R-Phycoerythrin Induces SGC-7901 Apoptosis by Arresting Cell Cycle at S Phase

R-Phycoerythrin (R-PE), one of the chemical constituents of red algae, could produce singlet oxygen upon excitation with the appropriate radiation and possibly be used in photodynamic therapy (PDT) for cancer. Documents reported that R-PE could inhibit cell proliferation in HepG2 and A549 cells, which was significative for cancer therapy. This is due to the fact that R-PE could kill cancer cells directly as well as by PDT. However, little is known about the cytotoxicity of R-PE to the SGC-7901 cell. In this study, it has been found that R-PE could inhibit SGC-7901 proliferation and induce cell apoptosis, which was achieved by arresting the SGC-7901 cell at S phase. CyclinA, CDK2 and CDC25A are proteins associated with the S phase, and it was found that R-PE could increase the expression of cyclin A protein and decrease the expression of CDK2 and CDC25A proteins. Thus, it was concluded that R-PE reduced the CDK2 protein activated through decreasing the CDC25A factor, which reduced the formation of Cyclin-CDK complex. The reduction of Cyclin-CDK complex made the SGC-7901 cells arrest at the S phase. Therefore, R-PE induced apoptosis by arresting the SGC-7901 cell at S phase was successful, which was achieved by the expression of the CDC25A protein, which reduced the CDK2 protein actived and the formation of Cyclin-CDK complex.


Introduction
Phycoerythrin (PE) is one of four classes of phycobili protein (phycoerythrin, phycocyanin, phycoerythrocyanin and allophycocyanin) [1], which are divided into four main classes on the basis of their absorption spectra, R-phycoerythrin (R-PE, peaks at 499, 565 nm and a shoulder peak at 545 nm), C-phycoerythrin (C-PE, peak at 565 nm), B-phycoerythrin (B-PE, peaks at 545 nm, 565 nm and a shoulder at 499 nm), and CU-phycoerythrin (CU-PE, peaks at about 498 nm, 540 nm and/or 565 nm) [1][2][3][4]. R-phycoerythrin (R-PE), molecular weight 240,000 daltons, is a phycobiliprotein isolated from proprietary red algae. R-PE is composed of a carrier protein connecting open-chain tetrapyrrole compound with thioetherbond. The protein has an (alpha-beta) 6 gamma composition. Both alpha-and beta-subunits are approximately 20,000 daltons, and the gamma-subunits approximately 30,000 daltons ( Figure 1a). The purified R-PE showed three absorption peaks at 498 nm, 538 nm (shoulder peak), 566 nm and one fluorescent emission maximum at 577 nm (Figure 1b) [5]. R-PE is commonly used as a fluorescent label probe [6][7][8][9], as biological and clinical assays [10] and for in-flow cytometry [8,11]. It is also used as a pigment or natural food dye [12]. R-PE can produce singlet oxygen upon excitation with appropriate radiation and hence could be useful in photodynamic therapy (PDT) for cancer [13]. Literature states numerous biological activities of R-PE, such as antioxidant activity [14], anti-cancer properties [15], anti-carcinogenic nature [13,16], and antiaging activity [17] et al. It was observed that it also shows proliferation inhibition on the SGC-7901 cell by arresting cell cycle phase, which was not reported by other research.

Effect of R-PE on SGC-7901 Cell Viability
To confirm the inhibitory effect of R-PE on SGC-7901 human gastric cancer cell growth, cell growth was measured using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As showed in Table 1, R-PE proved that it could inhibit the cell proliferation in SGC-7901 human gastric cancer cells, and the IC50 value of cell growth inhibition was 2.55 μM.

Effect of R-PE on SGC-7901 Cell Morphology and Cell Nuclei Morphology
An inverted light microscope was adopted to observe the effect that R-PE had on the cellular morphology. The SGC-7901 cells of the control group grew well, and the skeletons were clear, with most of the cells being polygonous (Figure 2(A1)). Cells treated with R-PE showed morphological changes of apoptosis, such as detachment from substratum and cell rounding, cell shrinkage and decrease in cell number (Figure 2(A3-5)).
The morphology of the nuclei was observed under a fluorescent microscope. The dye used was Hoechst 33258 and was evenly distributed in the cells of control group, which indicated that the chromatin was equally distributed in the nucleus. The cells treated with R-PE presented the morphological features of apoptotic cells, such as emitting brighter fluorescence (nuclear R-PE is commonly used as a fluorescent label probe [6][7][8][9], as biological and clinical assays [10] and for in-flow cytometry [8,11]. It is also used as a pigment or natural food dye [12]. R-PE can produce singlet oxygen upon excitation with appropriate radiation and hence could be useful in photodynamic therapy (PDT) for cancer [13]. Literature states numerous biological activities of R-PE, such as antioxidant activity [14], anti-cancer properties [15], anti-carcinogenic nature [13,16], and anti-aging activity [17] et al. It was observed that it also shows proliferation inhibition on the SGC-7901 cell by arresting cell cycle phase, which was not reported by other research.

Effect of R-PE on SGC-7901 Cell Viability
To confirm the inhibitory effect of R-PE on SGC-7901 human gastric cancer cell growth, cell growth was measured using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As showed in Table 1, R-PE proved that it could inhibit the cell proliferation in SGC-7901 human gastric cancer cells, and the IC 50 value of cell growth inhibition was 2.55 µM.

Effect of R-PE on SGC-7901 Cell Morphology and Cell Nuclei Morphology
An inverted light microscope was adopted to observe the effect that R-PE had on the cellular morphology. The SGC-7901 cells of the control group grew well, and the skeletons were clear, with most of the cells being polygonous (Figure 2(A1)). Cells treated with R-PE showed morphological changes of apoptosis, such as detachment from substratum and cell rounding, cell shrinkage and decrease in cell number (Figure 2(A3-5)).
The morphology of the nuclei was observed under a fluorescent microscope. The dye used was Hoechst 33258 and was evenly distributed in the cells of control group, which indicated that the chromatin was equally distributed in the nucleus. The cells treated with R-PE presented the morphological features of apoptotic cells, such as emitting brighter fluorescence (nuclear condensation identified by propidium iodide (PI) staining) [18,19] and nuclear membrane cracking (the nucleus dispersion) (Figure 2(B3-5)).

R-PE Induces Apoptosis Rate of SGC-7901 Cells
To quantify the apoptosis rate of R-PE on SGC-7901 cells, the PI staining and flow cytometry assay was employed. The data showed that R-PE induced cell apoptosis of SGC-7901 cells in a dosedependent manner ( Figure 2C,D and Table 2). When the cells were treated with 1.3, 2.6, 5.2 μM R-PE for 48 h, the average proportion of sub-G0 phase cells (apoptosis cells) significantly increased from 4.03% ± 2.64% in control to 12.37% ± 4.62%, 18.67% ± 3.06%, 24.50% ± 1.64%, respectively (Table 2, Figure 2C). Figure 2D shows the graphic representation of the increase in the apoptosis rate with an increase in the dose of R-PE.

R-PE Induces Apoptosis Rate of SGC-7901 Cells
To quantify the apoptosis rate of R-PE on SGC-7901 cells, the PI staining and flow cytometry assay was employed. The data showed that R-PE induced cell apoptosis of SGC-7901 cells in a dose-dependent manner ( Figure 2C,D and Table 2). When the cells were treated with 1.3, 2.6, 5.2 µM R-PE for 48 h, the average proportion of sub-G 0 phase cells (apoptosis cells) significantly increased from 4.03% ± 2.64% in control to 12.37% ± 4.62%, 18.67% ± 3.06%, 24.50% ± 1.64%, respectively (Table 2, Figure 2C). Figure 2D shows the graphic representation of the increase in the apoptosis rate with an increase in the dose of R-PE.

Effect of R-PE on the Cell Cycle in SGC-7901 Cells
Analysis of the cell cycle arrest in SGC-7901 cells was carried out using a flow cytometer assay ( Figure 3). The results showed that the number of cells in the S phase significantly increased from 23.17% ± 3.50% in the control group to 28.60% ± 1.66%, 33.43% ± 2.95% and 40.50% ± 2.66% in the R-PE treated groups (showed in Table 3), The Figure 3f showed the graphic representation of the increase of the S phase. The population of SGC-7901 cells at the G 2 /M phase was significantly reduced after R-PE treatment, which concurred that the R-PE could arrest the SGC-7901 cells at the S phase.
23.17% ± 3.50% in the control group to 28.60% ± 1.66%, 33.43% ± 2.95% and 40.50% ± 2.66% in the R-PE treated groups (showed in Table 3), The Figure 3f showed the graphic representation of the increase of the S phase. The population of SGC-7901 cells at the G2/M phase was significantly reduced after R-PE treatment, which concurred that the R-PE could arrest the SGC-7901 cells at the S phase.

Effect of R-PE on Cycle-Associated Protein Expression in SGC-7901 Cells
To further confirm the cell cycle arrest mediated by R-PE in SGC-7901 cancer cells, S phase cycle-related proteins (including cyclinA, CDK2, CDC25A) were examined. As shown in Figure 4a, R-PE significantly increased the protein expression of cyclin A and decreased the expression of CDK2 and CDC25A protein. The data in Figure 4b showed that R-PE affected the cyclin A, CDK2 and CDC25A protein expression in a dose-dependent manner.

Effect of R-PE on Cycle-Associated Protein Expression in SGC-7901 Cells
To further confirm the cell cycle arrest mediated by R-PE in SGC-7901 cancer cells, S phase cyclerelated proteins (including cyclinA, CDK2, CDC25A) were examined. As shown in Figure 4a, R-PE significantly increased the protein expression of cyclin A and decreased the expression of CDK2 and CDC25A protein. The data in Figure 4b showed that R-PE affected the cyclin A, CDK2 and CDC25A protein expression in a dose-dependent manner.

Discussion
Red algae is one of the oldest groups of eukaryotic algae [20] and also one of the largest phyla of algae, with more than 5000-6000 distinct species [21]. They are almost exclusively multicellular marine algae. Most red algae, such as dulse and laver, are also used as food throughout the world, especially in Asia. The red algae is also used to make other products such as agar [22], carrageenans [23] et al. Red algae get their color from a specific pigment called phycoerythrin. This pigment absorbs blue light and reflects red, hence the color. The R-PE studied in this paper was prepared from red algae.
Phycoerythrin is divided into four main classes on the basis of their absorption spectra: Rphycoerythrin (R-PE), C-phycoerythrin (C-PE), B-phycoerythrin (B-PE) and CU-phycoerythrin (CU-PE). CU-PE has similar chromophores and absorption properties as R-PE and used to be classified as R-PE. Literature states that C-PE and R-PE can inhibit cancer cell proliferation but none of B-PE. C-PE purified from marine Lyngbya sp. induces apoptosis in A549 human lung carcinoma cells. R-PE is the chemical constituent of red algaae and has been used to treat many types of cancer by Photodynamic Therapy (PDT). The R-PE subunit can easily accumulate in tumor cells because the tumor cells proliferate faster than normal tissue cells, which can be activated after 496 nm light

Discussion
Red algae is one of the oldest groups of eukaryotic algae [20] and also one of the largest phyla of algae, with more than 5000-6000 distinct species [21]. They are almost exclusively multicellular marine algae. Most red algae, such as dulse and laver, are also used as food throughout the world, especially in Asia. The red algae is also used to make other products such as agar [22], carrageenans [23] et al. Red algae get their color from a specific pigment called phycoerythrin. This pigment absorbs blue light and reflects red, hence the color. The R-PE studied in this paper was prepared from red algae.
Phycoerythrin is divided into four main classes on the basis of their absorption spectra: R-phycoerythrin (R-PE), C-phycoerythrin (C-PE), B-phycoerythrin (B-PE) and CU-phycoerythrin (CU-PE). CU-PE has similar chromophores and absorption properties as R-PE and used to be classified as R-PE. Literature states that C-PE and R-PE can inhibit cancer cell proliferation but none of B-PE. C-PE purified from marine Lyngbya sp. induces apoptosis in A549 human lung carcinoma cells. R-PE is the chemical constituent of red algaae and has been used to treat many types of cancer by Photodynamic Therapy (PDT). The R-PE subunit can easily accumulate in tumor cells because the tumor cells proliferate faster than normal tissue cells, which can be activated after 496 nm light irradiation. It can then destroy the tumor cell by photochemical reaction and can be applied in the treatment of liver cancer [13] and cervical cancer [16]. PDT using R-PE was proved to be effective in animal experiments [13,16]. In vitro experimentation showed that R-PE could induce the apoptosis in HepG2 cells and A549 cells by arresting the cell at G 2 /M phase [15]. The results of this study showed that R-PE could inhibit SGC-7901 cell proliferation and the IC 50 value recorded was 2.55 µM ( Table 1). The findings regarding the proliferation inhibition of R-PE are very significant for the treatment of cancer because R-PE is mainly used for PDT at present, and if the R-PE can kill cancer cell as well as be used in PDT, it has the effect of icing on the cake for cancer treatment.
It was also found that R-PE could destroy the morphology of cell and cell nucleus (Figure 2A,B). This showed R-PE may induce cell apoptosis. PI stain and flow cytometer assay were adopted to determine the apoptosis rate of R-PE in SGC-7901 cells, and the results showed that the apoptosis rate increased from 4.03% ± 2.64% in control to 12.37% ± 4.62%, 18.67% ± 3.06%, 24.50% ± 1.64% in 1.30 µM, 2.60 µM, and 5.20 µM R-PE group (Table 2, Figure 2C,D). This showed that R-PE could induce the apoptosis of SGC-7901 cells.
Cell cycle arrest is an important cause of cell apoptosis, Senthilkumar reported that R-PE could induce cell cycle arrest in HepG2 and A549 cells [15]. The effect of R-PE on cell cycle was observed, and it was found that R-PE arrested SGC-7901 cells at the S phase (Table 3, Figure 3), this is different from that of R-PE, which arrested the HepG2 and A549 cells at G2/M phase [15]. Therefore, the arresting effect was different in various types of cancer cell lines.
CDK and Cyclin are the core factors of endogenous regulation and control of cell cycle. CyclinA, CDK2 and CDC25A are cell factors associated with the S phase of cells. CDC25A could activate the CDK2, which in turn activates the Cyclin-CDK complex. This could promote the operation of the cell cycle and therefore high expression of CDC25A protein could cause rapid cell growth [31,32].
The results of this study showed that the R-PE can decrease the expression of CDC25A and CDK2 protein, and increase the expression of CyclinA (Figure 4). It was deduced that R-PE reduced the CDK2 protein activated through decreasing the CDC25A factor, which reduced the formation of Cyclin-CDK complex. The reduction of Cyclin-CDK complex made the SGC-7901 cells arrest at the S phase. Simultaneously, the expression of free Cyclin A protein would increase because of the reduction of Cyclin-CDK complex, which was confirmed in the experiments (showed in Figure 4). Therefore, R-PE induced apoptosis by arresting the SGC-7901 cell at S phase was achieved by decreasing the expression of the CDC25A protein, reducing the CDK2 protein activated and the formation of Cyclin-CDK complex.

Chemicals and Other Reagents
Phycoerythrin (R-PE, A max /A 280nm > 5.0) was purchased from Hongrui Biotech Company (Pan'an County, Zhejiang, China). MTT, trypsin, PI, RNase A, Tween-20, glycine, acrylamide, methylene diacrylamide and Tris were purchased from the Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 cell culture medium and trypsin were purchased from Gibco (Grand Island, NY, USA). Doxorubicin hydrochloride was purchased from Pfizer Pharmaceuticals Limited (Brooklyn, NY, USA). Fetal calf serum (FCS) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hoechst33258, penicillin-streptomycin, cell lysis buffer, and tetramethylethylenediamine (TEMED) were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). DAB Horseradish Peroxidase Color Development Kit was purchased from ZSGB-BIO (Beijing, China). All other chemicals and solvents used were the highest purity grade.

Cell Line and Culture Conditions
The human gastric carcinoma SGC-7901 cell line was obtained from the Institute of Cancer Prevention and Control of Harbin Medical University (Harbin, China). The cells were grown in RPMI-1640 medium containing 10% fetal bovine serum at 37 • C in a humidified atmosphere containing 5% CO 2 . Cells were harvested by trypsinization with 0.25 mg/mL trypsin solution and suspended in culture medium before use.

Cell Viability and Cytotoxicity
Exponentially growing cells were harvested using 0.25% (w/v) trypsin and dispensed into 96 well plates at 1.0 × 10 4 cells/well. After 24 h incubation, the cells were exposed to different concentrations of R-PE for 72 h. Then, the medium was discarded, 100 µL of MTT solution (1 mg/mL) was added and incubated for 4 h, and dimethyl sulfoxide (DMSO) was used to solubilize the water-insoluble purple formazan crystals. Then, the absorbance of the solution was read at a wavelength of 570 nm [33] using a microplate reader (Bio-Rad, Fitchburg, WI, USA). The percentage inhibition was calculated by comparing optical density (OD) value of drug-treated cells with that of untreated cells. All experiments were repeated six times in six wells of the microplate. The half maximal inhibitory concentration (IC 50 ) was calculated by the Logit method [34,35].

Cell Morphology and Cell Nuclear Morphology Observation (Hoechst 33258)
Cells were seeded in 6-well plates and after 24 h incubation, the medium was removed and replaced with a medium containing R-PE (1.3 µM, 2.6 µM, 5.2 µM) for 48 h. The cell morphology was observed by taking photos under an inverted microscope equipped with a digital camera. The cells were then fixed with ethanol/acetic acid (3:1) at 4 • C for 20 min, and stained with Hoechst 33,258 for 30 min in the dark. The nuclear morphology was observed under a fluorescence microscopy (Leica, Wetzlar, Germany).

Flow Cytometric Analysis of Apoptosis
Cells were seeded in 6-well plates, and incubated for 24 h, The medium was then removed and replaced with a medium containing the R-PE (1.3, 2.6, 5.2 µM) for 48 h. Cells were harvested and washed with phosphate buffer saline (PBS) twice, and then fixed in 1 mL cold 70% ethanol at 4 • C for 12 h. Fixed cells were washed with PBS and re-suspended in 1 mL PI staining solution (1 mg/mL sodium citrate, 50 µg/mL PI, 10 µg/mL RNase A, 0.5% Triton X-100). The cells were then incubated at 37 • C for 30 min in the dark. The data acquisition and analysis of the cell apoptotic rate was performed using MultiCycle software of flow cytometry (Beckman Coulter, XL, Indianapolis, IN, USA).

Flow Cytometric Analysis of Cell Cycle
Cells were seeded in 6-well plates, and after 24 h incubation, the medium was removed and replaced with a medium containing the R-PE (0.1625 µM, 0.325 µM, 0.65 µM) for 48 h. Cells were harvested and washed with PBS twice, and then fixed in 1 mL cold 70% ethanol at 4 • C for 12 h. Fixed cells were washed with PBS and re-suspended in 1 mL PI staining solution (1 mg/mL sodium citrate, 50 µg/mL PI, 10 µg/mL RNase A, 0.5% Triton X-100 ). The cells were finally incubated at 37 • C for 30 min in dark. The distribution of cells in the cell cycle was measured by MultiCycle software of flow cytometry (Beckman Coulter, XL, Indianapolis, IN, USA).

Total Protein Extraction and Western Blot Assay
SGC-7901 cells were treated with 0.1625 µM, 0.325 µM, 0.65 µM R-PE for 24 h. For isolation of total protein fractions [36], cells were harvested and washed twice with PBS, and then the cells were lysed with cell lysis buffer (50 mM Tris-Cl, pH 8.0, 120 mM NaCl, 50 mM NaF, 200 µM sodium vanadate, 0.5% NP-40, 10 mM phenylmethylsulfonyl fluoride (PMSF) and 2 µg/mL aprotinin 0.2 µL, 10 µg/mL Mar. Drugs 2016, 14, 166 8 of 10 leupeptin 10 µL). The lysates were centrifuged at 12,000× g for 10 min at 4 • C, and the supernatant was saved at −20 • C. Protein concentrations of cell lysates were detected by Bradford assay [37]. Total protein samples were separated by polyacrylamide gelelectrophoresis (SDS-PAGE). The separated proteins were transferred to nitrocellulose filter membrane. After being blocked with blocking solution (5% skim milk in TBS, 10 mM Tris-HCl, 150 mM NaCl, pH 7.5 plus 0.1% Tween-20) at room temperature for 2 h, each membrane was incubated with primary antibodies overnight at 4 • C. Afterwards, the membranes were probed with the appropriate horseradish-peroxidase conjugated secondary antibody for 2 h at room temperature. Detection was performed by the 3,3N-diaminobenzidine tertrahydrochloride (DAB) Horseradish Peroxidase Color Development kit (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer's instructions. Bands were recorded and relative density units of the bands were analyzed by a Gel Imaging System (Tanon, GIS-2019, Beijing, China). Densitometrical data of multiple experiments are shown.

Statistical Analysis
The data is presented as the mean ± SD. Statistical significance was calculated using one-way ANOVA. In addition, p-values of ≤5% were considered to indicate statistically significant differences.

Conclusions
The current data demonstrates that R-PE induces apoptosis by arresting the SGC-7901 cell at S phase, which was achieved by decreasing the expression of the CDC25A protein, reducing the CDK2 protein activated and the formation of Cyclin-CDK complex.