A Novel Polysaccharide Conjugate from Bullacta Exarata Induces G1-Phase Arrest and Apoptosis in Human Hepatocellular Carcinoma Hepg2 Cells

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Article A Novel Polysaccharide Conjugate from Bullacta exarata Induces G1-Phase Arrest and Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells

Ningbo Liao 1, Liang Sun 1, Jiang Chen 1,*, Jianjun Zhong 2, Yanjun Zhang 1 and Ronghua Zhang 1,*

1 Department of Nutrition and Food Safety, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China; [email protected] (N.L.); [email protected] (L.S.); [email protected] (Y.Z.) 2 College of Biosystem Engineering and Food Science, Zhejiang University, Hangzhou 310058, China; [email protected] * Correspondence: [email protected] (J.C.); [email protected] (R.Z.); Tel./Fax: +86-571-87115247 (J.C.); +86-571-87115214 (R.Z.)

Academic Editors: Quan-Bin Han, Sunan Wang and Shaoping Nie Received: 25 January 2017; Accepted: 25 February 2017; Published: 1 March 2017

Abstract: Bullacta exarata has been consumed in Asia, not only as a part of the normal diet, but also as a traditional Chinese medicine with liver- and kidney-benefitting functions. Several scientific investigations involving extraction of biomolecules from this mollusk and pharmacological studies on their biological activities have been carried out. However, little is known regarding the antitumor properties of polysaccharides from B. exarata, hence the polysaccharides from B. exarata have been investigated here. One polysaccharide conjugate BEPS-IA was isolated and purified from B. exarata. It mainly consisted of mannose and glucose in a molar ratio of 1:2, with an average molecular weight of 127 kDa. Thirteen general amino acids were identified to be components of the protein-bound polysaccharide. Methylation and NMR studies revealed that BEPS-IA is a heteropolysaccharide consisting of 1,4-linked-α-D-Glc, 1,6-linked-α-D-Man, 1,3,6-linked-α-D-Man, and 1-linked-α-D-Man residue, in a molar ratio of 6:1:1:1. In order to test the antitumor activity of BEPS-IA, we investigated its effect against the growth of human hepatocellular carcinoma cells HepG2 in vitro. The result showed that BEPS-IA dose-dependently exhibited an effective HepG2 cells growth inhibition with an IC50 of 112.4 µg/mL. Flow cytometry analysis showed that BEPS-IA increased the populations of both apoptotic sub-G1 and G1 phase. The result obtained from TUNEL assay corroborated apoptosis which was shown in flow cytometry. Western blot analysis suggested that BEPS-IA induced apoptosis and growth inhibition were associated with up-regulation of p53, p21 and Bax, down-regulation of Bcl-2. These findings suggest that BEPS-IA may serve as a potential novel dietary agent for hepatocellular carcinoma.

Keywords: Bullacta exarata; polysaccharide; human hepatocellular carcinoma HepG2 cells; apoptosis

1. Introduction Since ancient times, Bullacta exarata, a kind of shell mollusk native of Asia, has been consumed by humans, not only as a part of the normal diet, but also as a delicacy because they have a highly desirable taste and aroma. In addition, the nutritional, tonic, and medicinal properties of B. exarata have been recognized for a long time [1]. We previously reported that a B. exarata polysaccharide (BEPS-IB) showed much more effective antioxidant activity in scavenging superoxide radicals in vitro [2]. Furthermore, Zhang et al. showed that BEP-3, a bioactive polysaccharide, exhibited antitumor

Molecules 2017, 22, 384; doi:10.3390/molecules22030384 www.mdpi.com/journal/molecules Molecules 2017, 22, 384 2 of 14 activity against human pancreatic cancer SW1990 cells, human breast cancer Bcap37 cells and human HeLa cervical cancer cells [3]. Recently, some studies have also shown that the extracts of B. exarata possess a broad range of biological activities including hepato-protective, antioxidant, anticancer, antihypertension, and hypocholesterolemic effects [2–6]. However, the active constituents of B. exarata have not been studied extensively. Hepatocellular carcinoma (HCC) characteristically presents with a poor prognosis and is one of the major leading causes of cancer death worldwide, especially in Eastern Asia [7]. At present, HCC is the eighth most common malignancy in women, the fifth most common malignancy in men worldwide and the number of incident cases and deaths continues to increase year by year [8]. Although knowledge about HCC is expanding exponentially in recent years, treatment and prevention of HCC are still a big challenge. On diagnosis most HCC patients are terminal. Despite the fact that chemotherapy is a common therapeutic strategy after surgery, its application is hindered by growing multidrug resistance in tumor cells and limited due to its serious adverse effects. Therefore, finding new nontoxic chemo-preventive or chemotherapeutic agents with fewer side effects is essential. Polysaccharide-protein complexes are biopolymers comprised of the protein or polypeptide covalently linked together with carbohydrates as the side-chains. These structures can be linear or contain branched side chains. Polysaccharide-protein complexes were obtained from different sources, such as algae, plants, microorganisms, and animals. Previous studies had revealed that polysaccharide-protein complexes can inhibit the tumor cells growth in vitro by activating multiple signal pathways including cell cycle arrest, DNA damage, and alteration of death inhibitors or promoters expression [9]. In vitro experiments, Pleurotus abalonus polysaccharide-peptide LB-1b, Streptococcus pyogenes glycoprotein SAGP, Hypsizigus marmoreus glycoprotein HM3A and Lycium barbarum polysaccharide-protein complex could kill the tumor cell lines (liver, lymphoma, leukemia, stomach, and lung) directly via inducing cell cycle arrest, up-regulating Bax gene expression and/or down-regulating Bcl-2/Bcl-xl genes expression [10–14]. However, the antitumor effects of polysaccharide-protein complexes upon liver cancer have not yet been fully investigated. Moreover, the mechanism of direct inhibition of cell proliferation has also not been completely elucidated. In the present study, a novel polysaccharide–protein complex (BEPS-IA) was extracted and purified from B. exarata. Then the chemical structures were analyzed. Finally, we have investigated the antitumor activity of BEPS-IA against human hepatocellular carcinoma HepG2 cells, and its role on cell cycle arrest and apoptosis of HepG2 cells, and reveal a possible signal pathway for elucidating the BEPS-IA0 anticancer molecular mechanisms.

2. Results and Discussion

2.1. Composition Analyses and Image of BEPS-IA

The chemical compositions and contents of carbohydrate, protein, MW and sulfonic acid in BEPS-IA have been reported previously (Table S1) [2]. In this study, the AFM image of BEPS-IA is shown in Figure1. The ﬁber-like structure and small branches of BEPS-IA molecule can be observed. The chain length of the backbone was approximately 948–1032 nm and the height was 53–67 nm. Monosaccharide composition analysis, carried out by HPLC following acid hydrolysis and derivatization with PMP, showed that BEPS-IA was composed of primarily of glucose and mannose, with a molar ratio of about 2:1, whereas others (e.g., galactose (Gal), and arabinose) were in a minor content. The amino acid composition in the polysaccharide-protein complex was analyzed by the HPLC AccQ method. As shown in Table1, thirteen amino acids were identiﬁed to be components of the macromolecule. BEPS-IA was rich in glycine (22.14 mg/g), followed by asparagine (16.73 mg/g), glutamic acid (12.27 mg/g), alanine (9.26 mg/g) and valine (8.95 mg/g). Molecules 2017, 22, 384 3 of 14 Molecules 2017, 22, 384 3 of 15

Figure 1. Atomic force microscopy (AFM) image ofof B.B. exarataexarata polysaccharide-proteinpolysaccharide-protein complex BEPS-IA was obtained with a NanoNano ScopeScope IIIIII atomicatomic forceforce microscopemicroscope andand collectedcollected by tappingtapping mode atomic force microscopy. microscopy. The The BEPS-IA BEPS-IA concentration concentration was was 1 μ 1g/mL.µg/mL. (A) Topographic (A) Topographic image image of a 90 of min a 90 BEPS- min BEPS-IAIA sample sample taken takenby AFM by in AFM air at in room air at temperature; room temperature; (B) 3D (visualizationB) 3D visualization of BEPS-IA of BEPS-IA polymer polymer reveal revealthin and thin thick and polymer thick polymer portions portions corresponding corresponding to areas to highlighted areas highlighted with arrows with arrows in (A). in (A).

Table 1.1. The amino acid compositioncomposition ofof BEPS-IA.BEPS-IA. Amino Acid Concentration (mg/g) a a AminoAsparagine Acid 16.73Concentration ± 1.37 (mg/g) AsparagineSerine 5.3316.73 ± ±2.111.37 SerineGlutamic acid 12.275.33 ± ± 2.113.79 GlutamicGlycine acid 22.1412.27 ± 0.683.79 ± GlycineArginine 4.1222.14 ± 1.610.68 Arginine 4.12 ± 1.61 Threonine b 1.04 ± 2.14 Threonine b 1.04 ± 2.14 Alanine 9.26 ± 2.12 Alanine 9.26 ± 2.12 ProlineProline 5.12 ± 2.012.01 CysteineCysteine 4.28 ± 0.170.17 ValineValineb b 8.95 ± 1.421.42 MethionineMethionineb b 1.21 ± 2.042.04 LysineLysineb b 2.41 ± 0.260.26 LeucineLeucineb b 6.32 ± 0.240.24 TotalTotal amino amino acids acids 99.18 ± 6.436.43 ProportionProportion of of the the essential essential amino amino acids acids (%) (%) 20.09 ± 5.625.62 a Dataa Data are are shown shown as as mean mean ±± SD ((nn == 3);3);b bEssential Essential amino amino acids. acids.

2.2. UV and IR Spectrum of BEPS-IA 2.2. UV and IR Spectrum of BEPS-IA The absorption peak at 280 nm in the UV spectrum, indicated that BEPS-IA contained protein The absorption peak at 280 nm in the UV spectrum, indicated that BEPS-IA contained protein portions. The IR spectrum of BEPS-I exhibited some characteristic absorption peaks of polysaccharide. portions. The IR spectrum of BEPS-I exhibited some characteristic absorption peaks of polysaccharide. As shown in Figure 2, the peak at 3420.7 cm−1 would be due to the stretching vibration of O–H and/or As shown in Figure2, the peak at 3420.7 cm −1 would be due to the stretching vibration of O–H and/or N–H. The peak at 2934.1 cm−1 would be due to the stretching vibration of C–H. The band at 1654.8 cm−1 N–H. The peak at 2934.1 cm−1 would be due to the stretching vibration of C–H. The band at 1654.8 cm−1 was attributed to the stretching vibration of C–O and/or the variable-angle vibration of N–H. The was attributed to the stretching vibration of C–O and/or the variable-angle vibration of N–H. The band at 1481.6 cm−1–1372.2 cm−1 was attributed to the stretching vibration of C–O, and the band at band at 1481.6 cm−1–1372.2 cm−1 was attributed to the stretching vibration of C–O, and the band at 1341.1 cm−1–1235.5 cm−1 was attributed to the stretching vibration of O–H [15]. The characteristic 1341.1 cm−1–1235.5 cm−1 was attributed to the stretching vibration of O–H [15]. The characteristic absorption at 876.1–904.8 cm−1 would be due to the presence of β-glycosidic bonds in BEPS-IA [16]. absorption at 876.1–904.8 cm−1 would be due to the presence of β-glycosidic bonds in BEPS-IA [16]. Moreover, absorption bands at 820.2, and 842.4 cm−1 indicated the presence of mannose and glucose in Moreover, absorption bands at 820.2, and 842.4 cm−1 indicated the presence of mannose and glucose in BEPS-IA. A characteristic absorption at 854.6 cm−1 was also observed, indicating the α-configuration of BEPS-IA. A characteristic absorption at 854.6 cm−1 was also observed, indicating the α-conﬁguration of BEPS-IA units [16]. All these findings indicated the possibility that the polysaccharide BEPS-IA mainly BEPS-IA units [16]. All these ﬁndings indicated the possibility that the polysaccharide BEPS-IA mainly composed of mannose and glucose, containing both α-and β-glycosidic bonds, is a proteoheteroglycan. composed of mannose and glucose, containing both α-and β-glycosidic bonds, is a proteoheteroglycan.

Molecules 2017, 22, 384 4 of 14 Molecules 2017, 22, 384 4 of 15 Molecules 2017, 22, 384 4 of 15

Figure 2. FT-IR spectrum of B. exarata polysaccharide-protein complex BEPS-IA. Figure 2. FT-IR spectrum of B. exarata polysaccharide-protein complex BEPS-IA. Figure 2. FT-IR spectrum of B. exarata polysaccharide-protein complex BEPS-IA. 2.3. Structure Data from NMR 2.3. Structure Data from NMR 2.3. StructureNMR Data spectroscopy from NMR has become the most powerful technique for the structure analysis of NMRcarbohydrates. spectroscopy As shown has in becomeFigure 3A, the the most 1H spectrum powerful showed technique peaks in for the theanomeric structure region analysis at 4–6 of NMR spectroscopy has become the most powerful technique for the structure analysis of carbohydrates.ppm. In this Asspectrum, shown signals in Figure 4.82,3 4.91,A, the 5.011 andH spectrum 5.17 ppm, showedimplied that peaks the insugar the rings anomeric of BEPS-IA region at carbohydrates. As shown in Figure 3A, the 1H spectrum showed peaks in the anomeric region at 4–6 were pyranose rings and the sugar residues of BEPS-IA were connected by α- and β-configuration 4–6ppm. ppm. In this In this spectrum, spectrum, signals signals 4.82, 4.82, 4.91, 4.91, 5.01 5.01 and and 5.17 5.17 ppm, ppm, implied implied that that the the sugar sugar rings rings of of BEPS-IA BEPS-IA glycosidic bonds [16]. α β werewere pyranose pyranose rings rings andand thethe sugarsugar residuesresidues ofof BEPS-IABEPS-IA werewere connected by α-- and β-configuration-conﬁguration glycosidicglycosidic bonds bonds [ 16[16].].

Figure 3. NMR spectra of the B. exarata polysaccharide-protein complex BEPS-IA in D2O. (A) 1H-NMR and (B) 13C-NMR.

Figure 3. NMR spectra of the B. exarata polysaccharide-protein complex BEPS-IA in D2O. (A) 11H-NMR Figure 3. NMR spectra of the B. exarata polysaccharide-protein complex BEPS-IA in D2O. (A) H-NMR and (B) 13C-NMR. and (B) 13C-NMR.

Molecules 2017, 22, 384 5 of 14

There were also signals of other sugar protons at 3.30–4.31 ppm. According to Dore et al., 2014 [17], the chemical shifts correspond to C2 and C6. We also observed that the regions between 1.6 and 2.4 are related to the glucan-protein structure. These observations are in accord with other studies [17]. In addition, the 13C-NMR spectrum of BEPS-IA (Figure3B) had no signal at low ﬁeld from 160 to 180 ppm, which illustrated that it did not contain uronic acid. Based on the available data in the literature [18],Molecules the resonances 2017, 22, 384 of 99.28–105.06 ppm were attributed to the anomeric5 of 15 carbon atoms of mannopyranose (Manp) and glucopyranose (Glcp), respectively. Other important signals in the There were also signals of other sugar protons at 3.30–4.31 ppm. According to Dore et al., 2014 [17], spectra are thosethein chemical the 60–80 shifts correspond ppm range, to C2 and where C6. We they also areobserved related that the to regions C2 (73.71 between ppm), 1.6 andC3 2.4 (84.3 ppm), C4 (80.0 ppm), C5are (76.18 related ppm) to the glucan-protein and C6 (61.92 structure. ppm) These of observations that carbohydrate are in accord with [15 other,16]. studies The 47.8[17]. ppm signal is In addition, the 13C-NMR spectrum of BEPS-IA (Figure 3B) had no signal at low field from 160 to 180 ppm, related to the –CHwhich2 Nillustrated group that of it aminodid not contain acids. uronic The acid. presence Based on the of available additional data in peaksthe literature in the[18], range of 20–40 ppm may suggestthe resonances the presence of 99.28–105.06 of a glucan–protein ppm were attributed structureto the anomeric [18 carbon]. These atoms NMRof mannopyranose results were consistent (Manp) and glucopyranose (Glcp), respectively. Other important signals in the spectra are those in with those of thethe FTIR60–80 ppm spectrum. range, where they are related to C2 (73.71 ppm), C3 (84.3 ppm), C4 (80.0 ppm), C5 (76.18 ppm) and C6 (61.92 ppm) of that carbohydrate [15,16]. The 47.8 ppm signal is related to the 2.4. Structure Data–CH2 fromN group Methylation of amino acids. Analysis The presence of additional peaks in the range of 20–40 ppm may suggest the presence of a glucan–protein structure [18]. These NMR results were consistent with those The fully methylatedof the FTIR spectrum. BEPS-IA was hydrolyzed with acid, converted into alditol acetates, and analyzed by GC–MS.2.4. Structure As Data shown from Methylation in Table Analysis2, the presence of four major compounds, 2,3,6-Me 3-Glc, 2,4-Me2-Man, 2,3,4-MeThe fully3-Man methylated and BEPS-IA 2,3,4,6-Me was hydrolyzed4-Man, wi whichth acid, converted indicate into thealditol presence acetates, and of 4-linked Glc, 3,6-linked Man,analyzed 6-linked by GC–MS. Man, andAs shown terminal in Table Man, 2, the inpresence a molar of four ratio major of compounds, about 6:1:1:1. 2,3,6-Me The3-Glc, high proportion 2,4-Me2-Man, 2,3,4-Me3-Man and 2,3,4,6-Me4-Man, which indicate the presence of 4-linked Glc,→ 3,6-linked of 4-linked Glc indicatesMan, 6-linked that Man, the and main terminal consecutive Man, in a molar repeating ratio of about unit 6:1:1:1. of BEPS-IThe high isproportion (1 4)-linked of 4- Glc. On the basis of above-mentionedlinked Glc indicates results, that the main the consecutive monomer repeating of BEPS-IA unit of BEPS-I repeat is (1→ unit4)-linked was Glc. determined On the to be as illustrated in Figurebasis of4 above-mentionedbelow. results, the monomer of BEPS-IA repeat unit was determined to be as illustrated in Figure 4 below.

Figure 4. ProposedFigure 4. Proposed structural structural feature feature of the the BEPS-IA BEPS-IA isolated isolated from B. exarata from. B. exarata.

Table 2. GC-MS of alditol acetate derivatives from the methylated product of BEPS-IA. Table 2. GC-MS of alditol acetate derivatives from the methylated product of BEPS-IA. Methylated Sugar Molar Ratio Mass Fragment (m/z) b Type of Linkage 2,3,6-Tri-O-Me-Glc a 6.46 45, 87, 101, 113, 117, 161, 233 →4)-Glcp-(1→ Methylated2,4,-Di- SugarO-Me-Man Molar Ratio 1.13 Mass 71, 87, Fragment 99, 101, 189 ( m/z) b →3,6)-ManTypep-(1→ of Linkage → → 2,3,4,-Tri-aO-Me-Man 0.87 45, 71, 87, 101, 129, 161, 189 6)-Manp-(1 2,3,6-Tri-O2,3,4,6-Tetra--Me-Glc O-Me-Man 6.46 0.91 28, 43, 45, 71, 87, 87, 101, 117, 113, 129, 117, 145, 161, 161,205 233 Manp-(1→→ 4)-Glcp-(1→ 2,4,-Di-O-Me-Man 1.13 71, 87, 99, 101, 189 →3,6)-Manp-(1→ a 2,3,6-Tri-O-Me-Glc = 1,5,4-tri-O-acetyl-2,3,6-tri-O-methyl-D-glucitol. b Equipped with a OV1701 capillary → → 2,3,4,-Tri-Ocolumn-Me-Man (30 m × 0.25 mm 0.87internal diameter) using 45, a temperature 71, 87, 101, program 129, 161, from 189 150 °C (2 min) to 2506)-Man °C p-(1 2,3,4,6-Tetra-(5O min)-Me-Man at 3 °C·min−1. 0.91 28, 43, 71, 87, 101, 117, 129, 145, 161,205 Manp-(1→ a 2,3,6-Tri-O-Me-Glc = 1,5,4-tri-O-acetyl-2,3,6-tri-O-methyl-D-glucitol. b Equipped with a OV1701 capillary column (30 m × 0.252.5. mm Anti-Proliferation internal diameter) and Cytotoxicity using Assay a temperature program from 150 ◦C (2 min) to 250 ◦C (5 min) at ◦ −1 3 C·min . In this study, the anti-proliferation activity of BEPS-IA was tested on human hepatocellular carcinoma HepG2 Cells, and the common antitumor drug 5-FU was used as a positive control. Figure 5A 2.5. Anti-Proliferationshows that and BEPS-IA Cytotoxicity and 5-FU displayed Assay dose dependent inhibition effects on HepG2 cells. The amount of BEPS-IA and 5-FU required to inhibit the proliferation of HepG2 cells by 50% (IC50) was 112.4 ± 22.53 and 23.94 ± 5.17 μg/mL, respectively. BEPS-IA (from 0 to 200 μg/mL) dose- and time-dependently In this study,inhibited the the anti-proliferation proliferation of HepG2 cells activity (Figure 5). of With BEPS-IA 200 μg/mL was of BEPS-IA, tested the onproliferation human of hepatocellular carcinoma HepG2the HepG2 Cells, cells andwas inhibited the common by 56% after antitumor 72 h. To further drug evaluate 5-FU the anti wasproliferative used aseffects a of positive control. Figure5A showsBEPS-IA that BEPS-IAon human hepatocellular and 5-FU carcinoma displayed HepG2 dose cells, dependent the dose of BEPS-IA inhibition was increased effects from on HepG2 cells. 200 to 700 μg/mL. At concentrations >200 μg/mL, BEPS-IA also dose-dependently inhibited HepG2 The amount ofcell BEPS-IA proliferation. and At 5-FUthe highest required dose (700 to μg/mL), inhibit HepG2 the cell proliferation proliferation was of inhibited HepG2 by about cells by 50% (IC50) was 112.4 ± 22.5374.3% and (Figure 23.94 5A). However,± 5.17 µtheg/mL, study ofrespectively. cytotoxicity against BEPS-IA HepG2 cells (from by the BEPS-IA 0 to 200 showedµg/mL) dose- and time-dependently inhibited the proliferation of HepG2 cells (Figure5). With 200 µg/mL of BEPS-IA, the proliferation of the HepG2 cells was inhibited by 56% after 72 h. To further evaluate the antiproliferative effects of BEPS-IA on human hepatocellular carcinoma HepG2 cells, the dose of BEPS-IA was increased from 200 to 700 µg/mL. At concentrations >200 µg/mL, BEPS-IA also dose-dependently inhibited HepG2 cell proliferation. At the highest dose (700 µg/mL), HepG2 cell proliferation was inhibited by about 74.3% (Figure5A). However, the study of cytotoxicity against HepG2 cells by the BEPS-IA showed that the polysaccharide, above a dose of 200 µg/mL, exhibit cytotoxicity toward HepG2 cells Molecules 2017, 22, 384 6 of 14

Molecules 2017, 22, 384 6 of 15 in vitro (Cytotoxicitythat the polysaccharide, was determined above a dose by of a200 10% μg/mL, reduction exhibit cytotoxicity of absorbance toward atHepG2 570 nmcells readingin vitro for each concentration(Cytotoxicity compared was determined to the control), by a 10% reduction as shown of absorbance in Figure at S1. 570 Therefore,nm reading for the each low concentration doses of BEPS-IA (from 0 tocompared 200 µg/mL) to the were control), chosen as shown for thein Figure subsequent S1. Therefore, experiments. the low doses of BEPS-IA (from 0 to 200 μg/mL) were chosen for the subsequent experiments.

Figure 5. Growth inhibitory effect of B. exarata polysaccharide-protein complex BEPS-IA on HepG2 cells. Figure 5. Growth(A) HepG2 inhibitory cells were treated effect with of B.various exarata concentrations polysaccharide-protein of BEPS-IA for 72 h; ( complexB) The antiproliferative BEPS-IAon HepG2 cells. (A)activity HepG2 of BEPS-IA cells were (0, 50, treated100 and 200 with μg/mL, various respectively) concentrations on the HepG2 of cells BEPS-IA growth at for24, 48, 72 72 h; (B) The antiproliferativeand 96 h. activity Untreated of cells BEPS-IA (0 μg/mL) (0, group 50, 100was andused as 200 negativeµg/mL, control respectively) and 5-fluorouracil on the (5-FU) HepG2 cells growth atgroup 24, 48, was 72 used and as 96a positive h. Untreated control. The cells data (0weµreg/mL) expressed group as mean was ± SD used of three as negativeexperiments. control and Values marked with * are significantly different compared to the control (p < 0.05). 5-fluorouracil (5-FU) group was used as a positive control. The data were expressed as mean ± SD of three2.6. experiments. Effect of BEPS-IA Values on markedCell Cycle with Distribution * are significantly of HepG2 Cells different compared to the control (p < 0.05). To estimate the effect of BEPS-IA treatment on the distribution of cells in the cell cycle, we performed 2.6. EffectDNA of BEPS-IA cell cycle on analysis Cell Cycle by flow Distribution cytometry (Figure of HepG2 S2). CellsThe sub-G1 cell fraction was considered to represent apoptotic cells. As shown in Figure 6, the results revealed that BEPS-IA produced 2.1-, To estimate2.8-, and 6.5-fold the effect increases of BEPS-IAin the apoptosis treatment of HepG2 on cells the at concentrat distributionions of of50, 100, cells and in 200 the μg/mL, cell cycle, we performedrespectively, DNA cell compared cycle analysis with that induced by flow with cytometry 0 μg/mL BEPS-IA (Figure (Control). S2). The The sub-G1effects of BEPS-IA cell fraction was consideredtreatment to represent for 24 h on apoptotic the HepG2 cells. cell-cycle As phase shown distribution in Figure are also6, the observed. results Compared revealed with that the BEPS-IA control (41.2%), treatment with 50, 100, or 200 μg/mL BEPS-IA for 24 h increased the population of produced 2.1-, 2.8-, and 6.5-fold increases in the apoptosis of HepG2 cells at concentrations of 50, cells in G1 phase to 62.3%, 74.2%, or 46.8%, respectively. These results showed that BEPS-IA treatment 100, and 200 µg/mL, respectively, compared with that induced with 0 µg/mL BEPS-IA (Control).

The effects of BEPS-IA treatment for 24 h on the HepG2 cell-cycle phase distribution are also observed. Compared with the control (41.2%), treatment with 50, 100, or 200 µg/mL BEPS-IA for 24 h increased the population of cells in G1 phase to 62.3%, 74.2%, or 46.8%, respectively. These results showed that BEPS-IA treatment of HepG2 cells induced the accumulation of G1 phase cells. We also found that as the concentration of BEPS-IA increased, the sub-G1 apoptotic fraction of cells increased signiﬁcantly from 3.2% to 22.8%. Therefore, the induction of G1-phase arrest and an increased sub-G1 apoptotic fraction may be the major mechanisms by which the growth of HepG2 cell is inhibited. Molecules 2017, 22, 384 7 of 15

of HepG2 cells induced the accumulation of G1 phase cells. We also found that as the concentration of BEPS-IA increased, the sub-G1 apoptotic fraction of cells increased significantly from 3.2% to 22.8%. MoleculesTherefore,2017, 22, 384the induction of G1-phase arrest and an increased sub-G1 apoptotic fraction may be the7 of 14 major mechanisms by which the growth of HepG2 cell is inhibited.

Figure 6. BEPS-IA induced cell cycle arrest in HepG2 cells. Cells were cultured for 24 h with BEPS-IA Figure 6. BEPS-IA induced cell cycle arrest in HepG2 cells. Cells were cultured for 24 h with BEPS-IA (50, 100 and 200 μg/mL, respectively). Bar graph summarizes the data on cell cycle distribution from µ (50, 100three and independent 200 g/mL, experiments. respectively). Values Bar marked graph summarizeswith * are signific the dataantly on different cell cycle compared distribution to the from threecontrol independent (p < 0.05). experiments. Values marked with * are significantly different compared to the control (p < 0.05). 2.7. BEPS-IA Induced Apoptosis of HepG2 Cells In Vitro 2.7. BEPS-IATo further Induced evaluate Apoptosis the of effect HepG2 of BEPS-IA Cells In Vitroon the induction of apoptosis, the HepG2 cells were Toincubated further with evaluate different the concentrations effect of BEPS-IA of BEPS-I on theA, inductionand then the of cells apoptosis, were stained the HepG2 with methyl cells were green, followed by counting under light microscopy (magnification, ×40). As shown in Figure 7 and incubated with different concentrations of BEPS-IA, and then the cells were stained with methyl Figure S3, treatment with BEPS-IA increased the apoptotic staining in a dose-dependent manner green, followed by counting under light microscopy (magnification, ×40). As shown in Figure7 (4.1% ± 1.3% in the control, 12% ± 2.5% in the 50 μg/mL group, 16% ± 5.2% in the 100 μg/mL group, and Figure23% ± 2.3% S3, treatment in the 200 withμg/mL BEPS-IA group). These increased data corroborate the apoptotic the stainingBEPS-IA-induced in a dose-dependent sub-G1 apoptotic manner (4.1%fractions± 1.3% determinedin the control, with 12% flow± cytometry2.5% in theshown 50 µabove.g/mL These group, data 16% corrob± 5.2%orate inthe the BEPS-IA-induced 100 µg/mL group, 23% ±sub-G12.3% apoptoticin the 200 fractionsµg/mL determined group). These with dataflow corroboratecytometry shown the BEPS-IA-induced above. sub-G1 apoptotic fractions determined with flow cytometry shown above. These data corroborate the BEPS-IA-induced sub-G12.8. apoptotic Effect of BEPS-IA fractions on Protein determined Expression with of flowp53, p21 cytometry, Bcl-2 and shown Bax in above.HepG2 Cells To gain further information on the mechanism of anti-proliferation induced by BEPS-IA, the 2.8. Effectexpression of BEPS-IA of p53, on p21, Protein Bcl-2, Expression Bax were examined of p53, p21, in HepG2 Bcl-2 and cells. Bax The in levels HepG2 of Cells β-Actin served as an Tointernal gain control. further By information studying the results on the of western mechanism blot, we of observed anti-proliferation that the expression induced of Bcl-2 by BEPS-IA, was the expressiondown-regulated, of p53, but p21, the level Bcl-2, of Baxp53, werep21 and examined Bax were up-regulated, in HepG2 cells. compared The levels to the ofcontrolβ-Actin (Figure served 8). as Therefore, we postulated that the possibly mechanism of BEPS-IA-induced anti-proliferation in an internal control. By studying the results of western blot, we observed that the expression of Bcl-2 HepG2 cells by the increase of p53, p21 and the low ratio of Bcl-2 to Bax modulated. was down-regulated,Bullacta exarata but has the been level consumed of p53, in p21 Asia, and not Bax only were as a up-regulated,part of the normal compared diet, but to also the as control a (Figuretraditional8). Therefore, Chinese we medicine postulated with that liver- the and possibly kidney-benefitting mechanism functions of BEPS-IA-induced [3–6]. Human anti-proliferationhepatocellular in HepG2carcinomas cells byis theknown increase to be ofthe p53, most p21 common and the ma lowlignancy ratio ofand Bcl-2 the tothird Bax leading modulated. cause of cancer Bullactamortality exarata in peoplehas in beenboth deve consumedloped and in developing Asia, not onlycountries as a [ part19]. Polysaccharide-protein of the normal diet, butcomplex also as a traditionalis one Chineseof the major medicine active withcompounds liver- andof B. kidney-benefitting exarata, the antitumor functions activity [of3– 6polysaccharide-protein]. Human hepatocellular carcinomascomplex is has known never been to be reported the most before. common Our study malignancy was the first and to show the that third a polysaccharide-protein leading cause of cancer mortalitycomplex in people BEPS-IA in bothfrom developedB. exarata inhibited and developing the growth countries of human [19 hepatocellular]. Polysaccharide-protein carcinoma HepG2 complex cells. The results demonstrated that BEPS-IA could induce a significant dose-dependent inhibition of is one of the major active compounds of B. exarata, the antitumor activity of polysaccharide-protein complex has never been reported before. Our study was the first to show that a polysaccharide-protein complex BEPS-IA from B. exarata inhibited the growth of human hepatocellular carcinoma HepG2 cells. The results demonstrated that BEPS-IA could induce a significant dose-dependent inhibition of HepG2 cell growth (Figure2). The IC 50 of BEPS-IA was 112.4 µg/mL. Further analysis revealed BEPS-IA inhibited the growth of HepG2 cells mainly through blocking cell cycle progression at G1 phase as well as via inducing cell apoptosis. Molecules 2017, 22, 384 8 of 15

HepG2 cell growth (Figure 2). The IC50 of BEPS-IA was 112.4 μg/mL. Further analysis revealed BEPS- IA inhibited the growth of HepG2 cells mainly through blocking cell cycle progression at G1 phase Molecules 2017, 22, 384 8 of 15 as well as via inducing cell apoptosis.

HepG2 cell growth (Figure 2). The IC50 of BEPS-IA was 112.4 μg/mL. Further analysis revealed BEPS- Molecules 2017IA, 22 inhibited, 384 the growth of HepG2 cells mainly through blocking cell cycle progression at G1 phase 8 of 14 as well as via inducing cell apoptosis.

Figure 7. Apoptotic rate (%) of human hepatocellular carcinoma HepG2 cells treated with polysaccharide Figure 7. Apoptotic rate (%) of human hepatocellular carcinoma HepG2 cells treated with Figure 7. ApoptoticBEPS-IA. Cells rate were (%) cultured of human for 24he hpatocellular with BEPS-IA carcinoma (50, 100 and HepG2 200 g/mL, cells respectively). treated with Untreated polysaccharide polysaccharide BEPS-IA. Cells were cultured for 24 h with BEPS-IA (50, 100 and 200 g/mL, respectively). BEPS-IA. Cellscells (0 wereg/mL) culturedwere used asfor control. 24 h withValues BEPS-IA marked with (5 0,* are 100 significantly and 200 g/mL,different respectively). compared to the Untreated Untreatedcontrol cells (0(p < g/mL)0.05). were used as control. Values marked with * are signiﬁcantly different cells (0 g/mL) were used as control. Values marked with * are significantly different compared to the compared to the control (p < 0.05). control (p < 0.05).

Figure 8. Effects of B. exarata polysaccharide-protein complex BEPS-IA on the expression of p53, p21, Bcl-2 and Bax proteins associated with cell cycle and apoptosis in HepG2 cells. Western blot analysis was performed in triplicate per experimental point, and the relative expression of protein was quantified densitometrically (%); β-actin was used as reference control. Values marked with * are significantly different compared to the control (p < 0.05).

Furthermore, we explored the molecular mechanisms underlying the BEPS-IA induced cell cycle arresting in HepG2 cells. Previous studies showed that chemo preventive agents were capable of FigureFigure 8.8. EffectsEffects of of B.B. exarata exarata polysaccharide-proteinpolysaccharide-protein complex complex BEPS-IA BEPS-IA on the on theexpression expression of p53, of p53,p21, Bcl-2 andinducing Bax cellproteins cycle blockingassociated and with apoptosis cell cycle [20]. Itan isd also apoptosis widely recognizedin HepG2 thatcells. p53 Western serves asblot a key analysis p21, Bcl-2player and in mediating Bax proteins cell response associated to various with cellstresses, cycle exerting and apoptosis its function in mainly HepG2 through cells. inducing Western blot analysiswas performed was performed in triplicate in per triplicate experimental per experimental point, and the point, relative and expression the relative of expressionprotein was ofquantified protein

wasdensitometrically quantiﬁed densitometrically (%); β-actin was (%); usedβ-actin as reference was used control. as reference Values control. marked Values with marked* are significantly with * are signiﬁcantlydifferent compared different to compared the control to (p the < 0.05). control (p < 0.05).

Furthermore, we explored the molecular mechanisms underlying the BEPS-IA induced cell cycle Furthermore, we explored the molecular mechanisms underlying the BEPS-IA induced cell cycle arresting in HepG2 cells. Previous studies showed that chemo preventive agents were capable of arresting in HepG2 cells. Previous studies showed that chemo preventive agents were capable of inducing cell cycle blocking and apoptosis [20]. It is also widely recognized that p53 serves as a key inducing cell cycle blocking and apoptosis [20]. It is also widely recognized that p53 serves as a key player in mediating cell response to various stresses, exerting its function mainly through inducing player in mediating cell response to various stresses, exerting its function mainly through inducing or suppressing a number of transcriptional regulatory proteins involved in cell cycle arrest and apoptosis, such as the cyclin-dependent kinase (CDKs) inhibitor p21, for the cell cycle arrest and the proapoptotic protein Bax for inducing apoptosis [21]. P21 is a very important checkpoint transcriptional regulatory protein in the cell cycle, which is also regulated by the transcription of p53; it can repair damaged cells Molecules 2017, 22, 384 9 of 14 by stopping DNA synthesis and inactivate the nuclear antigen in proliferating cells [22]. Our data showed that BEPS-IA up-regulated p53 and p21 expression in a dose-dependent manner (Figure4). These data suggested that the cell cycle arrest induced by BEPS-IA might be mediated through the regulation of the cell cycle regulating factors, in particular p53. Alternatively, p53 can also induce apoptosis as a mediator through expression of Bcl-2 and Bax [23]. It has been shown previously that Bcl-2 family proteins are important regulators of the apoptosis [24]. Bax associates with Bcl-2 protein and down-regulation of Bcl-2 favors the formation of Bax homodimer that increases the rate of apoptosis and sensitivity of cells to apoptotic stimuli [25]. The Bax/Bcl-2 ratio was found to be elevated at the time when most prominent apoptosis occurred. Our data demonstrated that BEPS-IA treatment resulted in a significant decrease of Bcl-2 expression and an increase of Bax, thus the calculated Bax/Bcl-2 ratio was markedly higher in the BEPS-IA -treatment group compared to the control group. Increasing evidence demonstrated that the Bax/Bcl-2 ratio determines the susceptibility of cells to the induced apoptosis [24,25]. Therefore, we concur that the growth inhibition process occurring in in vitro cultured HepG2 cell lines after BEPS-IA treatment may be associated with the up-regulation of p53, p21 and bax expression and down-regulation of bcl-2 expression, suggesting that p53 was involved in cell cycle arrest and apoptosis regulation of BEPS-IA. The structure of the polysaccharide is strongly related to its antitumor activity. Previous research has mainly focused on the structure of main chain structure, branching degree, helical conformation, molecular weight, and the monosaccharide composition. Generally, polysaccharides of molecular weights between 400 and 800 kDa appeared to be more effective than those of low molecular weight in suppressing tumor cell growth [26]. In addition, the amount of protein groups has been reported to play role for antitumor activity of polysaccharide-protein complexes [12]. Moreover, according to the report, polysaccharides with β-glucan such as (1→3)-linked-β-D-glucose backbone chain exerted a relatively higher antiproliferation activity [27]. However, in practice, it is difficult to make a correlation between antitumor activity of purified polysaccharide-protein complex and their structures. There are some other antitumor polysaccharides with different chemical structures, such as α-glucan or low molecular weights [28]. Thus, the antitumor activity of tested polysaccharide-protein complex is not a function of a single factor but a combination a many factors such as amount of protein groups, monosaccharide residues ratio, and type of sugar residues. To our knowledge, there are few reports of polysaccharides with a structure similar to BEPS-IA displaying an antitumor effect and having apoptosis-inducing activity. Our research will facilitate the understanding of the structural basis of the polysaccharides with antitumor effects and their antitumor mechanisms.

3. Materials and Methods

3.1. Materials and Reagents Bullacta exarata snails were obtained from Rulong Seafood Co., Ltd. (Zhejiang, China). Monosaccharide standards (arabinose, rhamnose, fucose, xylose, galactose, glucose and mannose), disaccharide lactose, sodium dodecyl sulfate (SDS), ethylendiamine tetraacetic acid (EDTA), bovine serum albumin (BSA), papain, cysteine (Cys) and nonfat milk powder were obtained from Sigma Chemical Company (St. Louis, MO, USA). MTS was from Promega (Madison, WI, USA). Anti-Bax antibody (rabbit polyclonal) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-P53 antibody (mouse monoclonal) and anti-Bcl-2 antibody (mouse monoclonal) were purchased from Abcam (Cambridge, MA, USA). HRP-linked anti-mouse IgG secondary antibody was purchased from Cell Signaling Technologies (Danvers, MA, USA). All other chemicals used were analytical grade.

3.2. Polysaccharide-Protein Complex BEPS-IA Extraction The BEPS-IA was isolated and purified by our previously described procedure [2]. Fresh or deep-frozen Bullacta exarata (500 g) was defatted by extraction for 24 h using for acetone (3L). Samples of defatted, dried, and powdered Bullacta exarata were treated with papain (1200 U·g−1) for 24 h at 200 rpm Molecules 2017, 22, 384 10 of 14 at 60 ◦C. The extracts were combined, centrifugated, dialyzed, concentrated, and polysaccharides were precipitated with four volumes of 96% ethanol. The precipitates were washed with 96% ethanol and acetone and air-dried. The crude polysaccharide preparation was applied onto a DEAE column (2.5 cm × 30 cm, Bio-Rad, Hercules, CA, USA), followed with a Sephacryl S-300 gel filtration column (1.6 cm × 100 cm). The final two fractions, named as BEPS-IA and BEPS-IB, were desalted and lyophilized as previously reported [2]. In this study, the polysaccharide conjugate BEPS-IA, with an average molecular weight of about 127 kDa, was collected for further study.

3.3. Analytical Procedures Total carbohydrates were quantiﬁed using the phenol-sulfuric acid method [29]. The monosaccharide composition was analyzed by the PMP-HPLC method. 13 Sulfate group determination was carried out by the BaCl2/gelatin method and ion-exchange chromatography [30]. The contentration of proteins was quantiﬁed by the method of Lowry and the amino acid constituents were analyzed by the HPLC AccQ method [31]. Tryptophan can be obtained by alkaline hydrolysis and UV detection at 280 nm [32]. BEPS-IA was imaged by AFM method according to previously described procedure [33]. Topographic observations were performed using the Nano Scope III atomic force microscope (Digital Instruments, Santa Barbara, CA, USA) and collected by tapping mode atomic force microscopy (Tm AFM).

3.4. Methylation Analyses Methylation of BEPS-IA was carried out three times using the method described by Souza et al. [34]. The methylated polysaccharides were confirmed by FTIR spectroscopy. The complete methylated products were hydrolyzed, reduced and acetylated as described earlier. The resulting alditol acetates were subjected to GC and GC-MS analysis. Qualitative and quantitative analyses were performed on gas chromatography–mass spectrometry (GC-MS, Finnigan Trace, Thermo Electron Finnigan Co. Ltd., Waltham, MA, USA) with helium as carrier gas (2 mL/min). The GC column was an OV1701 (30 m, internal diameter 0.25 mm, film thickness 0.25 µm) at a temperature program from 140 to 280 ◦C with a rate of 3 ◦C/min. The rate of helium carrier gas was at a rate of 3.0 mL/min. The quantification for molar ratio for each O-methylated alditol acetate was calibrated using the peak area and response factor of the FID in GC.

3.5. FI-IR and NMR Analyses

1 13 Nuclear magnetic resonance (NMR) spectra ( H, C) for solutions of substances in D2O were obtained on a 500 MHz Avance™ 500 spectrometer (Bruker, Berlin, Germany) at 60 ◦C[35]. FTIR spectra (KBr disc) were recorded with a Nicolet 5700 FT-IR (Thermo Electron Corporation, Madison, WI, USA) spectrophotometer for detecting functional groups [36].

3.6. Cell Culture The human hepatocellular carcinoma HepG2 cell line (ATCC, Rockville, MD, USA) was grown in monolayer in minimum essential medium alpha medium (MEM) containing 50 units/mL penicillin, 50 µg/mL streptomycin, 100 µg/mL gentamicin, 10 mM Hepes, 5 µg/mL insulin, 2 µg/mL glucagon, 0.05 µg/mL hydrocortisone and 5% fetal bovine serum (Gibco, Life Technologies, Grand Island, NY, ◦ USA) in a humidiﬁed atmosphere containing 5% CO2 at 37 C[37].

3.7. Cytotoxicity Measurements To estimate cell cytotoxicity, cells (HepG2) were seeded (4.0 × 104/well) in 96-well ﬂat-bottom ◦ plate at 37 C in a 5% CO2 incubator. After 24 h, the medium was removed and replaced by a fresh medium containing the different concentrations of the polysaccharide BEPS-IA for additional 24 h ◦ at 37 C in a 5% CO2 incubator. Blank wells contained 100 µL of growth medium with no cells and Molecules 2017, 22, 384 11 of 14 control cultures (CK) received the extraction solution minus the extracts. Cytotoxicity was determined by the methylene blue assay and measured by a 10% reduction of absorbance at 570 nm reading for each concentration compared to the control [38,39].

3.8. Measurement of Cell Proliferation To estimate cell proliferation, cells (HepG2) were seeded (2.5 × 104/well) in 96-well ﬂat-bottom ◦ plate at 37 C in a 5% CO2 incubator. After 4 h, the medium was removed and replaced by a fresh medium containing the different concentrations of the polysaccharide BEPS-IA for additional 72 h ◦ at 37 C in a 5% CO2 incubator. Blank wells contained 100 µL of growth medium with no cells and control cultures (CK) received the extraction solution minus the extracts. The common antitumor drug 5-ﬂuorouracil (5-FU) was used as a positive control. Cell proliferation was determined by the methylene blue assay and measured by the absorbance at the 570 nm reading for each concentration compared to the control [39].

3.9. TUNEL Assay In vitro induction of apoptosis by BEPS-IA treatment was determined by the TUNEL assay, with an Apoptosis Detection Kit (Biovision Research Products, Mountain View, CA, USA). HepG2 cells were harvested and cytospun on slides with concentration of 1 × 105 cells/mL. TUNEL staining was performed using the detection kit according to the manufacturer’s instructions. The apoptotic cells were counted with a light microscope (Olympus, C5060-ADU, Tokyo, Japan) and the results are reported as the mean of the three experiments.

3.10. Cell Cycle Analysis Cells were cultured with BEPS-IA (50, 100, 200 µg/mL) for 24 h, while the control group received only growth medium. The cells were harvested and washed in ice-cold PBS for three times. Cells were then ﬁxed with ice-cold 70% methanol, and DNA was stained with propidium iodide (50 mg/mL). Cell cycle analysis was performed with ﬂow cytometer (BD FACSVerse™, BD Biosciences, Franklin Lakes, NJ, USA). ModFitL T V2.0 software was used to determine the cell cycle phase distribution after debris exclusion. The sub-G1/G0 cell fraction was considered representative of apoptotic cells.

3.11. Western Blotting Analysis BEPS-IA treated cells were lysed in solubilization buffer (50 mM Tris, pH 7.4; 1% Igepal; 150 mM sodium chloride; 1 mM EDTA) with protease inhibitors (1 µg/mL aprotinin; 1 µg/mL leupeptin; 1 µg/mL pepstain; 1 mM phenylmethylsulfonyl ﬂuoride (PMSF); 1 mM sodium orthovanadate; 1 mM sodium ﬂuoride) for 30 min at 4 ◦C, and insoluble materials was removed by centrifugation at 12,000× g for 10 min at 4 ◦C. The supernatant was collected and the protein concentration was determined by Sigma Diagnostics Micro Protein Determination Kit and Dynex Microplate Reader (Dynex Technologies, Chantilly, VA, USA). Equal amounts of protein (60 mg) were loaded in each lane and separated by 12% SDS-PAGE. The proteins were transferred to Immobilon-p transfer membranes. The membranes were blocked with 3% non-fat dry milk in TBS containing 0.1% Tween 20, and then incubated at room temperature for 1 h in the presence of each antibody (Anti-Bax antibody, Anti-P53 antibody and anti-Bcl-2 antibody). The membrane was washed three times with 0.1% Tween 20 in PBS and stained with HRP-linked anti-mouse IgG secondary antibody. β-Actin was used as internal control. The ECL system (Amersham Biosciences, Buckinghamshire, UK) was used for detection [40].

3.12. Statistical Analyses Experiments were performed at least three times with similar results. All results were presented as mean ± SD. Statistical differences were evaluated using SPSS software (SPSS Inc., Chicago, IL, USA) and considered to be signiﬁcant when p < 0.05. Molecules 2017, 22, 384 12 of 14

4. Conclusions In summary, a novel protein-bound polysaccharide BEPS-IA was extracted and purified from Bullacta exarata. The glycoprotein was mainly composed of mannose and Glc in a molar ratio of about 1:2, with an average molecular weight of about 127 kDa. The glycosyl residues of BEPS-IA mainly linked by α-configuration glycosidic bonds, and theirs repeating unit of structure were proposed as described above. Antitumor activity tests conducted in vitro revealed that BEPS-IA exhibited an effective HepG2 breast cancer cell growth inhibition. The inhibitory action, does-dependent manner, was mainly caused by G1 cell cycle arrest and apoptosis. And the mechanism was associated with up-regulation of p53, p21 and Bax, down-regulation of Bcl-2. Therefore, the present studies demonstrated that BEPS-IA would be a new adjuvant chemotherapeutic and chemo-preventive strategy against human hepatocellular carcinoma HepG2 cells in vitro. However, due to only one cell line in our present study, further investigations on more cell lines supporting the findings are currently in progress.

Supplementary Materials: Supplementary materials are available online. Acknowledgments: This research was sponsored the Public Projects of Zhejiang Province (No. 2015C32017), the Program of Health and Medicine Science in Zhejiang Province (No. 2015104235), the National Natural Science Foundation of China (No. 31371872) and Major Science and Technology Special Projects of Zhejiang, China (No. 2013C03045-1). Author Contributions: Ningbo Liao and Jianjun Zhong completed the extraction and purification of polysaccharides, and study the structure characterizations. Liang Sun accomplished antitumor activity of B. exarata polysaccharide-protein complex BEPS-IA. Yanjun Zhang prepared for the experimental materials. Jiang Chen and Ronghua Zhang instructed all the members to carry out the research. All authors read and approved the final version of the manuscript. Conflicts of Interest: The authors declared no conflict of interest.

Abbreviations

BEPS-IA puriﬁed Bullacta exarata polysaccharides of molecular weight 127 kDa AFM Atomic Force Microscopy NMR Nuclear Magnetic Resonance PMP 1-phenyl-3-methyl-5-pyrazolone CK Control culture IC50 inhibitory concentration representing the concentration at which cell viability was reduced by 50% HepG2 human hepatocellular carcinoma cell MEM Medium Alpha Medium Gal galactose Man mannose Glc glucose GlcUA glucuronic acid Arb arabinose Rha rhamnose Xyl xylose GalN galactosamine GlcN glucosamine Fuc fucose

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