Tumor Biol. (2014) 35:1641–1647 DOI 10.1007/s13277-013-1226-9

RESEARCH ARTICLE

Antitumor activity of a sulfated polysaccharide from Enteromorpha intestinalis targeted against hepatoma through mitochondrial pathway Xuxia Wang & Ying Chen & Jingjie Wang & Zhenxiong Liu & Shuguang Zhao

Received: 19 August 2013 / Accepted: 16 September 2013 / Published online: 2 October 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract A sulfated polysaccharide (EI-SP), extracted from Enteromorpha intestinalis that is a kind of algae, is found to have anticancer activity. This study was designed to investigate the anti-tumor effect of EI-SP on human hepatoma HepG2 cell line and its possible mechanisms. An MTT assay showed that EI-SP could specifically inhibit the growth of human hepatoma HepG2 cells in a dose-dependent manner. Analysis by flow cytometry indicated that the apoptosis of tumor cells increased after treatment with EI-SP in range of 100–400 μg/ml. Furthermore, Western blot analysis showed that EI-SP treatment led to decreased protein expression of Bcl-2 and an increase in Bax, cleaved caspase-3, cleaved caspase-9 and cleaved poly(ADP-ribose) polymerase (PARP). Moreover, it was found that EI-SP caused a loss of mitochondrial membrane potential (Δψ m) and the release of cytochrome c to the cytosol. Collectively, our results showed that the EI-SP induces apoptosis in HepG2 cells involving a caspases-mediated mitochondrial signalling pathway. Keywords Enteromorpha intestinalis . Sulfated polysaccharide . Antitumor . Human hepatoma HepG2 cells

Introduction In recent years, many novel bioactive compounds from marine resources have been extensively studied due to their varied biological activities [1, 2]. Among marine resources, X. Wang : J. Wang : Z. Liu : S. Zhao (*) Department of Gastroenterology, Tangdu Hosptial, Fourth Military Medical University, Xi’an 710038, China e-mail: [email protected] Y. Chen West China School of Medicine, Sichuan University, Chengdu 610041, China

marine algae are valuable sources of structurally diverse bioactive compounds with various biological activities [3–5]. Edible marine algae, normally referred as seaweeds, have aroused a special interest as good sources of nutrients and marine algae are rich in sulfated polysaccharides (SPs) such as fucoidans in brown algae, carrageenans in red algae and ulvans in green algae, the uses of which span from food, cosmetic and pharmaceutical industries to microbiology and biotechnology [6]. Recently, their importance as a source of novel bioactive substances is growing rapidly and researchers have revealed that marine algal originated SPs exhibit various biological activities, including anticoagulant [7], antiviral [8], antioxidative [9], anticancer [10] and anti-inflammation [11]. Many studies have documented that Enteromorpha alga is famous as a nutritious and low-calorie food that is rich in essential amino acids of human, fatty acids, vitamins, and many kinds of mineral matters [12–14]. In the past, it was used as edible and medical algae by residents of the coastal districts in China. Enteromorpha intestinalis is s a green alga, belonging to genus Enteromorpha and distributes widely in Southeast Asia water, which has a strong propagation capability and tremendous production. The shoots that grow from a common root are tubular, often gas filled, intestine like and devoid of branching. E. intestinalis has many biological effects, such as clearing away heat, detoxification and anti-inflammation [14]. Since the first report by Zhou et al. [15], the polysaccharide from E. intestinalis exhibits significant hypolipemic and anti-aging activities. In addition, Enteromorpha polysaccharide could promote both cellular and humoral immunity to inhibit the tumor growth [16–18]. However, there is still no available document regarding the antitumor potential of sulfated polysaccharide from E. intestinalis against human hepatoma. Therefore, in this study, we tested its inhibitory activity on aspects of tumor progression using relevant in vitro assays, in an attempt to determine whether the medicinal uses are supported by pharmacological effects.

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Materials and methods Materials Aprotinin, dithiothreitol (DTT) and phenylmethyl-sulfonylfluoride (PMSF) were obtained from Sigma. Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were purchased from Gibco Invitrogen. DEAE-sepharose CL-6B and Sepharose CL-6B were purchased from Amersham. All other chemicals were standard commercial products of analytical grade. Extraction and purification of sulfated polysaccharide The algae E. intestinalis was collected in Taizhou City of Zhejiang Province, China, in June 2012 and dried in an oven at 50 °C. Dried E. intestinalis were extracted thrice with distilled water at 100 °C for 2 h in each time and filtered through four sheets of gauze. The combined extraction solution was concentrated and precipitated by addition of 95 % ethanol (4 volumes). After centrifugation, the precipitate was dried by washing with ethanol, acetone and ether in turn to yield crude sulfated polysaccharides. The crude sulfated polysaccharides was dissolved in distilled water, freeze-thawed, centrifuged at 4 °C until no insoluble substance was visible, and deproteinated using the Sevag method [19]. The crude sulfated polysaccharides was dissolved in distilled water and filtered. After the filtering solution was loaded onto a column (3×30 cm) of DEAE-sepharose CL-6B, the column was first eluted with distilled water and then eluted successively with 0.15→2 M NaCl aqueous solution (pH=6–7) at 4 ml/min, and each tube fraction was combined according to the absorbance detected by Dubois's method at 490 nm. The NaCl-eluted fraction was collected and dialyzed. Then the sample was further purified on a Sepharose CL-6B column (2.6×100 cm) with 0.15 M NaCl at a flow rate of 1 ml/min to yield one fraction: EI-SP. The EI-SP was collected, dialyzed and lyophilized to give white purified polysaccharide fraction. Physicochemical property analysis Total sugar and uronic acid content was determined by phenol–sulfuric acid method [20] and m -hydroxydiphenyl analysis [21], respectively. Estimation of sulfate was measured by turbidimetry following hydrolysis of the corresponding fraction in 1 M HCl and addition of a gelatin/BaCl2 solution [22]. In addition, protein was measured by the Bradford's method using bovine serum albumin (BSA) as the standard [23]. The averaged molecular weight was determined by HPGPC [24], which was performed on a Shimadzu system with a TSK-G3000PWXL column (7.8 mm×30.0 cm) and a Shimadzu RID-10A detector. 0.7 % Na2SO4 was chosen as eluent buffer and the flow rate was 0.7 ml/min at 40 °C with

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1.6 mPa. The averaged molecular weight was estimated according to a calibration curve made from a set of dextran standards (T 130, 80, 50, 20, 10). The identification and quantification of the monosaccharides of EI-SP (10 mg) was achieved by gas chromatography (GC) analysis. EI-SP (10 mg) was hydrolyzed with 2 M TFA at 100 °C for 2 h. The monosaccharides were conventionally converted into the alditol acetates as described previously [24] and were analyzed by GC. Cell lines and MTT assay Human cervical cancer cells (HeLA), human renal adenocarcinoma cell line (ACHN), human hepatoma cell lines (Bel7402 and HepG2), human breast cancer cells (MCF-7) and human colon cancer cells (HT-29) and human normal liver cell line (L-O2) were purchased from the China Center for Type Culture Collection. They all were cultured in RPMI1640 supplemented with 10 % FCS, penicillin (100 IU/ml) and streptomycin (100 μg/ml) in a humidified atmosphere of 5 % CO2 at 37 until confluent. In vitro antitumor activity of EI-SP was determined using six kinds of tumor cells (HeLA, ACHN, Bel-7402, HepG2, MCF-7 and HT-29), together with a normal human liver cell line L-O2. Various cells were seeded in 96-well flat-bottomed plates and allowed to adhere for 24 h at 37 °C with 5 % CO2 atmosphere. For MCF-7, HepG2 and L-O2, 1×104 cells were incubated in 96-well plates containing 100 μl of the growth medium per well; while for HeLa, Bel-7402, HT-29 and ACHN, 5×103 cells were seeded per well. Polysaccharide solutions (100 μl), at concentrations of 25, 50, 100, 200, 400, or 800 μg/ml were added to the wells, and the cells were cultured for 48 h. After this incubation, 20 μl of the MTT (pH4.7) was added to each well, and the solution was further incubated for 4 h at 37 °C. The supernatant fluid was then removed, 100 μl/well DMSO was added and samples were shaken for 15 min. Absorbance at 570 nm was measured with a microplate reader (Bio-Rad, Richmond, CA, USA) using wells without cells as blanks. Three independent experiments were performed. The IC50 value was determined as the concentration that caused 50 % inhibition of cell proliferation [25]. Acridine orange/ethidium bromide (AO/EB) staining assay AO/EB double fluorescent dyes were used to qualitatively observe apoptotic morphology of individual cells in a cell population as described previously [26]. AO can emit a green fluorescence if it passes through the complete cell membrane and embeds in nuclear DNA while EB can mark nuclear DNA of damaged cells and emit a red-orange fluorescence [27]. Briefly, HepG2 cells were treated with vehicle or EI-SP at 100, 200 and 400 μg/ml for 48 h and then immediately stained

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with AO/EB. Morphological changes were observed using fluorescent microscope in a blinded manner.

fraction. Cytosolic fraction was stored at −80 °C until ready for Western blot analysis.

Assay for cell apoptosis by annexin V/propidium iodide (PI) double staining

Western blot analysis

HepG2 cells (2×105 cells per well) were incubated in a 12-well plate for 48 h in the presence of indicated concentrations of EI-SP. After the incubation, the cells were washed with PBS and used for determining apoptosis. Apoptotic cell death was identified by double supravital staining with recombinant FITC (fluorescein isothiocyanate) conjugated annexin V and PI, using the annexin V–FITC Apoptosis Detection kit (Becton Dickinson, Frankly Lakes, NJ, USA) according to the manufacturer's specifications. Flow cytometric analysis was performed immediately after the staining. Data acquisition and analysis were performed in a Becton Dickinson FACSCalibur flow cytometer using CellQuest software. In each analysis, 10,000 events were recorded. Apoptotic rate was calculated as the relative number of apoptotic cells compared to the total number. Assay for change of mitochondrial membrane potential (Δψ m) The changes in Δψ m were estimated using the fluorescent cationic dye rhodamine123 (Rh123), which accumulates in mitochondria as a direct function of the membrane potential and is released upon membrane depolarization. Briefly, 1 × 10 6 cells/ml HepG2 cells were plated in a 6-well plate, exposed to different concentrations of EI-SP in the presence of 100, 200 and 400 μg/ml EI-SP for 48 h, respectively, or left untreated, washed with PBS and incubated with 10 μM Rh-123 at 37 °C for 30 min in the dark. Cells were then washed twice with PBS and suspended in PBS (0.1 M, pH 7.8) prior to flow cytometry. The percentage of cells that had lost Δψ m was calculated using CellQuest software. Preparation of cytosolic and mitochondrial extract The subcellular fractions were prepared as described previously [28]. The harvested pellets were suspended in 100 μl of buffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM DTT, 1 μg/ml aprotinin, 100 μg/ml PMSF, and 250 mM sucrose). After incubation on ice for 10 min, homogenize cells in an ice-cold dounce tissue grinder (45 strokes) until 70–80 % of the nuclei did not have the shiny ring and centrifuge at 700×g for 10 min at 4 °C. The supernatant was collected and further centrifuged at 10,000×g for 30 min at 4 °C to isolate cytosolin

An equal amount of protein (30 μg) was separated electrophoretically by 8 % to 12 % SDS-PAGE and transferred onto nitrocellulose membranes. The blots were probed with the adequate concentration of the primary antibodies specific to Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, cytochrome c , or β-actin, respectively. The membrane was then incubated with appropriate HRP-conjugated secondary antibody, and the protein expression was detected by enhanced chemiluminescence reagents. Each membrane was stripped and reprobed with anti-β-actin antibody to ensure equal protein loading. Statistical analysis All the results were expressed as the mean ± SD. The data were analyzed statistically by ANOVA. Significance of any differences between groups was evaluated using Student's t -test. P values of less than 0.05 were considered significant.

Results and discussion Isolation and characterization of sulfated polysaccharide EI-SP The crude sulfated polysaccharide was prepared from the algae E. intestinalis by hot-water extraction, EtOH precipitation and protein removal by Sevag method and dialysis. After crude sulfated polysaccharide was subjected to a DEAE-sepharose CL-6B chromatography, one fraction eluted by 0.15–2 M NaCl was collected for further purification on a Sepharose CL-6B column to give a white purified polysaccharide fraction, named EI-SP. As determined by the phenol–sulfuric acid method, the Bradford's method and m hydroxydiphenyl analysis, the content of total sugar, protein and uronic acid of EI-SP were 84.76 %, 2.16 % and 6.24 %, respectively. In addition, EI-SP was a sulfated heteropolysaccharide, as indicted by the 16.05 % sulfate content (Table 1). A UV scan in the region of 200–400 nm showed strong absorbance at about 200 nm and weak absorbance at 280 nm, which further indicated that LRGP1 was a glycoconjugate. The sugar composition determined by GC showed that EI-SP was composed mainly of rhamnose, xylose, galactose, glucose, and glucuronic acid in a molar ratio of 6.5:1.2:0.4:0.2:0.9. The HPGPC profile of EI-SP

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Table 1 The content of total sugar, protein and uronic acid of EI-SP and its monosaccharide composition Sample

EI-SP

Monosaccharide composition (molar ratio) Rhamnose

Xylose

Galactose

Glucose

Glucuronic acid

6.5

1.2

0.4

0.2

0.9

(Fig. 1) showed a single and symmetrically sharp peak, indicating that EI-SP was a homogeneous polysaccharide, with a weight-average molecular weight of ~5.35 × 105 Da. Cytotoxicity of EI-SP The cytotoxicity of EI-SP on various cancer cells was evaluated by an MTT assay and the IC50 values were derived from the dose–response curves (Table 2). A 48h exposure to EI-SP had the greatest cytotoxicity on human hepatoma cell lines Bel-7402 (IC50 =108.0 μg/ ml) and HepG2 (IC50 = 98.5 μg/ml) than other cancer cell lines (IC50 > 300 μg/ml). Of the cancer cells, the human hepatoma cell line HepG2 was most susceptible to EI-SP treatment. To test the safety of EI-SP in normal cells, we added EI-SP to human normal liver cell line L-O2. In contrast, the IC50 value of L-O2 cells was more than 500 μg/ml. Thus, EI-SP seemed to have a lower toxicity against normal cells than against human hepatoma cells. Together, these data indicated that EI-SP may possess relative selective cytotoxicity to human hepatoma cells, especially for HepG2 cells. Therefore HepG2 cell line was used in the following experiments to elucidate the antitumor mechanism of EI-SP. EI-SP induces apoptotic death of HepG2 cells To determine whether HepG2 cells treated with EI-SP underwent apoptosis, EI-SP-treated cells were stained

Fig. 1 HPGPC Profile of EI-SP

Carbohydrate (%)

Uronic acid (%)

Protein (%)

Sulfate (%)

84.76

2.16

6.24

16.05

with annexin V–FITC and PI, and then subsequently analyzed by flow cytometry. As indicated by the flow cytometry, with the increase in the concentrations of EI-SP (100–400 μg/ml), apoptotic death increased approximately from 30.2 % to 62.0 % (Fig. 2). Furthermore, the dose-dependence of EI-SP inducing apoptosis in HepG2 cells was also examined using AO/EB staining (Fig. 3). The cell morphology treated by EI-SP displayed typical apoptotic features including chromatin condensation and nuclear fragmentation [29]. These results suggest that HER-induced cell death is mainly due to apoptosis. Effect of EI-SP on Bax and Bcl-2 protein expression in HepG2 cells Mitochondrial integrity is regulated by pro-apoptotic and anti-apoptotic members of the Bcl-2 group of proteins such as Bcl-2 (anti-apoptotic) and Bax (proapoptotic) [30]. Since the Bcl-2 family proteins play a crucial role in regulating the mitochondrial-mediated apoptosis pathway; we next studied the effect of EISP on the expression of the pro-apoptotic and antiapoptotic Bcl-2 proteins in HepG2 cells. Western blot analysis revealed that EI-SP induced an increase in the level of the pro-apoptotic Bax protein in HepG2 cells in a concentration-dependent manner (Fig. 4). In contrast, the level of the anti-apoptotoic Bcl-2 protein decreased upon EI-SP treatment. Thus, an increase in the pro-apoptotic/ antiapoptotic ratio of Bax to Bcl-2 was significantly observed after treatment with EI-SP, suggesting the

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Table 2 Comparison of cytotoxic activity of EI-SP on the seven cell lines by MTT assay Cell lines

IC50 (μg/ml)

Human cervical cancer cells (HeLA) Human renal adenocarcinoma cells (ACHN) Human hepatoma cells (Bel-7402) Human hepatoma cells (HepG2)

338.8 >500 108.0 98.5

Human breast cancer cells (MCF-7) Human colon cancer cells (HT-29) Human normal liver cells (L-O2)

>500 326.7 >500

involvement of Bcl-2 family proteins in EI-SP-induced apoptosis in HepG2 cells. Effect of EI-SP on the loss of mitochondria membrane potential (Δψ m) and the release of cytochrome c in HepG2 cells A decrease in Δψ m disrupts the outer mitochondrial membrane, followed by the release of cytochrome c ,

Fig. 2 Flow cytometric analysis of EI-SP-induced apoptosis in HepG2 cells using annexin V–FITC/PI. The data represent the mean ± SD of three independent experiments. ***P