absorption spectrum. (C), electrophoretogram. Left, SDS-PAGE. Right, native-PAGE. (a)–(f), crude extraction; (g)–(h), purified PE or PC. (a), crude extract before precipitation with ammonia sulfate; (b), after the first precipitation; (c), after the second precipitation; (d), after the third precipitation; (e), after the fourth precipitation; (f), after centrifugation at 19000 r min-1and 4℃ for 30 min. (g), purified PE after twice hydroxylapatites chromatography; (h), purified PC after twice hydroxylapatites chromatography.

Continuous precipitation with ammonium sulfate gradually increased the purity of PE and PC from 0.38 and 0.21 of raw extract to 1.49 and 0.70, 392% and 333% of the raw extract, respectively. The yield of PE and PC was 1.06% and 0.65% of dryPorphyra, respectively (Fig.1A). Further purificationviaHAP chromatography increased the purity of PE and PC to 5.50 and 5.10, respectively (Fig.1B). Both PE and PC were high purity as were indicated by one or two bands in electrophoresis (Fig.1C). The yield of high purity PE and PC were 0.21% and 0.09% of dryP. yezoensisblade, respectively(Fig.1A).

3.2 Inhibition of Cancer Cells Proliferation by PE and PC

The growth of SMMC-7721 cells treated with 60, 120 or 250 mg L-1PE or PC for 72 h, respectively, was partially inhibited. It was found that the inhibition ratio of cells treated with 250 mg L-1PE and irradiated at 100 J cm-2reached 80%, and that of cells treated with PC at 250 mg L-1and irradiated at 35 J cm-2reached 59% (Fig.2).

Fig.2 Photodynamic cytotoxicity of PE (left) and PC (right) on SMMC-7221 cells (n=12).

3.3 Apoptosis Induction of PE and PC

The apoptotic rates of cells treated with 100 mg L-1laser-irradiated (100 J cm-2) PE for 4, 8 and 12 h was 25.6%, 31.5% and 31.2%, respectively, and the apoptotic rates of cells treated with 100 mg L-1laserirradiated (35 J cm-2) PC for 4, 8 and 12 h was 27.6%, 32.5%, 29.6%, respectively, indicating that both PE and PC can induce cancer cell apoptosis after laser excitation (Fig.3).

Fig.3 Photodynamic cytotoxicity of PE (left) and PC (right) on the apoptosis of SMMC-7221 cells. PE1, cells irradiated with argon ion laser at 100 J cm-2, and cultured for 8 h. PC1, cells irradiated with QHP He-Ne laser at 35 J cm-2, and cultured for 8 h. PE2 or PC2, cells treated with100 mg L-1PE or PC for 8 h. PE3 through 5, cells treated with PE for 18h before being irradiated with argon ion laser at 100 J cm-2, and cultured for 4, 8 and 12 h. PC3 through 5, cells treated with PC for 18 h before being irradiated with QHP He-Ne laser at 35 J cm-2, and cultured for 4, 8 and 12 h, respectively.

4 Discussion

An efficient protocol for the extraction of PC from microalgaSpirulina platensisdescribed early (Moraeset al., 2010; Silveiraet al., 2007) could be used for large-scale purification. PBPs formGracilaria verrucosa(Wang, 2002),Palmaria palmate(Wanget al., 2002),Polysiphonia urceolataGrev (Niuet al., 2006),P. haitanensis(Niuet al., 2007) andP. yezoensisUeda (Niuet al., 2009) have been prepared successfullyviahydrophobic chromatography. We improved here the crude extraction procedure of PBPs fromP. yezoensis, increased the efficiency of column chromatographic purification.

It is speculated that the principle of ammonium sulfate precipitation was the change of protein conformation at high salt concentration and consequent non-covalent interactions of hydrophobic groups, by which proteins were separated from hydrophilic molecules. Except for ammonium sulfate, precipitation regents used priviously include also activated carbon (Cohenet al., 1993) and rivanol (Minkovaet al., 2007); unfiortunately these two regents were not effective for extracting PBPs fromP. yezoensis(Caiet al., 2011). In this study, we developed a four step ammonium sulfate precipitation procedure of crude PBPs extraction, which purified PE in crude extraction to a purity of 1.94, higher than the reported (1.74) with DEAE-52 chromatography (Maet al., 2007).

The anti-cancer potential of PC isolated from Chlorophyta is well known. Their function includes the interference of DNA synthesis (Wanget al., 2001), the activation of caspase-dependent cell apoptosis pathway (Royet al., 2007), the suppression of tumor cell growth by membrane destruction (El-Baky, 2003) and the improvement of host immune performance (Liet al., 2010). The PE from red algaPolysiphonia urceolata(Liet al., 2001) andPorphyra haitanensis(Chenet al., 2005) was found to inhibit the growth of 8113 (human oral epithelial), S180 (mice ascitic hepatoma) and Bcap-3 (human breast cancer) cell lines significantlyviaunidentified pathways. In this study, PE and PC purified fromP. yezoensiswere found to be able to survive most SMMC-7221 cells irradiated with either argon ion laser or He-Ne laser, and induce programmed cell death of more than 30% cells, indicating that apoptosis was one of the mechanisms of PDT. Apoptosis is regulated by various external stimuli including radiation, heat and some drugs under certain conditions and genetic regulations (Vaux and Strasser, 1996). Because apoptosis is a dynamic process including initiation, affection and degradation stages, further studies at more time points were appreciated in order to elucidate the photodynamic mechanism of algal PBPs precisely.

5 Conclusions

The present work developed a suitable method for the extraction of PE and PC from the red algaP. yezoensis. The developed included the immersion with 5-fold extraction buffer followed by precipitations with ammonium sulfate for 4 times and chromatography separation using hydroxylapatite for two times. Under these conditions, PE with a purity ratio (A564:A280) exceeding 5.50 and yield of 0.21%, and PC with a purity ratio (A615:A280) of 5.10 and yield of 0.09% were extracted. When treated with 250 mg L-1PBPs, purified PE and PC inhibited hepatocellular carcinoma cell growth in 24 h after laser-irradiation by 80% and 59%, respectively, and 100 mg L-1purified PE and PC induced the apoptosis of 31.54% and 32.54% of the cells, respectively, 8 h after photodynamic therapy.

Acknowledgements

This research was financially supported by the National Key Technology R&D Program (2012BAC07B03), Shanghai Universities First-class Disciplines Project, Discipline name: Marine Science and Shanghai Municipal Education Commission (Preponderant Subject Program #S30701), and Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, P. R. China, Shanghai Engineering Research Center of Aquaculture, Shanghai University Knowledge Service Platform, Shanghai Ocean University Aquatic Animal Breeding Center (ZF1206).

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(Edited by Qiu Yantao)

(Received September 4, 2012; revised October 23, 2012; accepted September 29, 2013)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2014

* Corresponding author. Tel: 0086-021-61900467

E-mail: pmhe@shou.edu.cn