- Email: [email protected]
The effect of polydisperse fucoidans from Fucus vesiculosus on Hep G2 and Chang liver cells
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Contents lists available at ScienceDirect
Bioactive Carbohydrates and Dietary Fibre journal homepage: http://www.elsevier.com/locate/bcdf
The effect of polydisperse fucoidans from Fucus vesiculosus on Hep G2 and Chang liver cells Elena V. Zhurishkina a, b, Sergey I. Stepanov a, Olga N. Ayrapetyan a, b, c, Yury A. Skorik d, Elena N. Vlasova d, Igor V. Kruchina-Bogdanov e, Dmitry V. Lebedev a, f, Anna A. Kulminskaya a, b, Irina M. Lapina a, b, * a
Petersburg Nuclear Physics Institute named by B.P. Konstantinov of National Research Centre «Kurchatov Institute», Gatchina, Russia Kurchatov Genome Center – PNPI, Gatchina, Russia Saint Petersburg National Research University of Information Technologies, Mechanics and Optics, St. Petersburg, Russia d Institute of Macromolecular Compounds of the Russian Academy of Sciences, St. Petersburg, Russia e Analytics. Materials. Technologies Ltd, St. Petersburg, Russia f National Research Centre «Kurchatov Institute», Moscow, Russia b c
A R T I C L E I N F O
A B S T R A C T :
Keywords: Fucoidan Chang liver Hep G2
The effect of polydisperse fucoidans from brown algae Fucus vesiculosus on Hep G2 and Chang liver cells was studied. Despite the high molecular weight and polydispersity, these fucoidans exhibited antitumor activity, whereas the difference in the inhibitory effects on cell proliferation between the native and the partially hy drolyzed fucoidans was not statistically significant. Fucoidans were internalized by cells of both cell lines through endocytosis, with Chang liver cells metabolizing fucopolysaccharides more rapidly compared to the Hep G2. In both cell lines treatment with fucoidans was shown to induce apoptosis and autophagy, with apoptosis more pronounced in Hep G2 cells, whereas autophagy - in Chang liver cells.
1. Introduction Sulfated fucopolysaccharides (fucoidans) contained in the cell walls of brown algae demonstrate a variety of biological activities: antiviral (Pavliga, Kompanets, & Tsygankov, 2016; Trinchero et al., 2009), antibacterial (Marudhupandi & Kumar, 2013), anticoagulant (Durig et al., 1997; Jin, Zhang, Wang, & Zhang, 2013), immunostimulating (Cho, Lee, Kim, & You, 2014; Kima & Joo, 2008), anti-inflammatory (Li & Ye, 2015) and antitumor (Kwak, 2014; Senthilkumar, Manivasagan, Venkatesan, & Kim, 2013). Fucoidan properties attract a close attention of researchers around the world for decades. Its potential in medicine is due to the selective cytotoxicity as malignant tumor cells have been shown to have significantly greater sensitivity to fucoidan than the normal ones (Usoltseva, Anastyuk, et al., 2018 Abudabbus, Badmus, Shalaweh, Bauer, & Hiss, 2017; Usoltseva, Shevchenko et al., 2018; Kim & Nam, 2018; Park, Kim, Nam, Kim, & Choi, 2011; Zhang et al., 2016). Fucoidan is well known to induce apoptosis in cells. However, until now it remains unclear how an interaction of the cells with sulfated poly saccharides is triggering mechanisms for its self-destruction. Even the
basic aspects of the observed cytotoxicity, i.e. whether fucoidans have to penetrate into the cell, act by an external contact with receptors, or affect the intercellular contacts, are not firmly established. There are several reports that fucoidan binds to various types of scavenger re ceptors including classes A, B and F-SR (Berwin, Delneste, Lovingood, Post, & Pizzo, 2004; Peiser & Gordon, 2001; Tamura et al., 2003), as well as enters the cells through endocytosis. It has been shown that the SR-A-fucoidan complex is internalized both through clathrin and caveolin-dependent pathways of endocytosis (Zhu et al., 2011). Deux reported internalization of low molecular weight fucoidan in the peri nuclear vesicles of rabbit smooth muscle cells (Deux et al., 2002). It was also shown that low molecular weight fucoidan is internalized by HUVEC mainly through the clathrin-mediated endocytosis pathway (Marinval et al., 2016). Regardless of the mechanism of internalization, fluorescently-labeled sulfated polysaccharides can be used to visualize the interaction of fucoidan and the cell (Marinval et al., 2016; Oliveira et al., 2017). The heterogeneity of the composition and the large size of the fucoidan molecules both complicate establishing the relationship
* Corresponding author. Petersburg Nuclear Physics Institute named by B.P. Konstantinov of National Research Centre «Kurchatov Institute», Gatchina, Russia. E-mail address: [email protected] (I.M. Lapina). https://doi.org/10.1016/j.bcdf.2019.100209 Received 1 July 2019; Received in revised form 16 December 2019; Accepted 17 December 2019 Available online 19 December 2019 2212-6198/© 2019 Published by Elsevier Ltd.
Please cite this article as: Elena V. Zhurishkina, Bioactive Carbohydrates and Dietary Fibre, https://doi.org/10.1016/j.bcdf.2019.100209
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
between the structure of sulfated fucopolysaccharides and their bio logical activity, which is a necessary step for their potential application in medicine. A number of publications report biological activity of low molecular weight fucoidans obtained by acidic, radical or enzymatic hydrolysis (reviewed in (Morua, Kim, & Kim, 2012)). However, there are only few studies comparing the cytotoxicity of fucopolysaccharides with different degrees of polydispersity (Anastyuk et al., 2012; Oliveira et al., 2017; Yang et al., 2008). In our previous study of biological activity of fucoidans from Fucus vesiculosus (Zhurishkina et al. 2015, 2017) we compared the cytotoxicity of crude fucoidan and its fractions obtained by anion-exchange chro matography, showing that the unfractioned fucoidan had higher toxicity toward Hep G2 cells than its most active fraction with similar degree of sulfation. In this work we investigate the effect of two preparations of polydispersed sulfated fucopolysaccharides with different content of high and low molecular weight fractions on the Hep G2 and Chang liver cell lines.
followed by dialysis (pore size of 3.5 kDa, Spektra/Por, Canada) against the distilled water for 12 h. The sulfate content in fucoidans was determined by the turbidimetric method after acidic hydrolysis of the samples with a 1 N HCl solution at 100 � C for 6 h using K2SO4 as a standard (Dodgson, 1961). The content of fucose was determined by the method described in (Dische, Shettles, & Osnos, 1949). Molecular mass distribution of the fucoidan samples was evaluated by HPLC on a column 7.5 x 600 mm TSKGel 3000SW (Toyo Soda, Japan). A 10 mM phosphate buffer at 0.9 ml/min as the mobile phase with high pressure pump CN 4000 (Milton Roy, USA) and refractometric detector RI Model 504 (Gasukuro Kogyo/Japan) were used. Dextrans with molecular weights from 15 to 200 kDa were used for MW calibration. For low pressure gel chromatography were used 0.3% ammonium sulfate solution as eluent, column 15.7 x 182 mm, Sepharose 4B (flow rate 11 ml/h) and column 10.3 x 160 mm, Sephadex G15 (flow rate 15.2 ml/h), peristaltic pump NP 1M (Russia), and the same refractometric detector. Dextrans with molecular weights from 40 kDa to 2MDa with the former column and glucose–saccharose–raffinose–Blue dextran with the latter column were used for MW calibration. Monosaccharide composition of low and high MW fractions of fucoidans F and FG isolated by HPLC was quantified with gas-liquid chromatography of trimethylsilyl derivatives after the acid hydrolysis of polysaccharides with 2M trifluoroacetic acid for 5 h and 105 � C. Monosaccharide mixture obtained was treated in pyridine with 1,1,1,3,3,3-hexamethyldisilazane in the presence of trifluoroacetic acid at 60 � C for 1 h (Orata, 2012). Analysis conditions: chromatograph Shimadzu 2010 (Shimadzu, Japan), CBP5-25 column (25 m x 0.25 mm x 0.2 μm); carrier gas N2, 20 cm/s; sample volume 2 μl; injector temper ature 270� С; flame ionization detector, temperature 325� С. FTIR spectra were obtained using a Vertex-70 spectrometer (Bruker, Germany) equipped with a Pike attenuated total reflectance accessory (a ZnSe prism). The Bruker OPUS spectroscopy software was used for data acquisition and analysis. The average molecular weight was estimated by a viscometric method (Torres et al., 2007) and calculated from the viscosity data by the Mark-Houwink equation [η] ¼ KMα, where [η] is the intrinsic vis cosity, M is the average molecular weight; K, α are empirical constants. In this case, the intrinsic viscosity was determined as [η]¼(ηsp/c)c→0, where c is the polymer concentration, g/100 ml; ηsp is the specific vis cosity, defined as ηsp¼(η– ηsolv)/ηsolv, where ηsolv is the viscosity of the solvent, η is the measured viscosity of the polymer solution. The vis cosity was measured on SV-10A tuning fork vibration viscometer (A&D, Japan) at 20 � C. Bidistilled water with a conductivity of less than 0.3 μS and a viscosity of 1.00 mPa*s was used as a solvent. Solutions of dex trans having a molecular weight in a range from 200 kDa to 2000 kDa were used for the calibration.
2. Materials and methods All reagents were purchased from Sigma-Aldrich (Germany) and Acros Organics (USA), unless otherwise specified. Confocal microscopy was performed using Leica SP5 (Leica Microsystems, Germany) micro scope. Flow cytometry measurements were done using an in-house flow cytometer built in the radiobiology and medicine group of the Peters burg Institute of Nuclear Physics (Stepanov, Konyshev, Kotlovanova, & Roganov, 1996). 2.1. Cell culture Cells of human hepatoblastoma (Hep G2 cell line) were purchased at the Cellular Center of the Institute of Cytology RAS (St. Petersburg). The cells of normal human liver tissue (Chang liver cell line) were purchased at the Scientific Research Institute of Virology named after D.I. Iva novsky (Moscow). Hep G2 and Chang liver cells were cultured in Carrel vials in MEM medium (L-glutamine) (ICN, USA) containing 10% of bovine serum and 1% of a mixture of penicillin and streptomycin (Bio lot, St. Petersburg) at 37 � C. To construct the growth curves and deter mine the size of the Sub-G1 population, the cells were grown for 24 h, then fucoidans F and FG were added to the culture medium so that the final concentration was 180 μg/ml. The concentration of 180 μg/ml was chosen in a series of preliminary experiments as the minimal concen tration at which cytotoxic effect of fucoidan was observed reliably on both cell lines (data not shown). After 12, 24, 48 and 72 h of incubation, the cells were stained with propidium iodide and counted on a flow cytometer. 2.2. Preparation and analysis of fucoidans
2.3. The production of fucoidans with a fluorescent label
As a source of fucoidan, the mechanically ground brown algae F. vesiculosus (Alganika LLC, St. Petersburg) was used. A crude fraction of sulfated polysaccharides F was isolated from the dry ground algae F. vesiculosus according to a modified method described in (Bilan et al., 2010; Usov & Bilan, 2009). Dry ground algae F. vesiculosus (100 g) was treated with a 4: 2: 1 mixture of methanol-chloroform-water to remove lipids and pigments, centrifuged and dried. One liter of 2% CaCl2 solu tion was mixed with the resulted dried algae (90 g) and stirred at 85 � C for 5 h. The extract was separated by centrifugation at 5500 rpm and ethanol (1.2 L) was poured into 700 ml of the supernatant. The pre cipitate was separated by centrifugation at 5500 rpm, dissolved in 200 ml of a 2% CaCl2 solution, and dialyzed with a 3.5 kDa pore dialysis membrane (Spektra/Por, Canada) against distilled water for 12 h. The insoluble part was removed by centrifugation, the solution was lyophi lized and, as a result, 2.9 g of fucoidan F was obtained. A sample of partially hydrolyzed fucoidan (FG) was prepared by acidic hydrolysis with 0.01 N HCl for 5 min according to the procedure (Yang et al., 2008),
Samples of labeled fucoidans Fflu and FGflu were synthesized ac cording to the procedure described in (Glabe, Harty, & Rosen, 1983). The degree of hydroxyl groups substitution with fluoresceinamine was determined using a F-4010 fluorescence spectrophotometer (Hitachi, Japan). 2.4. Confocal microscopy Hep G2 and Chang liver cells were seeded in Glass Bottom cups (NEST Scientific, USA). After the cells were attached (about 6 h), the fucoidans Fflu and FGflu were added to the culture medium to the final concentration of 180 μg/ml. Measurements were conducted on a confocal microscope Leica SP5 for 22 h, taking the image every hour. Throughout the observation period, the cells were incubated in 5% CO2 atmosphere and 37 � C. Oil immersion 63-x objective with aperture 1.4 was used, fluorescence excitation was done by an argon laser at 488 nm, 2
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
emission was detected in the 500–550 nm range, LAS AF program (Leica Microsystems, Germany) was used to image processing.
3. Results
2.5. Flow cytometry with labeled fucoidans
The native fucoidan F and partially depolymerized FG sample ob tained from F. vesiculosus brown algae contained 45.6% and 53.2% of fucose and 14.6% and 14.2% sulfates, respectively. Sulfo group position in sulfated polysaccharides F and FG was resolved with infrared analysis (Fig. 1S Supplementary materials). IR spectra of both the native fucoidan F and partially hydrolyzed fucoidan – O bond at 1219 FG had intensive absorption bands corresponding to S– and 1215 сm 1, respectively. When analyzing the peak bands at 840 cm 1 for F and 841 cm 1 for FG, we found that they can be described by two components at 846 and 819 cm 1. In the case of F, the ratio of S846/ S819 was 1.3, which indicates the prevailing content of sulfo groups in the axial position, whereas in the IR spectrum of FG the S846/S819 ratio was 1.1, which indicated that the content of sulfo groups in the axial position was only slightly higher than that in the equatorial position. According to SEC-HPLC (Fig. 1a), the molecular weight profile of native fucoidan F had three peaks, the first one corresponded to HMW fraction (MW > 200 kDa), and its peak maxima both for 200 nm and for RI curves coincided in retention times. Two peaks of lower MW fractions (MW < 15 kDa) were poorly resolved on the refractometer curve, but the obviously overwhelming UV absorbance at 200 nm made it possible to pinpoint the maximum of the broad former one at 22.4 min, whereas on the RI curve this fraction appeared as a close shoulder on a front of the latter, narrower peak with maximum at 24.1 min. The ratio between areas of HMW and LMW fractions can be estimated by refractometer
3.1. Characterization of fucoidans F and FG
Hep G2 and Chang liver cells were seeded in Karel vials at a con centration of 60,000 cells/ml. After 24 h, the labeled fucoidans Fflu and FGflu were added at the final concentration 180 μg/ml and incubated for 4, 22 and 43 h. The cells were then rinsed with PBS and harvested with a Versene solution, centrifuged at 700 rpm, resuspended in the buffer and analyzed on a flow cytometer. Excitation with argon laser for green fluorescence at a wavelength of 488 nm, detection of emission at 535 nm. At least 10,000 cells were analyzed in each sample. 2.6. Determination of apoptotic cells Cells of Hep G2 and Chang liver were cultivated in culture flasks. After 24 h, fucoidans F and FG were added to reach the final concen tration of 180 μg/ml. After 24 and 48 h the combined staining with Membrane Permeability/Dead Cell Apoptosis Kit with Yo-Pro®1 and PI for Flow Cytometry (Invitrogen, USA) was performed according to the manufacturer’s protocol. Cells were analyzed with a flow cytometer. Each experiment was performed three times, at least 10,000 cells were analyzed in each sample. 2.7. Staining with acridine orange Cytometry. Hep G2 and Chang liver cells were seeded in culture flasks. After 24 h, fucoidans F and FG were added to the final concen tration of 180 μg/ml. After growth for the specific time period (43, 30, 24, 22, 18, 14, 8 and 4 h) cells were harvested using a Versene solution, centrifuged at 700 rpm, culture medium containing acridine orange at a concentration of 1 μg/ml was added for 15 min. Then the medium was removed, cells were washed with PBS and analyzed with a flow cy tometer. Green fluorescence was excited with argon laser at a wave length of 488 nm, emission was detected at 535 nm. Red fluorescence excitation was at a wavelength of 630 nm, detection of emission at 650 nm. At least 10,000 cells were analyzed in each sample. Flatbed analyzer. Hep G2 and Chang liver cells were seeded in 96-well black opaque plates (Thermo Fisher Scientific, Denmark) (5000 cells per well) and grown for 24 h in a damp atmosphere at 37 � C and 5% CO2. Fucoidans F and FG were added to the final concentration of 180 μg/ml and incubated for 4, 8, 14, 18, 22, 24, 30, 43 and 48 h. The medium was then removed by aspiration, cells were washed with PBS and stained with acridine orange in a concentration of 1 μg/ml for 15 min. The cells were washed with buffer and analyzed on a multimode plate analyzer En Spire 2300 (PerkinElmer, USA). The analysis of the obtained data was carried out using the manufacturer’s software. Microscopy. Hep G2 and Chang liver cells were grown on coverslips in culture flasks. After 24 h, fucoidans F and FG were added at a concen tration of 180 μg/ml and incubated for 8 h. The medium was then removed, cells were washed with PBS and stained with acridine orange at a concentration of 1 μg/ml for 15 min. The cells were washed with buffer and analyzed on EVOS FLoid microscope (Life technologies, USA). 2.8. Statistical processing
Fig. 1. Molecular weight profile of fucoidans F (a) and FG (b), HPLC method. Output curves (pink - refractometer, blue - photometer 200 nm). The bottom axis is the output volume, ml; the left axis is the refractometer signal, mV; the right axis is the signal of the photometer, mV. (For interpretation of the ref erences to colour in this figure legend, the reader is referred to the Web version of this article.)
For statistical data processing, plotting charts and diagrams, Excel 2010 and OriginPro 16 were used. The graphs show average values of at least 3 independent experiments, bars represent the standard errors. The statistical significance of the observed differences was assessed using Student’s t-test. 3
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
data at 57:43. The molecular weight profile of the partially depolymerized fucoidan FG (Fig. 1b) is presented as three peaks of the same retention times as fucoidan F with a ratio of HMW to LMW areas as 83.6:7.3:9.1. A monosaccharide composition of the both the high and low mo lecular weight fractions F and FG obtained by GC is presented in Table 1 Supplementary materials. Molecular mass distribution of the fucoidans F and FG by gelfiltration on Sepharose 4B is presented in the Table 1. The effective mean molecular weight of F and FG fucopoly saccharides, estimated by viscometric method (Torres et al., 2007), was 1730 kDa and 2502 kDa, respectively. The apparent increase in the mean molecular weight in the partially hydrolyzed samples should likely be attributed to a higher rate of hydrolysis of fucoidans in the lowest molecular weight fraction (<40 kDa) with the subsequent removal of hydrolysis products with MW < 3.5 kDa during dialysis, as demon strated by SEC-HPLC and gel filtration data. The molecular weight of the LMW fractions of fucoidans F and FG, estimated by gel-filtration on Sephadex G15, was 490 Da and 530 Da, respectively. 3.2. Effect of fucoidans F and FG on the proliferation and death of Hep G2 and Chang liver cells Analysis of the growth rates of Hep G2 and Chang liver cells showed that fucoidans F and FG at a dose of 180 μg/ml inhibited the prolifera tion of both cell lines after 48 h. Hep G2 cells were more sensitive to both fucoidans than Chang liver cells (Fig. 2). According to flow cytometry data, treatment of Hep G2 and Chang liver cells with sulfated fucopolysaccharides F and FG at a concentration of 180 μg/ml for 24 and 48 h resulted in an increase in the percentage of dead cells compared to the control (Fig. 3). The effect of partially hy drolyzed fucoidan FG on cells appears to be stronger than the native one, but the difference was not statistically significant. When applied to Hep G2 cells, the effect of both F and FG was more pronounced after 48 h of treatment as compared to 24 h. Fig. 2. The antiproliferative effect of F and FG fucoidans against Hep G2 (a) and Chang liver (b) cells at concentration 180 μg/ml. Live cells were counted by flow cytometry using propidium iodide staining, as described in Methods. (* different from control, p < 0.01; ** - different from control, p < 0.001).
3.3. Internalization of fucoidans Fflu and FGflu by Hep G2 and Chang liver cells To investigate the interaction of cells with fucoidans, the fluorescently-labeled fucoidans Fflu and FGflu were synthesized. Using fluorescence spectrophotometry, it was found that the degree of hy droxyl groups substitution with fluoresceinamine was approximately 2 molecules of the dye per 100 monomers of fucose. After the addition of Fflu and FGflu at a concentration of 180 μg/ml to the culture medium, Hep G2 cells and the Chang liver cells were monitored by a confocal microscopy for 22 h, recording the images every hour. It was found that fucoidans were internalized by the cells of both cell lines (Fig. 4). Labeled fucoidan was visualized inside the cells as small bright in clusions (Fig. 5 a,b). The number of vesicular structures containing fluorescently labeled fucoidan was higher in Chang liver cells. A visual comparison of the fluorescence from the labeled sulfated
polysaccharides within the cells after 22 h has revealed that in both cell types the overall brightness of inclusion spots appears to be higher when non-hydrolyzed fucoidan Fflu was used for treatment as compared the partially hydrolyzed FGflu. While both Fflu and FGflu caused timedependent death of Hep G2 tumor cells, the death of non-malignant Chang liver cells was practically not observed. Interestingly, the accu mulation of strong fluorescence intensity was observed in the remnants of dead cells, apparently due to direct uptake after the loss of integrity of the cell walls during cell death. To quantify the dynamics of fucoidan accumulation, Hep G2 and Chang liver cells were treated with the labeled fucoidans Fflu and FGflu for 4, 22 and 43 h and analyzed on a flow cytometer. The green fluo rescence of the acquired labeled fucoidans in cells was detected. A timedependent increase in fluorescence was observed for the cells of both cell lines. There was no significant difference between F and FG uptake rates, although the uptake level of the former appeared to be slightly higher. A significant absorption of fucoidan was observed by 22 h, with accumulation in Chang liver cells of both Fflu and FGflu fucoidans by the end of 43 h treatment exceeding that in Hep G2 cells (Fig. 5c).
Table 1 Molecular mass distribution of the fucoidans F and FG, %, (low pressure gel chromatography on Sepharose 4B). MW, kDa
>4000 2000–4000 1000–2000 500–1000 40–500 <40
Fucoidan F
FG
2,46 22,4 12,1 7,9 26,5 28,7
0,62 19,6 18,8 10,9 36,0 14,0
4
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Fig. 3. Effect of fucoidans F and FG on the death of Hep G2 (a) and Chang liver (b) cells after 24 and 48 h. Dead cells were counted by flow cytometry using propidium iodide staining, as described in Methods. (* - different from control, p < 0.01).
3.4. Fucoidans F and FG induce apoptotic death of Hep G2 cells and Chang liver
there was no significant difference in the effect between fucoidans F and FG on the degree of acidity or the volume of the cellular compartments.
To determine whether fucoidans F and FG induced apoptotic death of Hep G2 and Chang liver cells, double staining with Yo-Pro dyes and propidium iodide was carried out. From the results presented in Fig. 6, it follows that the number of cells in the stage of early apoptosis (stained with Yo-Pro and not stained with PI) after 24-h cultivation with fucoi dans F and FG exceeded the control values by 8.7 and 2.5%, respec tively, in Chang liver cells, and by 6.9 and 4.7% in Hep G2 cells. After 48 h of cultivation with fucoidans, the number of cells in the stage of late apoptosis (stained with both Yo-Pro and PI) increases significantly: in Chang liver cells by more than 20% compared to the control, in Hep G2 by more than 50%, whereas the percentage of cells in the stage of early apoptosis practically did not increase in Chang liver cells, and even decreased in Hep G2 cells. There was no noticeable difference in this effect between fucoidans F and FG.
4. Discussion The key factors affecting the antitumor activity of fucoidans are thought to be the degree of sulfation of polysaccharides, high fucose content and location of sulfate groups in the fucose residue (Cho, Lee, & You, 2011; Zhurishkina et al. 2015, 2017). Molecular weight undoubt edly plays an important role in the biological activity of fucopoly saccharides (Senthilkumar et al., 2013), however, as far as their antitumor effect, it can not be unequivocally predicted, whether fucoi dans with a greater or smaller molecular weight will be more effective. For example, Yang et al. report that acid-hydrolyzed fucoidan from Undaria pinnatifida exhibits greater antitumor activity than native. Anastyuk (Anastyuk et al., 2012) have shown that native fucoidan iso lated from Fucus evanescens algae inhibits the proliferation and growth of colonies of human melanoma cells SK-MEL-28 and SK-MEL-5, whereas a low molecular weight polysaccharide obtained under mild hydrolysis conditions hardly exhibits an effect on proliferation in both cell lines. Ustyuzhanina (Ustyuzhanina et al., 2014) reported that high-molecular fucoidan has an anti-angiogenic effect, whereas low-molecular weight fractions exhibited a pro-angiogenic effect. When analyzing the literature on the properties of fucoidans, it should be noted that the characterization of sulfated polysaccharides by molecular weight is associated with significant problems. A review by Holtkamp (Holtkamp, Kelly, Ulber, & Lang, 2009) summarizes the data where the molecular weight of fucoidans varies from 13 to 950 kDa, depending on the algal species from which the polysaccharides are isolated, mentioning the difficulties in determination of the molecular weight of fucopolysaccharides by the gel filtration method, in particular, the absence of markers, sulfated polysaccharides similar to fucoidans in structure. There is also no single consistent classification of poly saccharides by weight. The review of Senthilkumar (Senthilkumar et al., 2013) differentiate fucoidans into low (<10kDa), medium (10–10000 kDa) and high (>10000 kDa) molecular weight. However, in the article (Matsubara et al., 2005), which is cited in this review, a polysaccharide with a mass of 166 kDa is designated as high molecular weight. In another review (Kwak, 2014) when discussing the antitumor activity of fucoidans in vivo polysaccharides with a weight of 490 kDa (from the algae Undaria pinnatifida) and 6.4–10 kDa (Cladosiphon okamuranus) are
3.5. Fucoidans F and FG increase the number of acidic vesicular organelles (AVO) in Hep G2 and Chang liver cells In order to determine whether the treatment with sulfated fucopo lysaccharides caused an increase in the number of acidic vesicular or ganelles (AVO) in the Hep G2 and Chang liver cells, their staining with acridine orange was performed. As a lysosomotropic agent acridine or ange freely penetrates the membranes and accumulates in acidic com partmets, where, at low pH values, it forms aggregates with red fluorescence, the intensity of which is directly proportional to the degree of acidity and volume of the cell compartments (Paglin et al., 2001). According to flow cytometry data, the treatment of Hep G2 cells and Chang liver with fucoidans F and FG caused a noticeable increase in red fluorescence, as compared to the control (Fig. 7a). From the results shown in Fig. 7, it follows that fucoidans F and FG induced an increase in the number of acidic vesicular organelles (AVO) in the cells of both lines. In cells stained with acridine orange, the cytoplasm and nucleolus fluoresced light green, while acidic compartmets were bright red (Paglin et al., 2001). Comparing the ratio of red and green fluorescence in cells treated with fucoidans F and FG for 48 h using a plate analyzer, it was found that the number of intracellular acidic vesicles increased with the duration of treatment with fucoidans, and after 14 h this value for Chang liver cells began to exceed that for Hep G2 (Fig. 7b). At the same time, 5
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Fig. 4. Interaction of fucoidans Fflu and FGflu with Hep G2 and Chang liver cells. Confocal microscopy (Leica SP5, oil immersion 63-x objective with aperture 1.4). Concentration of fucoidans Fflu and FGflu in the medium was 180 μg/ml. The cells were maintained in 5% CO2 atmosphere at 37 � C for the entire duration of measurements. Fluorescence excitation by an argon laser at 488 nm, emission detection in the 500–550 nm range. Bar size is 50 μm.
referred to as LMW when compared with native fucoidan weighing 5100 kDa and HMW 300–330 kDa, respectively. Polydispersity of fucoidans is also often overlooked. A review by Fitton (Fitton, Stringer, & Karpiniec, 2015) indicates that native fucoidans are a mixture of molecules that vary greatly in size but, as a rule, only average molecular weights of polysaccharides are given or the range of their values without specifying the proportions of polysaccharide fractions (Cumashi et al., 2007; Fitton et al., 2015; Kwak, 2014). In this work we investigated fucoidans with different distribution of molecular weight of sulfated fucopolysaccharides. In order to save the
original structure of the polysaccharide as much as possible, we used a method of acid-free isolation from algae. The chosen method of extracting polysaccharides from the cell walls of brown algae allows the polysaccharides retain their original molecular weight (see materials and methods) (Bilan et al., 2010; Usov & Bilan, 2009), while the weakly acidic solutions, ordinarily used in the extraction of polysaccharides from the algae, contribute to their partial hydrolysis (Zayed et al., 2016; Anastyuk et al., 2010; Ale et al., 2011). We obtained SEC-HPLC data showed that while in the native fucoidan the low molecular weight and high molecular weight fractions are present in approximately the same 6
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
cells is retained by polydisperse fucopolysaccharides that were isolated from F. vesiculosus using a simplified method by acid-free extraction (Figs. 2 and 3). Information on the intracellular localization of fucoidans was limited to the reports on the internalization of LMW fucoidans (Deux et al., 2002; Marinval et al., 2016; Zhu et al., 2011). To investigate how fucopolysaccharides interact with cells, and how this process differs between cells different сell lines, fucoidans F and FG have been labeled with a fluorescent FITC label. That allowed us to observe in dynamics how fucopolysaccharides of different degree of polydispersity are internalized by cells (Fig. 4) and visualize their distribution in and around the cells (Fig. 5a and b). Confocal microscopy images taken throughout 22 h of incubation showed that the fucoidans were inter nalized by the cells of both cell lines. Labeled fucoidan was visualized inside the cells as small bright inclusions, consistent with endocytic vesicles formed during endocytosis. No observable increase in fluores cence at the cell walls that could indicate persistent fucoidan binding was seen. The native Fflu seemed to be taken up faster by both types of cells as compared to the partially hydrolyzed FGflu (Figs. 4 and 5c). This observation can likely be explained by the fact that at the same dosage (180 μg/ml) of fucoidans added to the medium, the percentage of LMW fraction of polysaccharides in the Fflu composition was greater than in FGflu (Fig. 1). It therefore appears that it is the polysaccharide fraction with low molecular weight that is readily internalized by the cells. These results are consistent with the flow cytometry data characterizing the dynamics of uptake of labeled fucoidans by cells: after 22 h, fluorescence is more detectable in the cells of both lines that have been incubated with Fflu (Fig. 5). A visual comparison of the absorption of fucoidans shows that the accumulation of both native and partially hydrolyzed fucoidans inside Chang liver cells is higher than in Hep G2 cells (Fig. 4). Further, according to flow cytometry measurements after 43 h of incubation, the fluorescence in the Chang liver cells of both Fflu and FGflu fucoidans exceeds that in Hep G2 cells (Fig. 5). According to the study of the absorption and binding of iron oxide nanoparticles by breast cells (Zhang et al., 2008), non-malignant MCF10A cells assimilate nanoparticles by vesicular transport much faster than the tumor MCF7. Thus, it can be inferred that the endocytosis of sulfated polysaccharides may also be faster in normal cells than in tumor cells. The antitumor activity of fucoidans is associated with the induction of apoptosis in cells as shown in a large number of publications devoted to the detection of fucoidan-induced apoptosis (Aisa et al., 2005; Kwak, 2014; Senthilkumar et al., 2013). By double staining of cells with Yo-Pro dyes and propidium iodide, we showed a slight increase in the number of Chang liver and Hep G2 cells in the early apoptosis stage after 24 h of cultivation with fucoidans F and FG. After 48 h, the number of cells in the stage of late apoptosis increased significantly, and the excess per centage of Hep G2 tumor cells stained with both Yo-Pro and PI was about 30% as compared to non-malignant Chang liver (Fig. 6). Recently, there has been a growing number of reports that sulfated fucopolysaccharides induce another type of cell response, autophagy. Park et al. showed that, in addition to apoptosis, fucoidan induces autophagy in AGS human adenocarcinoma cells (Park et al., 2011). Zhang et al. reported that treatment of breast tumor cells MDA-MB-231 and MCF-7 with an extract from F. vesiculosus also causes both apoptosis and autophagy (Zhang et al., 2016). However, Li et al. pointed out that, on the contrary, fucoidan from F. vesiculosus inhibits the formation of autophagosomes and autophagolysomes in liver tissues of fibrotic-affected mice in vivo (Li et al., 2016). In our study, staining cells with acridine orange showed that after treatment with sulfated fuco polysaccharides F and FG a time-dependent formation of vesicles with acidic content, presumably autophagolysomes, was observed in both Chang liver and Hep G2 cells. The presence of vesicles with acidic contents detected by acridine orange staining is considered to indicate the presence of autophagy (Paglin et al., 2001). Regardless of the type of fucoidan used, the number of vesicle-like structures in Chang liver cells exceeds that in Hep G2 cells, inferring that, while in tumor cells fucoidan
Fig. 5. Uptake of the fluorescently labeled fucoidan Fflu by Chang liver cells at (a) approximately 1 h of incubation and (b) 20 h of incubation. (c) Flow cytometry measurements of absorption of labeled fucoidans Fflu and FGflu by Hep G2 and Chang liver cells. Concentration of fucoidans Fflu and FGflu in the medium was 180 μg/ml.
amounts, after acidic hydrolysis and subsequent dialysis the content of the low molecular weight fractions significantly decreased (Fig. 1, Table 1). The average molecular weight of native fucoidan F and partially hydrolyzed FG was 1730 kDa and 2502 kDa, respectively, when estimated by a viscometric method. The observed increase in the mean molecular weight of the partially hydrolyzed sample compared to the native fucoidan is obviously due to the fact that a significant portion of the low molecular weight fractions was removed by dialysis after hy drolysis. Thus, at a high degree of polydispersity of polysaccharides, the average molecular weight apparently cannot be considered an adequate parameter to describe the properties of fucoidans. In our previous study of the antitumor activity of fucoidans from algae F. vesiculosus (Zhurishkina et al. 2015, 2017) we show that such an activity of the native fucoidan comes from its major fraction charac terized by the highest fucose content and degree of sulfation. The sensitivity of Chang liver cells, which are widely used by researchers as models of normal liver cells, to the action of fucoidans from brown alga F. vesiculosus was significantly lower than of hepatoma Hep G2 cells. Selective toxicity toward the malignant tumor cells is the most impor tant characteristic for the potential medical applications for sulfated polysaccharides (Li et al., 2017; Vishchuk, Tarbeeva, Ermakova, & Zvyagintseva, 2012; Yamasaki-Miyamoto, Yamasaki, Tachibana, & Yamada, 2009). In the present work we compared the effect of two polydisperse fucoidan preparations isolated from the same algae species on Hep G2 and Chang liver cells. In both cell lines the difference in the inhibitory effect on cell proliferation between the partially hydrolyzed and native fucoidans was very small and not statistically significant (Fig. 2). Our results also indicate that selective toxicity for the tumor 7
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Fig. 6. Distribution of apoptotic and viable cells: Chang liver (a) and Hep G2 (b). Flow cytometry with double staining of Yo-Pro1/propidium iodide after cultivation with fucoidans 180 μg/ml F or FG for 24 and 48 h. Intensity of fluorescence Yo-Pro1 is shown on the abscissa axis; fluorescence intensity PI (rela tive units) is on ordinate: 1st quadrant (top left) cells negative for Yo-Pro1 and positive for PI - ne crosis; 2nd quadrant (top right) - cells positive for PI and Yo-Pro1 - late stage of apoptosis; 3rd quadrant (bottom left) - cells negative for PI and Yo-Pro1 viable cells; 4th quadrant (bottom right) - cells pos itive for Yo-Pro1 and negative for PI - early stage of apoptosis.
induce apoptosis to a greater degree, in non-malignant ones it primarily induces autophagy. Autophagy is considered to be a physiological process of cell selfrenewal, which can serve protective function under stressful in fluences, or, under certain conditions, can lead to cell death (Paglin et al., 2001, Wilson et al., 2011; Potapnev, 2014). It is logical to assume that the reason for the difference in the effect of fucopolysaccharides on tumor and non-malignant cells is that after fucoidan is internalized, different scenarios of intracellular events develop in different cell types. The toxicity of fucoidans to human hepatoma Hep G2 cells may be related to such features of this cell line such as low expression of cyto chrome P450, an enzyme responsible for intracellular metabolic pro �mez-Lecho �n, 2012). cesses (Tolosa, Donato, P�erez-Cataldo, Castell, & Go This could result in accumulation of toxic products within Hep G2 cells after the uptake of fucoidans, leading ultimately to their death. Obvi ously, different mechanisms are possible for other cells. Targeted acti vation of various complementary forms of cell death can serve as a new strategy to combat various diseases (Kopeina, Senichkin, & Zhivotovsky, 2017).
5. Conclusions The high molecular weight and polydispersity of fucoidans isolated from F. vesiculosus by acid-free extraction do not appear to adversely affect their antitumor activity. Fucoidans are internalized by cells, most likely via endocytosis, with Chang liver cells more rapidly absorbing the polysaccharide as compared to Hep G2 cells. Treatment with fucoidans induces different types of response depending on the type of cells: pri marily autophagy in Chang liver cells, whereas in Hep G2 cells apoptosis was more pronounced and lower autophagy was detected. The observed biological activity of polydisperse fucoidans obtained from brown algae by a simplified procedure, rather than the labor-intensive multi-stage purification of fractions, could help practical application of these unique polysaccharides. Acknowledgments The work has been performed as part of the Genome Research Center development program “Kurchatov Genome Center - PNPI” (agreement No. 075-15-2019-1663).
8
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Fig. 7. Detection of acidic vesicular organ elles (AVO) in Hep G2 and Chang liver cells with acridine orange staining. (a) -changes in red fluorescence after 8 h after treatment with fucoidans F and FG, flow cytometry; (b) - red:green fluorescence ratio in Hep G2 and Chang liver cells treated with fucoidans F and FG for 48 h (data shown with control subtracted); (c) - morphological changes in Chang liver and Hep G2 cells 43 h after treatment with fucoidans F and FG. Bar size is 200 μm. (For interpretation of the refer ences to colour in this figure legend, the reader is referred to the Web version of this article.)
9
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Author contribution
Glabe, C. G., Harty, P. K., & Rosen, S. D. (1983). Preparation and properties of fluorescent polysaccharides. Analytical Biochemistry, 130, 287–294. Holtkamp, A. D., Kelly, S., Ulber, R., & Lang, S. (2009). Fucoidans and fucoidanases focus on techniques for molecular structure elucidation and modification of marine polysaccharides. Applied Microbiology and Biotechnology, 82, 1–11. https://doi.org/ 10.1007/s00253-008-1790-x. Jin, W., Zhang, Q., Wang, J., & Zhang, W. (2013). A comparative study of the anticoagulant activities of eleven fucoidans. Carbohydrate Polymers, 91, 1–6. https:// doi.org/10.1016/j.carbpol.2012.07.067. Kima, M. H., & Joo, H. G. (2008). Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cell. Immunology Letters, 115, 138–143. https://doi.org/ 10.1016/j.imlet.2007.10.016. Kim, I. H., & Nam, T. J. (2018). Fucoidan downregulates insulin-like growth factor-I receptor levels in HT-29 human colon cancer cells. Oncology Reports, 39, 1516–1522. https://doi.org/10.3892/or.2018.6193. Kopeina, G. S., Senichkin, V. V., & Zhivotovsky, B. (2017). Caloric restriction - a promising anti-cancer approach: From molecular mechanisms to clinical trials. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 1867, 29–41. https://doi. org/10.1016/j.bbcan.2016.11.002. Kwak, J. Y. (2014). Fucoidan as a marine anticancer agent in preclinical development. Marine Drugs, 12, 851–870. https://doi.org/10.3390/md12020851. Li, J., Chen, K., Li, S., Feng, J., Liu, T., Wang, F., et al. (2016). Protective effect of fucoidan from Fucus vesiculosus on liver fibrosis via the TGF-β1/Smad pathwaymediated inhibition of extracellular matrix and autophagy. Drug Design, Development and Therapy, 10, 619–630. https://doi.org/10.2147/DDDT.S98740. Li, X. J., & Ye, Q. F. (2015). Fucoidan reduces inflammatory response in a rat model of hepatic ischemia-reperfusion injury. Canadian Journal of Physiology and Pharmacology, 93, 999–1005. https://doi.org/10.1139/cjpp-2015-0120. Li, J., Zhang, Q., Li, S., Dai, W., Feng, J., Wu, L., et al. (2017). The natural product fucoidan ameliorates hepatic ischemia-reperfusion injury in mice. Biomedicine & Pharmacotherapy, 94, 687–696. https://doi.org/10.1016/j.biopha.2017.07.109. Marinval, N., Saboural, P., Haddad, O., Maire, M., Bassand, K., Geinguenaud, F., et al. (2016). Identification of a pro-angiogenic potential and cellular uptake mechanism of a LMW highly sulfated fraction of fucoidan from Ascophyllum nodosum. Marine Drugs, 14, E185. https://doi.org/10.3390/md14100185. pii. Marudhupandi, T., & Kumar, T. T. A. (2013). Antibacterial effect of fucoidan from Sargassum wightii against the chosen human bacterial pathogens. International Current Pharmaceutical Journal, 2, 156–158. https://doi.org/10.3329/icpj.v2i10.16408. Matsubara, K., Xue, C., Zhao, X., Mori, M., Sugawara, T., & Hirata, T. (2005). Effects of middle molecular weight fucoidans on in vitro and ex vivo angiogenesis of endothelial cells. Int. J. Molec. Medicine, 15, 695–699. Morua, V. K., Kim, J., & Kim, E. K. (2012). Algal fucoidan: Structural and size-dependent bioactivities and their perspectives. Applied Microbiology and Biotechnology, 93, 71–82. https://doi.org/10.1007/s00253-011-3666-8. Oliveira, C., Ferreira, A. S., Novoa-Carballal, R., Nunes, C., Pashkuleva, I., Neves, N. M., et al. (2017). The key role of sulfation and branching on fucoidan antitumor activity. Macromolecular Bioscience. https://doi.org/10.1002/mabi.201600340. Orata, F. (2012). Derivatization reactions and reagents for gas chromatography analysis. In M. Mohd (Ed.), Advanced gas chromatography — progress in agricultural, biomedical and industrial applications (p. 94). InTech, ISBN 978-953-51-0298-4. Available from: http://www.intechopen.com/books/advanced-gas-chromatography-progress-in-ag ricultural-biomedical-and-industrial-applications/derivatization-reactions-and-reag ents-for-gas-chromatography-analysis. Paglin, S., Hollister, T., Delohery, T., Hackett, N., McMahill, M., Sphicas, E., et al. (2001). A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Research, 15, 439–444. Park, H. S., Kim, G. Y., Nam, T. J., Kim, N. D., & Choi, Y. H. (2011). Antiproliferative activity of fucoidan was associated with the induction of apoptosis and autophagy in AGS human gastric cancer cells. Journal of Food Science, 76, 77–83. https://doi.org/ 10.1111/j.1750-3841.2011.02099.x. Pavliga, S. N., Kompanets, G. G., & Tsygankov, V. Y. (2016). The experimental research (in vitro) of carrageenans and fucoidans to decrease activity of Hantavirus. Food Environ. Virol., 8, 120–124. https://doi.org/10.1007/s12560-016-9233-9. Peiser, L. S., & Gordon, S. (2001). The function of scavenger receptors expressed by macrophages and their role in the regulation of inflammation. Microbes and Infection, 3, 149–159. Potapnev, M. P. (2014). Autophagy, apoptosis, necrosis and immune recognition of self and nonself. Immunology, 2, 95–102 (In Russ.). Senthilkumar, K., Manivasagan, P., Venkatesan, J., & Kim, S. K. (2013). Brown seaweed fucoidan: Biological activity and apoptosis, growth signaling mechanism in cancer. International Journal of Biological Macromolecules, 60, 366–374. https://doi.org/ 10.1016/j.ijbiomac.2013.06.030. Stepanov, S. I., Konyshev, V. N., Kotlovanova, L. V., & Roganov, A. P. (1996). Karyotyping of individual cells with flow cytometry. Cytometry, 23, 279–283. https://doi.org/10.1002/(SICI)1097-0320(19960401)23:4<279::AID-CYTO3>3.0. CO;2-C. Tamura, Y., Adachi, H., Osuga, J., Ohashi, K., Yahagi, N., Sekiya, M., et al. (2003). FEEL1 and FEEL-2 are endocytic receptors for advanced glycation end products. Journal of Biological Chemistry, 278, 12613–12617. https://doi.org/10.1074/jbc.M210211200. Tolosa, L., Donato, M. T., P�erez-Cataldo, G., Castell, J. V., & G� omez-Lech� on, M. J. (2012). Upgrading cytochrome P450 activity in Hep G2 cells co-transfected with adenoviral vectors for drug hepatotoxicity assessment. Toxicology in Vitro, 26, 1272–1277. https://doi.org/10.1016/j.tiv.2011.11.008. Torres, M. A., Sousa, A. P., Silva Filho, E. A., Melo, D. F., Feitosa, J. P., de Paula, R. C., et al. (2007). Extraction and physicochemical characterization of Sargassum vulgare
Elena V. Zhurishkina: Investigation, Methodology, Formal analysis; Sergey I. Stepanov: Investigation; Olga N. Ayrapetyan: Visualization; Yury A. Skorik: Investigation, Methodology, Formal analysis; Elena N. Vlasova: Investigation; Igor V. Kruchina-Bogdanov: Investigation, Methodology, Formal analysis; Dmitry V. Lebedev: Investigation, Writing - Review & Editing; Anna A. Kulminskaya: Supervision, Re sources, Funding acquisition; Irina M. Lapina: Conceptualization, Investigation, Writing - Original Draft. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.bcdf.2019.100209. References Abudabbus, A., Badmus, J. A., Shalaweh, S., Bauer, R., & Hiss, D. (2017). Effects of fucoidan and chemotherapeutic agent combinations on malignant and nonmalignant breast cell lines. Current Pharmaceutical Biotechnology, 18, 748–757. https://doi.org/10.2174/1389201018666171115115112. Aisa, Y., Miyakawa, Y., Nakazato, T., Shibata, H., Saito, K., Ikeda, Y., et al. (2005). Fucoidan induces apoptosis of human HS-Sultan cells accompanied by activation of caspase-3 and down-regulation of ERK pathways. American Journal of Hematology, 78, 7–14. https://doi.org/10.1002/ajh.20182. Ale, M. T., Mikkelsen, J. D., & Meyer, A. S. (2011). Important determinants for fucoidan bioactivity: A critical review of structure-function relations and extraction methods for fucose-containing sulfated polysaccharides from brown seaweeds. Marine Drugs, 9, 2106–2130. https://doi.org/10.3390/md9102106. Anastyuk, S. D., Shevchenko, N. M., Ermakova, S. P., Vishchuk, O. S., Nazarenko, E. L., Dmitrenok, P. S., et al. (2012). Anticancer activity in vitro of a fucoidan from the brown alga Fucus evanescens and its low-molecular fragments, structurally characterized by tandem mass-spectrometry. Carbohydrate Polymers, 87, 186–194. Anastyuk, S. D., Shevchenko, N. M., Nazarenko, E. L., Imbs, T. I., Gorbach, V. I., Dmitrenok, P. S., et al. (2010). Structural analysis of a highly sulfated fucan from the brown alga Laminaria cichorioides by tandem MALDI and ESI mass spectrometry. Carbohydrate Research, 345, 2206–2212. https://doi.org/10.1016/j. carres.2010.07.043. Berwin, B., Delneste, Y., Lovingood, R. V., Post, S. R., & Pizzo, S. V. (2004). SREC-I, a type F scavenger receptor, is an endocytic receptor for calreticulin. Journal of Biological Chemistry, 279, 51250–51257. https://doi.org/10.1074/jbc.M406202200. Bilan, M. I., Grachev, A. A., Shashkov, A. S., Kelly, M., Sanderson, C. J., Nifantiev, N. E., et al. (2010). Further studies on the composition and structure of a fucoidan preparation from the brown alga Saccharina latissima. Carbohydrate Research, 345, 2038–2047. https://doi.org/10.1016/j.carres.2010.07.009. Cho, M. L., Lee, D. J., Kim, J. K., & You, S. (2014). Molecular characterization and immunomodulatory activity of sulfated fucans from Agarum cribrosum. Carbohydrate Polymers, 113, 507–514. https://doi.org/10.1016/j.carbpol.2014.07.055. Cho, M. L., Lee, B. Y., & You, S. (2011). Relationship between oversulfation and conformation of low and high molecular weight fucoidans and evaluation of their in vitro anticancer activity. Molecules, 16, 291–297. https://doi.org/10.3390/ molecules16010291. Cumashi, A., Ushakova, N. A., Preobrazhenskaya, M. E., D’Incecco, A., Piccoli, A., Totani, L., et al. (2007). A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic and antiadhesive activities of nine different fucoidans from brown seaweeds. Glycobiology, 17, 541–552. https://doi.org/10.1093/glycob/ cwm014. Deux, J. F., Meddahi-Pell� e, A., Le Blanche, A. F., Feldman, L. J., Colliec-Jouault, S., Br� ee, F., et al. (2002). Low molecular weight fucoidan prevents neointimal hyperplasia in rabbit iliac artery in-stent restenosis model. Arteriosclerosis, Thrombosis, and Vascular Biology, 22, 1604–1609. Dische, Z., Shettles, L. B., & Osnos, M. (1949). New specific color reactions of hexoses and spectrophotometric micro-metods for their determination. Archives of Biochemistry, 22, 169–184. Dodgson, K. S. (1961). Determination of inorganic sulphate in studies on the enzymatic and non-enzymatic hydrolysis of carbohydrate and other sulphate esters. Biochemical Journal, 78, 312–319. Durig, J., Bruhn, T., Zurborn, K. H., Gutensohn, K., Bruhn, H. D., & Beress, L. (1997). Anticoagulant fucoidan fraction from Fucus vesiculosus induce platelet activation in vitro. Thrombosis Research, 85, 479–491. Fitton, J. H., Stringer, D. N., & Karpiniec, S. S. (2015). Therapies from fucoidan: An update. Marine Drugs, 13, 5920–5946. https://doi.org/10.3390/md13095920.
10
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx cells. Journal of Agricultural and Food Chemistry, 57, 8677–8682. https://doi.org/ 10.1021/jf9010406. Yang, C., Chung, D., Shin, I., Lee, H., Kim, J., Lee, Y., et al. (2008). Effects of molecular weight and hydrolysis conditions on anticancer activity of fucoidans from sporophyll of Undaria pinnatifida. International Journal of Biological Macromolecules, 43, 433–437. https://doi.org/10.1016/j.ijbiomac.2008.08.006. Zayed, A., Muffler, K., Hahn, T., Rupp, S., Finkelmeier, D., Burger-Kentischer, A., et al. (2016). Physicochemical and biological characterization of fucoidan from Fucus vesiculosus purified by dye affinity chromatography. Marine Drugs, 14, E79. https:// doi.org/10.3390/md14040079. pii. Zhang, J., Riby, J. E., Conde, L., Grizzle, W. E., Cui, X., & Skibola, C. F. (2016). A Fucus vesiculosus extract inhibits estrogen receptor activation and induces cell death in female cancer cell lines. BMC Complementary and Alternative Medicine, 16, 151. https://doi.org/10.1186/s12906-016-1129-6. Zhang, Y., Yang, M., Portney, N. G., Cui, D., Budak, G., Ozbay, E., et al. (2008). Zeta potential: A surface electrical characteristic to probe the interaction of nanoparticles with normal and cancer human breast epithelial cells. Biomedical Microdevices, 10, 321–328. https://doi.org/10.1007/s10544-007-9139-2. Zhurishkina, E. V., Lapina, I. M., Ivanen, D. R., Stepanov, S. I., Shvetsova, S. V., Shavarda, A. L., et al. (2015). Effect of fucoidans isolated from seaweeds Laminaria digitata and Fucus vesiculosis on cell lines HeLa G-63, ECV 304 and PC 12. Tsitologiya, 57, 727–735. Zhurishkina, E. V., Stepanov, S. I., Shvetsova, S. V., Kulminskaya, A. A., & Lapina, I. M. (2017). A comparison of the effect of fucoidan from alga Fucus vesiculosus and its fractions obtained by anion-exchange chromatography on HeLa G-63, Hep G2, and Chang liver cells. Cell and Tissue Biology, 11, 242–249. Zhu, X. D., Zhuang, Y., Ben, J. J., Qian, L. L., Huang, H. P., Bai, H., et al. (2011). Caveolae-dependent endocytosis is required for class A macrophage scavenger receptor-mediated apoptosis in macrophages. Journal of Biological Chemistry, 286, 8231–8239. https://doi.org/10.1074/jbc.M110.145888.
alginate from Brazil. Carbohydrate Research, 342, 2067–2074. https://doi.org/ 10.1016/j.carres.2007.05.022. Trinchero, J., Ponce, N. M. A., Cordoba, O. L., Flores, M. L., Pampuro, S., Stortz, C. A., et al. (2009). Antiretroviral activity of fucoidans extracted from the brown seaweed Adenocystis utricularis. Phytotherapy Research, 23, 707–712. https://doi.org/10.1002/ ptr.2723. Usoltseva, R. V., Anastyuk, S. D., Ishina, I. A., Isakov, V. V., Zvyagintseva, T. N., Thinh, P. D., et al. (2018). Structural characteristics and anticancer activity in vitro of fucoidan from brown alga Padina boryana. Carbohydrate Polymers, 15, 260–268. https://doi.org/10.1016/j.carbpol.2017.12.071. Usoltseva, R. V., Shevchenko, N. M., Malyarenko, O. S., Ishina, I. A., Ivannikova, S. I., & Ermakova, S. P. (2018). Structure and anticancer activity of native and modified polysaccharides from brown alga Dictyota dichotoma. Carbohydrate Polymers, 180, 21–28. https://doi.org/10.1016/j.carbpol.2017.10.006. Usov, A. I., & Bilan, M. I. (2009). Fucoidans — sulfated polysaccharides of brown algae. Russian Chemical Reviews, 78, 785–799. https://doi.org/10.1070/ RC2009v078n08ABEH004063. Ustyuzhanina, N. E., Bilan, M. I., Ushakova, N. A., Usov, A. I., Kiselevskiy, M. V., & Nifantiev, N. E. (2014). Fucoidans: Pro- or antiangiogenic agents? Glycobiology, 24, 1265–1274. https://doi.org/10.1093/glycob/cwu063. Vishchuk, O. S., Tarbeeva, D. V., Ermakova, S. P., & Zvyagintseva, T. N. (2012). Structural characteristics and biological activity of fucoidans from the brown algae Alaria sp. and Saccharina japonica of different reproductive status. Chemistry and Biodiversity, 9, 817–828. https://doi.org/10.1002/cbdv.201100266. Wilson, E. N., Bristol, M. L., Di, X., Maltese, W. A., Koterba, K., Beckman, M. J., et al. (2011). A switch between cytoprotective and cytotoxic autophagy in the radio sensitization of breast tumor cells by chloroquine and vitamin D. Horm Cancer, 2, 272–285. https://doi.org/10.1007/s12672-011-0081-7. Yamasaki-Miyamoto, Y., Yamasaki, M., Tachibana, H., & Yamada, K. (2009). Fucoidan induces apoptosis through activation of caspase-8 on human breast cancer MCF-7
11
Contents lists available at ScienceDirect
Bioactive Carbohydrates and Dietary Fibre journal homepage: http://www.elsevier.com/locate/bcdf
The effect of polydisperse fucoidans from Fucus vesiculosus on Hep G2 and Chang liver cells Elena V. Zhurishkina a, b, Sergey I. Stepanov a, Olga N. Ayrapetyan a, b, c, Yury A. Skorik d, Elena N. Vlasova d, Igor V. Kruchina-Bogdanov e, Dmitry V. Lebedev a, f, Anna A. Kulminskaya a, b, Irina M. Lapina a, b, * a
Petersburg Nuclear Physics Institute named by B.P. Konstantinov of National Research Centre «Kurchatov Institute», Gatchina, Russia Kurchatov Genome Center – PNPI, Gatchina, Russia Saint Petersburg National Research University of Information Technologies, Mechanics and Optics, St. Petersburg, Russia d Institute of Macromolecular Compounds of the Russian Academy of Sciences, St. Petersburg, Russia e Analytics. Materials. Technologies Ltd, St. Petersburg, Russia f National Research Centre «Kurchatov Institute», Moscow, Russia b c
A R T I C L E I N F O
A B S T R A C T :
Keywords: Fucoidan Chang liver Hep G2
The effect of polydisperse fucoidans from brown algae Fucus vesiculosus on Hep G2 and Chang liver cells was studied. Despite the high molecular weight and polydispersity, these fucoidans exhibited antitumor activity, whereas the difference in the inhibitory effects on cell proliferation between the native and the partially hy drolyzed fucoidans was not statistically significant. Fucoidans were internalized by cells of both cell lines through endocytosis, with Chang liver cells metabolizing fucopolysaccharides more rapidly compared to the Hep G2. In both cell lines treatment with fucoidans was shown to induce apoptosis and autophagy, with apoptosis more pronounced in Hep G2 cells, whereas autophagy - in Chang liver cells.
1. Introduction Sulfated fucopolysaccharides (fucoidans) contained in the cell walls of brown algae demonstrate a variety of biological activities: antiviral (Pavliga, Kompanets, & Tsygankov, 2016; Trinchero et al., 2009), antibacterial (Marudhupandi & Kumar, 2013), anticoagulant (Durig et al., 1997; Jin, Zhang, Wang, & Zhang, 2013), immunostimulating (Cho, Lee, Kim, & You, 2014; Kima & Joo, 2008), anti-inflammatory (Li & Ye, 2015) and antitumor (Kwak, 2014; Senthilkumar, Manivasagan, Venkatesan, & Kim, 2013). Fucoidan properties attract a close attention of researchers around the world for decades. Its potential in medicine is due to the selective cytotoxicity as malignant tumor cells have been shown to have significantly greater sensitivity to fucoidan than the normal ones (Usoltseva, Anastyuk, et al., 2018 Abudabbus, Badmus, Shalaweh, Bauer, & Hiss, 2017; Usoltseva, Shevchenko et al., 2018; Kim & Nam, 2018; Park, Kim, Nam, Kim, & Choi, 2011; Zhang et al., 2016). Fucoidan is well known to induce apoptosis in cells. However, until now it remains unclear how an interaction of the cells with sulfated poly saccharides is triggering mechanisms for its self-destruction. Even the
basic aspects of the observed cytotoxicity, i.e. whether fucoidans have to penetrate into the cell, act by an external contact with receptors, or affect the intercellular contacts, are not firmly established. There are several reports that fucoidan binds to various types of scavenger re ceptors including classes A, B and F-SR (Berwin, Delneste, Lovingood, Post, & Pizzo, 2004; Peiser & Gordon, 2001; Tamura et al., 2003), as well as enters the cells through endocytosis. It has been shown that the SR-A-fucoidan complex is internalized both through clathrin and caveolin-dependent pathways of endocytosis (Zhu et al., 2011). Deux reported internalization of low molecular weight fucoidan in the peri nuclear vesicles of rabbit smooth muscle cells (Deux et al., 2002). It was also shown that low molecular weight fucoidan is internalized by HUVEC mainly through the clathrin-mediated endocytosis pathway (Marinval et al., 2016). Regardless of the mechanism of internalization, fluorescently-labeled sulfated polysaccharides can be used to visualize the interaction of fucoidan and the cell (Marinval et al., 2016; Oliveira et al., 2017). The heterogeneity of the composition and the large size of the fucoidan molecules both complicate establishing the relationship
* Corresponding author. Petersburg Nuclear Physics Institute named by B.P. Konstantinov of National Research Centre «Kurchatov Institute», Gatchina, Russia. E-mail address: [email protected] (I.M. Lapina). https://doi.org/10.1016/j.bcdf.2019.100209 Received 1 July 2019; Received in revised form 16 December 2019; Accepted 17 December 2019 Available online 19 December 2019 2212-6198/© 2019 Published by Elsevier Ltd.
Please cite this article as: Elena V. Zhurishkina, Bioactive Carbohydrates and Dietary Fibre, https://doi.org/10.1016/j.bcdf.2019.100209
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
between the structure of sulfated fucopolysaccharides and their bio logical activity, which is a necessary step for their potential application in medicine. A number of publications report biological activity of low molecular weight fucoidans obtained by acidic, radical or enzymatic hydrolysis (reviewed in (Morua, Kim, & Kim, 2012)). However, there are only few studies comparing the cytotoxicity of fucopolysaccharides with different degrees of polydispersity (Anastyuk et al., 2012; Oliveira et al., 2017; Yang et al., 2008). In our previous study of biological activity of fucoidans from Fucus vesiculosus (Zhurishkina et al. 2015, 2017) we compared the cytotoxicity of crude fucoidan and its fractions obtained by anion-exchange chro matography, showing that the unfractioned fucoidan had higher toxicity toward Hep G2 cells than its most active fraction with similar degree of sulfation. In this work we investigate the effect of two preparations of polydispersed sulfated fucopolysaccharides with different content of high and low molecular weight fractions on the Hep G2 and Chang liver cell lines.
followed by dialysis (pore size of 3.5 kDa, Spektra/Por, Canada) against the distilled water for 12 h. The sulfate content in fucoidans was determined by the turbidimetric method after acidic hydrolysis of the samples with a 1 N HCl solution at 100 � C for 6 h using K2SO4 as a standard (Dodgson, 1961). The content of fucose was determined by the method described in (Dische, Shettles, & Osnos, 1949). Molecular mass distribution of the fucoidan samples was evaluated by HPLC on a column 7.5 x 600 mm TSKGel 3000SW (Toyo Soda, Japan). A 10 mM phosphate buffer at 0.9 ml/min as the mobile phase with high pressure pump CN 4000 (Milton Roy, USA) and refractometric detector RI Model 504 (Gasukuro Kogyo/Japan) were used. Dextrans with molecular weights from 15 to 200 kDa were used for MW calibration. For low pressure gel chromatography were used 0.3% ammonium sulfate solution as eluent, column 15.7 x 182 mm, Sepharose 4B (flow rate 11 ml/h) and column 10.3 x 160 mm, Sephadex G15 (flow rate 15.2 ml/h), peristaltic pump NP 1M (Russia), and the same refractometric detector. Dextrans with molecular weights from 40 kDa to 2MDa with the former column and glucose–saccharose–raffinose–Blue dextran with the latter column were used for MW calibration. Monosaccharide composition of low and high MW fractions of fucoidans F and FG isolated by HPLC was quantified with gas-liquid chromatography of trimethylsilyl derivatives after the acid hydrolysis of polysaccharides with 2M trifluoroacetic acid for 5 h and 105 � C. Monosaccharide mixture obtained was treated in pyridine with 1,1,1,3,3,3-hexamethyldisilazane in the presence of trifluoroacetic acid at 60 � C for 1 h (Orata, 2012). Analysis conditions: chromatograph Shimadzu 2010 (Shimadzu, Japan), CBP5-25 column (25 m x 0.25 mm x 0.2 μm); carrier gas N2, 20 cm/s; sample volume 2 μl; injector temper ature 270� С; flame ionization detector, temperature 325� С. FTIR spectra were obtained using a Vertex-70 spectrometer (Bruker, Germany) equipped with a Pike attenuated total reflectance accessory (a ZnSe prism). The Bruker OPUS spectroscopy software was used for data acquisition and analysis. The average molecular weight was estimated by a viscometric method (Torres et al., 2007) and calculated from the viscosity data by the Mark-Houwink equation [η] ¼ KMα, where [η] is the intrinsic vis cosity, M is the average molecular weight; K, α are empirical constants. In this case, the intrinsic viscosity was determined as [η]¼(ηsp/c)c→0, where c is the polymer concentration, g/100 ml; ηsp is the specific vis cosity, defined as ηsp¼(η– ηsolv)/ηsolv, where ηsolv is the viscosity of the solvent, η is the measured viscosity of the polymer solution. The vis cosity was measured on SV-10A tuning fork vibration viscometer (A&D, Japan) at 20 � C. Bidistilled water with a conductivity of less than 0.3 μS and a viscosity of 1.00 mPa*s was used as a solvent. Solutions of dex trans having a molecular weight in a range from 200 kDa to 2000 kDa were used for the calibration.
2. Materials and methods All reagents were purchased from Sigma-Aldrich (Germany) and Acros Organics (USA), unless otherwise specified. Confocal microscopy was performed using Leica SP5 (Leica Microsystems, Germany) micro scope. Flow cytometry measurements were done using an in-house flow cytometer built in the radiobiology and medicine group of the Peters burg Institute of Nuclear Physics (Stepanov, Konyshev, Kotlovanova, & Roganov, 1996). 2.1. Cell culture Cells of human hepatoblastoma (Hep G2 cell line) were purchased at the Cellular Center of the Institute of Cytology RAS (St. Petersburg). The cells of normal human liver tissue (Chang liver cell line) were purchased at the Scientific Research Institute of Virology named after D.I. Iva novsky (Moscow). Hep G2 and Chang liver cells were cultured in Carrel vials in MEM medium (L-glutamine) (ICN, USA) containing 10% of bovine serum and 1% of a mixture of penicillin and streptomycin (Bio lot, St. Petersburg) at 37 � C. To construct the growth curves and deter mine the size of the Sub-G1 population, the cells were grown for 24 h, then fucoidans F and FG were added to the culture medium so that the final concentration was 180 μg/ml. The concentration of 180 μg/ml was chosen in a series of preliminary experiments as the minimal concen tration at which cytotoxic effect of fucoidan was observed reliably on both cell lines (data not shown). After 12, 24, 48 and 72 h of incubation, the cells were stained with propidium iodide and counted on a flow cytometer. 2.2. Preparation and analysis of fucoidans
2.3. The production of fucoidans with a fluorescent label
As a source of fucoidan, the mechanically ground brown algae F. vesiculosus (Alganika LLC, St. Petersburg) was used. A crude fraction of sulfated polysaccharides F was isolated from the dry ground algae F. vesiculosus according to a modified method described in (Bilan et al., 2010; Usov & Bilan, 2009). Dry ground algae F. vesiculosus (100 g) was treated with a 4: 2: 1 mixture of methanol-chloroform-water to remove lipids and pigments, centrifuged and dried. One liter of 2% CaCl2 solu tion was mixed with the resulted dried algae (90 g) and stirred at 85 � C for 5 h. The extract was separated by centrifugation at 5500 rpm and ethanol (1.2 L) was poured into 700 ml of the supernatant. The pre cipitate was separated by centrifugation at 5500 rpm, dissolved in 200 ml of a 2% CaCl2 solution, and dialyzed with a 3.5 kDa pore dialysis membrane (Spektra/Por, Canada) against distilled water for 12 h. The insoluble part was removed by centrifugation, the solution was lyophi lized and, as a result, 2.9 g of fucoidan F was obtained. A sample of partially hydrolyzed fucoidan (FG) was prepared by acidic hydrolysis with 0.01 N HCl for 5 min according to the procedure (Yang et al., 2008),
Samples of labeled fucoidans Fflu and FGflu were synthesized ac cording to the procedure described in (Glabe, Harty, & Rosen, 1983). The degree of hydroxyl groups substitution with fluoresceinamine was determined using a F-4010 fluorescence spectrophotometer (Hitachi, Japan). 2.4. Confocal microscopy Hep G2 and Chang liver cells were seeded in Glass Bottom cups (NEST Scientific, USA). After the cells were attached (about 6 h), the fucoidans Fflu and FGflu were added to the culture medium to the final concentration of 180 μg/ml. Measurements were conducted on a confocal microscope Leica SP5 for 22 h, taking the image every hour. Throughout the observation period, the cells were incubated in 5% CO2 atmosphere and 37 � C. Oil immersion 63-x objective with aperture 1.4 was used, fluorescence excitation was done by an argon laser at 488 nm, 2
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
emission was detected in the 500–550 nm range, LAS AF program (Leica Microsystems, Germany) was used to image processing.
3. Results
2.5. Flow cytometry with labeled fucoidans
The native fucoidan F and partially depolymerized FG sample ob tained from F. vesiculosus brown algae contained 45.6% and 53.2% of fucose and 14.6% and 14.2% sulfates, respectively. Sulfo group position in sulfated polysaccharides F and FG was resolved with infrared analysis (Fig. 1S Supplementary materials). IR spectra of both the native fucoidan F and partially hydrolyzed fucoidan – O bond at 1219 FG had intensive absorption bands corresponding to S– and 1215 сm 1, respectively. When analyzing the peak bands at 840 cm 1 for F and 841 cm 1 for FG, we found that they can be described by two components at 846 and 819 cm 1. In the case of F, the ratio of S846/ S819 was 1.3, which indicates the prevailing content of sulfo groups in the axial position, whereas in the IR spectrum of FG the S846/S819 ratio was 1.1, which indicated that the content of sulfo groups in the axial position was only slightly higher than that in the equatorial position. According to SEC-HPLC (Fig. 1a), the molecular weight profile of native fucoidan F had three peaks, the first one corresponded to HMW fraction (MW > 200 kDa), and its peak maxima both for 200 nm and for RI curves coincided in retention times. Two peaks of lower MW fractions (MW < 15 kDa) were poorly resolved on the refractometer curve, but the obviously overwhelming UV absorbance at 200 nm made it possible to pinpoint the maximum of the broad former one at 22.4 min, whereas on the RI curve this fraction appeared as a close shoulder on a front of the latter, narrower peak with maximum at 24.1 min. The ratio between areas of HMW and LMW fractions can be estimated by refractometer
3.1. Characterization of fucoidans F and FG
Hep G2 and Chang liver cells were seeded in Karel vials at a con centration of 60,000 cells/ml. After 24 h, the labeled fucoidans Fflu and FGflu were added at the final concentration 180 μg/ml and incubated for 4, 22 and 43 h. The cells were then rinsed with PBS and harvested with a Versene solution, centrifuged at 700 rpm, resuspended in the buffer and analyzed on a flow cytometer. Excitation with argon laser for green fluorescence at a wavelength of 488 nm, detection of emission at 535 nm. At least 10,000 cells were analyzed in each sample. 2.6. Determination of apoptotic cells Cells of Hep G2 and Chang liver were cultivated in culture flasks. After 24 h, fucoidans F and FG were added to reach the final concen tration of 180 μg/ml. After 24 and 48 h the combined staining with Membrane Permeability/Dead Cell Apoptosis Kit with Yo-Pro®1 and PI for Flow Cytometry (Invitrogen, USA) was performed according to the manufacturer’s protocol. Cells were analyzed with a flow cytometer. Each experiment was performed three times, at least 10,000 cells were analyzed in each sample. 2.7. Staining with acridine orange Cytometry. Hep G2 and Chang liver cells were seeded in culture flasks. After 24 h, fucoidans F and FG were added to the final concen tration of 180 μg/ml. After growth for the specific time period (43, 30, 24, 22, 18, 14, 8 and 4 h) cells were harvested using a Versene solution, centrifuged at 700 rpm, culture medium containing acridine orange at a concentration of 1 μg/ml was added for 15 min. Then the medium was removed, cells were washed with PBS and analyzed with a flow cy tometer. Green fluorescence was excited with argon laser at a wave length of 488 nm, emission was detected at 535 nm. Red fluorescence excitation was at a wavelength of 630 nm, detection of emission at 650 nm. At least 10,000 cells were analyzed in each sample. Flatbed analyzer. Hep G2 and Chang liver cells were seeded in 96-well black opaque plates (Thermo Fisher Scientific, Denmark) (5000 cells per well) and grown for 24 h in a damp atmosphere at 37 � C and 5% CO2. Fucoidans F and FG were added to the final concentration of 180 μg/ml and incubated for 4, 8, 14, 18, 22, 24, 30, 43 and 48 h. The medium was then removed by aspiration, cells were washed with PBS and stained with acridine orange in a concentration of 1 μg/ml for 15 min. The cells were washed with buffer and analyzed on a multimode plate analyzer En Spire 2300 (PerkinElmer, USA). The analysis of the obtained data was carried out using the manufacturer’s software. Microscopy. Hep G2 and Chang liver cells were grown on coverslips in culture flasks. After 24 h, fucoidans F and FG were added at a concen tration of 180 μg/ml and incubated for 8 h. The medium was then removed, cells were washed with PBS and stained with acridine orange at a concentration of 1 μg/ml for 15 min. The cells were washed with buffer and analyzed on EVOS FLoid microscope (Life technologies, USA). 2.8. Statistical processing
Fig. 1. Molecular weight profile of fucoidans F (a) and FG (b), HPLC method. Output curves (pink - refractometer, blue - photometer 200 nm). The bottom axis is the output volume, ml; the left axis is the refractometer signal, mV; the right axis is the signal of the photometer, mV. (For interpretation of the ref erences to colour in this figure legend, the reader is referred to the Web version of this article.)
For statistical data processing, plotting charts and diagrams, Excel 2010 and OriginPro 16 were used. The graphs show average values of at least 3 independent experiments, bars represent the standard errors. The statistical significance of the observed differences was assessed using Student’s t-test. 3
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
data at 57:43. The molecular weight profile of the partially depolymerized fucoidan FG (Fig. 1b) is presented as three peaks of the same retention times as fucoidan F with a ratio of HMW to LMW areas as 83.6:7.3:9.1. A monosaccharide composition of the both the high and low mo lecular weight fractions F and FG obtained by GC is presented in Table 1 Supplementary materials. Molecular mass distribution of the fucoidans F and FG by gelfiltration on Sepharose 4B is presented in the Table 1. The effective mean molecular weight of F and FG fucopoly saccharides, estimated by viscometric method (Torres et al., 2007), was 1730 kDa and 2502 kDa, respectively. The apparent increase in the mean molecular weight in the partially hydrolyzed samples should likely be attributed to a higher rate of hydrolysis of fucoidans in the lowest molecular weight fraction (<40 kDa) with the subsequent removal of hydrolysis products with MW < 3.5 kDa during dialysis, as demon strated by SEC-HPLC and gel filtration data. The molecular weight of the LMW fractions of fucoidans F and FG, estimated by gel-filtration on Sephadex G15, was 490 Da and 530 Da, respectively. 3.2. Effect of fucoidans F and FG on the proliferation and death of Hep G2 and Chang liver cells Analysis of the growth rates of Hep G2 and Chang liver cells showed that fucoidans F and FG at a dose of 180 μg/ml inhibited the prolifera tion of both cell lines after 48 h. Hep G2 cells were more sensitive to both fucoidans than Chang liver cells (Fig. 2). According to flow cytometry data, treatment of Hep G2 and Chang liver cells with sulfated fucopolysaccharides F and FG at a concentration of 180 μg/ml for 24 and 48 h resulted in an increase in the percentage of dead cells compared to the control (Fig. 3). The effect of partially hy drolyzed fucoidan FG on cells appears to be stronger than the native one, but the difference was not statistically significant. When applied to Hep G2 cells, the effect of both F and FG was more pronounced after 48 h of treatment as compared to 24 h. Fig. 2. The antiproliferative effect of F and FG fucoidans against Hep G2 (a) and Chang liver (b) cells at concentration 180 μg/ml. Live cells were counted by flow cytometry using propidium iodide staining, as described in Methods. (* different from control, p < 0.01; ** - different from control, p < 0.001).
3.3. Internalization of fucoidans Fflu and FGflu by Hep G2 and Chang liver cells To investigate the interaction of cells with fucoidans, the fluorescently-labeled fucoidans Fflu and FGflu were synthesized. Using fluorescence spectrophotometry, it was found that the degree of hy droxyl groups substitution with fluoresceinamine was approximately 2 molecules of the dye per 100 monomers of fucose. After the addition of Fflu and FGflu at a concentration of 180 μg/ml to the culture medium, Hep G2 cells and the Chang liver cells were monitored by a confocal microscopy for 22 h, recording the images every hour. It was found that fucoidans were internalized by the cells of both cell lines (Fig. 4). Labeled fucoidan was visualized inside the cells as small bright in clusions (Fig. 5 a,b). The number of vesicular structures containing fluorescently labeled fucoidan was higher in Chang liver cells. A visual comparison of the fluorescence from the labeled sulfated
polysaccharides within the cells after 22 h has revealed that in both cell types the overall brightness of inclusion spots appears to be higher when non-hydrolyzed fucoidan Fflu was used for treatment as compared the partially hydrolyzed FGflu. While both Fflu and FGflu caused timedependent death of Hep G2 tumor cells, the death of non-malignant Chang liver cells was practically not observed. Interestingly, the accu mulation of strong fluorescence intensity was observed in the remnants of dead cells, apparently due to direct uptake after the loss of integrity of the cell walls during cell death. To quantify the dynamics of fucoidan accumulation, Hep G2 and Chang liver cells were treated with the labeled fucoidans Fflu and FGflu for 4, 22 and 43 h and analyzed on a flow cytometer. The green fluo rescence of the acquired labeled fucoidans in cells was detected. A timedependent increase in fluorescence was observed for the cells of both cell lines. There was no significant difference between F and FG uptake rates, although the uptake level of the former appeared to be slightly higher. A significant absorption of fucoidan was observed by 22 h, with accumulation in Chang liver cells of both Fflu and FGflu fucoidans by the end of 43 h treatment exceeding that in Hep G2 cells (Fig. 5c).
Table 1 Molecular mass distribution of the fucoidans F and FG, %, (low pressure gel chromatography on Sepharose 4B). MW, kDa
>4000 2000–4000 1000–2000 500–1000 40–500 <40
Fucoidan F
FG
2,46 22,4 12,1 7,9 26,5 28,7
0,62 19,6 18,8 10,9 36,0 14,0
4
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Fig. 3. Effect of fucoidans F and FG on the death of Hep G2 (a) and Chang liver (b) cells after 24 and 48 h. Dead cells were counted by flow cytometry using propidium iodide staining, as described in Methods. (* - different from control, p < 0.01).
3.4. Fucoidans F and FG induce apoptotic death of Hep G2 cells and Chang liver
there was no significant difference in the effect between fucoidans F and FG on the degree of acidity or the volume of the cellular compartments.
To determine whether fucoidans F and FG induced apoptotic death of Hep G2 and Chang liver cells, double staining with Yo-Pro dyes and propidium iodide was carried out. From the results presented in Fig. 6, it follows that the number of cells in the stage of early apoptosis (stained with Yo-Pro and not stained with PI) after 24-h cultivation with fucoi dans F and FG exceeded the control values by 8.7 and 2.5%, respec tively, in Chang liver cells, and by 6.9 and 4.7% in Hep G2 cells. After 48 h of cultivation with fucoidans, the number of cells in the stage of late apoptosis (stained with both Yo-Pro and PI) increases significantly: in Chang liver cells by more than 20% compared to the control, in Hep G2 by more than 50%, whereas the percentage of cells in the stage of early apoptosis practically did not increase in Chang liver cells, and even decreased in Hep G2 cells. There was no noticeable difference in this effect between fucoidans F and FG.
4. Discussion The key factors affecting the antitumor activity of fucoidans are thought to be the degree of sulfation of polysaccharides, high fucose content and location of sulfate groups in the fucose residue (Cho, Lee, & You, 2011; Zhurishkina et al. 2015, 2017). Molecular weight undoubt edly plays an important role in the biological activity of fucopoly saccharides (Senthilkumar et al., 2013), however, as far as their antitumor effect, it can not be unequivocally predicted, whether fucoi dans with a greater or smaller molecular weight will be more effective. For example, Yang et al. report that acid-hydrolyzed fucoidan from Undaria pinnatifida exhibits greater antitumor activity than native. Anastyuk (Anastyuk et al., 2012) have shown that native fucoidan iso lated from Fucus evanescens algae inhibits the proliferation and growth of colonies of human melanoma cells SK-MEL-28 and SK-MEL-5, whereas a low molecular weight polysaccharide obtained under mild hydrolysis conditions hardly exhibits an effect on proliferation in both cell lines. Ustyuzhanina (Ustyuzhanina et al., 2014) reported that high-molecular fucoidan has an anti-angiogenic effect, whereas low-molecular weight fractions exhibited a pro-angiogenic effect. When analyzing the literature on the properties of fucoidans, it should be noted that the characterization of sulfated polysaccharides by molecular weight is associated with significant problems. A review by Holtkamp (Holtkamp, Kelly, Ulber, & Lang, 2009) summarizes the data where the molecular weight of fucoidans varies from 13 to 950 kDa, depending on the algal species from which the polysaccharides are isolated, mentioning the difficulties in determination of the molecular weight of fucopolysaccharides by the gel filtration method, in particular, the absence of markers, sulfated polysaccharides similar to fucoidans in structure. There is also no single consistent classification of poly saccharides by weight. The review of Senthilkumar (Senthilkumar et al., 2013) differentiate fucoidans into low (<10kDa), medium (10–10000 kDa) and high (>10000 kDa) molecular weight. However, in the article (Matsubara et al., 2005), which is cited in this review, a polysaccharide with a mass of 166 kDa is designated as high molecular weight. In another review (Kwak, 2014) when discussing the antitumor activity of fucoidans in vivo polysaccharides with a weight of 490 kDa (from the algae Undaria pinnatifida) and 6.4–10 kDa (Cladosiphon okamuranus) are
3.5. Fucoidans F and FG increase the number of acidic vesicular organelles (AVO) in Hep G2 and Chang liver cells In order to determine whether the treatment with sulfated fucopo lysaccharides caused an increase in the number of acidic vesicular or ganelles (AVO) in the Hep G2 and Chang liver cells, their staining with acridine orange was performed. As a lysosomotropic agent acridine or ange freely penetrates the membranes and accumulates in acidic com partmets, where, at low pH values, it forms aggregates with red fluorescence, the intensity of which is directly proportional to the degree of acidity and volume of the cell compartments (Paglin et al., 2001). According to flow cytometry data, the treatment of Hep G2 cells and Chang liver with fucoidans F and FG caused a noticeable increase in red fluorescence, as compared to the control (Fig. 7a). From the results shown in Fig. 7, it follows that fucoidans F and FG induced an increase in the number of acidic vesicular organelles (AVO) in the cells of both lines. In cells stained with acridine orange, the cytoplasm and nucleolus fluoresced light green, while acidic compartmets were bright red (Paglin et al., 2001). Comparing the ratio of red and green fluorescence in cells treated with fucoidans F and FG for 48 h using a plate analyzer, it was found that the number of intracellular acidic vesicles increased with the duration of treatment with fucoidans, and after 14 h this value for Chang liver cells began to exceed that for Hep G2 (Fig. 7b). At the same time, 5
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Fig. 4. Interaction of fucoidans Fflu and FGflu with Hep G2 and Chang liver cells. Confocal microscopy (Leica SP5, oil immersion 63-x objective with aperture 1.4). Concentration of fucoidans Fflu and FGflu in the medium was 180 μg/ml. The cells were maintained in 5% CO2 atmosphere at 37 � C for the entire duration of measurements. Fluorescence excitation by an argon laser at 488 nm, emission detection in the 500–550 nm range. Bar size is 50 μm.
referred to as LMW when compared with native fucoidan weighing 5100 kDa and HMW 300–330 kDa, respectively. Polydispersity of fucoidans is also often overlooked. A review by Fitton (Fitton, Stringer, & Karpiniec, 2015) indicates that native fucoidans are a mixture of molecules that vary greatly in size but, as a rule, only average molecular weights of polysaccharides are given or the range of their values without specifying the proportions of polysaccharide fractions (Cumashi et al., 2007; Fitton et al., 2015; Kwak, 2014). In this work we investigated fucoidans with different distribution of molecular weight of sulfated fucopolysaccharides. In order to save the
original structure of the polysaccharide as much as possible, we used a method of acid-free isolation from algae. The chosen method of extracting polysaccharides from the cell walls of brown algae allows the polysaccharides retain their original molecular weight (see materials and methods) (Bilan et al., 2010; Usov & Bilan, 2009), while the weakly acidic solutions, ordinarily used in the extraction of polysaccharides from the algae, contribute to their partial hydrolysis (Zayed et al., 2016; Anastyuk et al., 2010; Ale et al., 2011). We obtained SEC-HPLC data showed that while in the native fucoidan the low molecular weight and high molecular weight fractions are present in approximately the same 6
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
cells is retained by polydisperse fucopolysaccharides that were isolated from F. vesiculosus using a simplified method by acid-free extraction (Figs. 2 and 3). Information on the intracellular localization of fucoidans was limited to the reports on the internalization of LMW fucoidans (Deux et al., 2002; Marinval et al., 2016; Zhu et al., 2011). To investigate how fucopolysaccharides interact with cells, and how this process differs between cells different сell lines, fucoidans F and FG have been labeled with a fluorescent FITC label. That allowed us to observe in dynamics how fucopolysaccharides of different degree of polydispersity are internalized by cells (Fig. 4) and visualize their distribution in and around the cells (Fig. 5a and b). Confocal microscopy images taken throughout 22 h of incubation showed that the fucoidans were inter nalized by the cells of both cell lines. Labeled fucoidan was visualized inside the cells as small bright inclusions, consistent with endocytic vesicles formed during endocytosis. No observable increase in fluores cence at the cell walls that could indicate persistent fucoidan binding was seen. The native Fflu seemed to be taken up faster by both types of cells as compared to the partially hydrolyzed FGflu (Figs. 4 and 5c). This observation can likely be explained by the fact that at the same dosage (180 μg/ml) of fucoidans added to the medium, the percentage of LMW fraction of polysaccharides in the Fflu composition was greater than in FGflu (Fig. 1). It therefore appears that it is the polysaccharide fraction with low molecular weight that is readily internalized by the cells. These results are consistent with the flow cytometry data characterizing the dynamics of uptake of labeled fucoidans by cells: after 22 h, fluorescence is more detectable in the cells of both lines that have been incubated with Fflu (Fig. 5). A visual comparison of the absorption of fucoidans shows that the accumulation of both native and partially hydrolyzed fucoidans inside Chang liver cells is higher than in Hep G2 cells (Fig. 4). Further, according to flow cytometry measurements after 43 h of incubation, the fluorescence in the Chang liver cells of both Fflu and FGflu fucoidans exceeds that in Hep G2 cells (Fig. 5). According to the study of the absorption and binding of iron oxide nanoparticles by breast cells (Zhang et al., 2008), non-malignant MCF10A cells assimilate nanoparticles by vesicular transport much faster than the tumor MCF7. Thus, it can be inferred that the endocytosis of sulfated polysaccharides may also be faster in normal cells than in tumor cells. The antitumor activity of fucoidans is associated with the induction of apoptosis in cells as shown in a large number of publications devoted to the detection of fucoidan-induced apoptosis (Aisa et al., 2005; Kwak, 2014; Senthilkumar et al., 2013). By double staining of cells with Yo-Pro dyes and propidium iodide, we showed a slight increase in the number of Chang liver and Hep G2 cells in the early apoptosis stage after 24 h of cultivation with fucoidans F and FG. After 48 h, the number of cells in the stage of late apoptosis increased significantly, and the excess per centage of Hep G2 tumor cells stained with both Yo-Pro and PI was about 30% as compared to non-malignant Chang liver (Fig. 6). Recently, there has been a growing number of reports that sulfated fucopolysaccharides induce another type of cell response, autophagy. Park et al. showed that, in addition to apoptosis, fucoidan induces autophagy in AGS human adenocarcinoma cells (Park et al., 2011). Zhang et al. reported that treatment of breast tumor cells MDA-MB-231 and MCF-7 with an extract from F. vesiculosus also causes both apoptosis and autophagy (Zhang et al., 2016). However, Li et al. pointed out that, on the contrary, fucoidan from F. vesiculosus inhibits the formation of autophagosomes and autophagolysomes in liver tissues of fibrotic-affected mice in vivo (Li et al., 2016). In our study, staining cells with acridine orange showed that after treatment with sulfated fuco polysaccharides F and FG a time-dependent formation of vesicles with acidic content, presumably autophagolysomes, was observed in both Chang liver and Hep G2 cells. The presence of vesicles with acidic contents detected by acridine orange staining is considered to indicate the presence of autophagy (Paglin et al., 2001). Regardless of the type of fucoidan used, the number of vesicle-like structures in Chang liver cells exceeds that in Hep G2 cells, inferring that, while in tumor cells fucoidan
Fig. 5. Uptake of the fluorescently labeled fucoidan Fflu by Chang liver cells at (a) approximately 1 h of incubation and (b) 20 h of incubation. (c) Flow cytometry measurements of absorption of labeled fucoidans Fflu and FGflu by Hep G2 and Chang liver cells. Concentration of fucoidans Fflu and FGflu in the medium was 180 μg/ml.
amounts, after acidic hydrolysis and subsequent dialysis the content of the low molecular weight fractions significantly decreased (Fig. 1, Table 1). The average molecular weight of native fucoidan F and partially hydrolyzed FG was 1730 kDa and 2502 kDa, respectively, when estimated by a viscometric method. The observed increase in the mean molecular weight of the partially hydrolyzed sample compared to the native fucoidan is obviously due to the fact that a significant portion of the low molecular weight fractions was removed by dialysis after hy drolysis. Thus, at a high degree of polydispersity of polysaccharides, the average molecular weight apparently cannot be considered an adequate parameter to describe the properties of fucoidans. In our previous study of the antitumor activity of fucoidans from algae F. vesiculosus (Zhurishkina et al. 2015, 2017) we show that such an activity of the native fucoidan comes from its major fraction charac terized by the highest fucose content and degree of sulfation. The sensitivity of Chang liver cells, which are widely used by researchers as models of normal liver cells, to the action of fucoidans from brown alga F. vesiculosus was significantly lower than of hepatoma Hep G2 cells. Selective toxicity toward the malignant tumor cells is the most impor tant characteristic for the potential medical applications for sulfated polysaccharides (Li et al., 2017; Vishchuk, Tarbeeva, Ermakova, & Zvyagintseva, 2012; Yamasaki-Miyamoto, Yamasaki, Tachibana, & Yamada, 2009). In the present work we compared the effect of two polydisperse fucoidan preparations isolated from the same algae species on Hep G2 and Chang liver cells. In both cell lines the difference in the inhibitory effect on cell proliferation between the partially hydrolyzed and native fucoidans was very small and not statistically significant (Fig. 2). Our results also indicate that selective toxicity for the tumor 7
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Fig. 6. Distribution of apoptotic and viable cells: Chang liver (a) and Hep G2 (b). Flow cytometry with double staining of Yo-Pro1/propidium iodide after cultivation with fucoidans 180 μg/ml F or FG for 24 and 48 h. Intensity of fluorescence Yo-Pro1 is shown on the abscissa axis; fluorescence intensity PI (rela tive units) is on ordinate: 1st quadrant (top left) cells negative for Yo-Pro1 and positive for PI - ne crosis; 2nd quadrant (top right) - cells positive for PI and Yo-Pro1 - late stage of apoptosis; 3rd quadrant (bottom left) - cells negative for PI and Yo-Pro1 viable cells; 4th quadrant (bottom right) - cells pos itive for Yo-Pro1 and negative for PI - early stage of apoptosis.
induce apoptosis to a greater degree, in non-malignant ones it primarily induces autophagy. Autophagy is considered to be a physiological process of cell selfrenewal, which can serve protective function under stressful in fluences, or, under certain conditions, can lead to cell death (Paglin et al., 2001, Wilson et al., 2011; Potapnev, 2014). It is logical to assume that the reason for the difference in the effect of fucopolysaccharides on tumor and non-malignant cells is that after fucoidan is internalized, different scenarios of intracellular events develop in different cell types. The toxicity of fucoidans to human hepatoma Hep G2 cells may be related to such features of this cell line such as low expression of cyto chrome P450, an enzyme responsible for intracellular metabolic pro �mez-Lecho �n, 2012). cesses (Tolosa, Donato, P�erez-Cataldo, Castell, & Go This could result in accumulation of toxic products within Hep G2 cells after the uptake of fucoidans, leading ultimately to their death. Obvi ously, different mechanisms are possible for other cells. Targeted acti vation of various complementary forms of cell death can serve as a new strategy to combat various diseases (Kopeina, Senichkin, & Zhivotovsky, 2017).
5. Conclusions The high molecular weight and polydispersity of fucoidans isolated from F. vesiculosus by acid-free extraction do not appear to adversely affect their antitumor activity. Fucoidans are internalized by cells, most likely via endocytosis, with Chang liver cells more rapidly absorbing the polysaccharide as compared to Hep G2 cells. Treatment with fucoidans induces different types of response depending on the type of cells: pri marily autophagy in Chang liver cells, whereas in Hep G2 cells apoptosis was more pronounced and lower autophagy was detected. The observed biological activity of polydisperse fucoidans obtained from brown algae by a simplified procedure, rather than the labor-intensive multi-stage purification of fractions, could help practical application of these unique polysaccharides. Acknowledgments The work has been performed as part of the Genome Research Center development program “Kurchatov Genome Center - PNPI” (agreement No. 075-15-2019-1663).
8
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Fig. 7. Detection of acidic vesicular organ elles (AVO) in Hep G2 and Chang liver cells with acridine orange staining. (a) -changes in red fluorescence after 8 h after treatment with fucoidans F and FG, flow cytometry; (b) - red:green fluorescence ratio in Hep G2 and Chang liver cells treated with fucoidans F and FG for 48 h (data shown with control subtracted); (c) - morphological changes in Chang liver and Hep G2 cells 43 h after treatment with fucoidans F and FG. Bar size is 200 μm. (For interpretation of the refer ences to colour in this figure legend, the reader is referred to the Web version of this article.)
9
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx
Author contribution
Glabe, C. G., Harty, P. K., & Rosen, S. D. (1983). Preparation and properties of fluorescent polysaccharides. Analytical Biochemistry, 130, 287–294. Holtkamp, A. D., Kelly, S., Ulber, R., & Lang, S. (2009). Fucoidans and fucoidanases focus on techniques for molecular structure elucidation and modification of marine polysaccharides. Applied Microbiology and Biotechnology, 82, 1–11. https://doi.org/ 10.1007/s00253-008-1790-x. Jin, W., Zhang, Q., Wang, J., & Zhang, W. (2013). A comparative study of the anticoagulant activities of eleven fucoidans. Carbohydrate Polymers, 91, 1–6. https:// doi.org/10.1016/j.carbpol.2012.07.067. Kima, M. H., & Joo, H. G. (2008). Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cell. Immunology Letters, 115, 138–143. https://doi.org/ 10.1016/j.imlet.2007.10.016. Kim, I. H., & Nam, T. J. (2018). Fucoidan downregulates insulin-like growth factor-I receptor levels in HT-29 human colon cancer cells. Oncology Reports, 39, 1516–1522. https://doi.org/10.3892/or.2018.6193. Kopeina, G. S., Senichkin, V. V., & Zhivotovsky, B. (2017). Caloric restriction - a promising anti-cancer approach: From molecular mechanisms to clinical trials. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 1867, 29–41. https://doi. org/10.1016/j.bbcan.2016.11.002. Kwak, J. Y. (2014). Fucoidan as a marine anticancer agent in preclinical development. Marine Drugs, 12, 851–870. https://doi.org/10.3390/md12020851. Li, J., Chen, K., Li, S., Feng, J., Liu, T., Wang, F., et al. (2016). Protective effect of fucoidan from Fucus vesiculosus on liver fibrosis via the TGF-β1/Smad pathwaymediated inhibition of extracellular matrix and autophagy. Drug Design, Development and Therapy, 10, 619–630. https://doi.org/10.2147/DDDT.S98740. Li, X. J., & Ye, Q. F. (2015). Fucoidan reduces inflammatory response in a rat model of hepatic ischemia-reperfusion injury. Canadian Journal of Physiology and Pharmacology, 93, 999–1005. https://doi.org/10.1139/cjpp-2015-0120. Li, J., Zhang, Q., Li, S., Dai, W., Feng, J., Wu, L., et al. (2017). The natural product fucoidan ameliorates hepatic ischemia-reperfusion injury in mice. Biomedicine & Pharmacotherapy, 94, 687–696. https://doi.org/10.1016/j.biopha.2017.07.109. Marinval, N., Saboural, P., Haddad, O., Maire, M., Bassand, K., Geinguenaud, F., et al. (2016). Identification of a pro-angiogenic potential and cellular uptake mechanism of a LMW highly sulfated fraction of fucoidan from Ascophyllum nodosum. Marine Drugs, 14, E185. https://doi.org/10.3390/md14100185. pii. Marudhupandi, T., & Kumar, T. T. A. (2013). Antibacterial effect of fucoidan from Sargassum wightii against the chosen human bacterial pathogens. International Current Pharmaceutical Journal, 2, 156–158. https://doi.org/10.3329/icpj.v2i10.16408. Matsubara, K., Xue, C., Zhao, X., Mori, M., Sugawara, T., & Hirata, T. (2005). Effects of middle molecular weight fucoidans on in vitro and ex vivo angiogenesis of endothelial cells. Int. J. Molec. Medicine, 15, 695–699. Morua, V. K., Kim, J., & Kim, E. K. (2012). Algal fucoidan: Structural and size-dependent bioactivities and their perspectives. Applied Microbiology and Biotechnology, 93, 71–82. https://doi.org/10.1007/s00253-011-3666-8. Oliveira, C., Ferreira, A. S., Novoa-Carballal, R., Nunes, C., Pashkuleva, I., Neves, N. M., et al. (2017). The key role of sulfation and branching on fucoidan antitumor activity. Macromolecular Bioscience. https://doi.org/10.1002/mabi.201600340. Orata, F. (2012). Derivatization reactions and reagents for gas chromatography analysis. In M. Mohd (Ed.), Advanced gas chromatography — progress in agricultural, biomedical and industrial applications (p. 94). InTech, ISBN 978-953-51-0298-4. Available from: http://www.intechopen.com/books/advanced-gas-chromatography-progress-in-ag ricultural-biomedical-and-industrial-applications/derivatization-reactions-and-reag ents-for-gas-chromatography-analysis. Paglin, S., Hollister, T., Delohery, T., Hackett, N., McMahill, M., Sphicas, E., et al. (2001). A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Research, 15, 439–444. Park, H. S., Kim, G. Y., Nam, T. J., Kim, N. D., & Choi, Y. H. (2011). Antiproliferative activity of fucoidan was associated with the induction of apoptosis and autophagy in AGS human gastric cancer cells. Journal of Food Science, 76, 77–83. https://doi.org/ 10.1111/j.1750-3841.2011.02099.x. Pavliga, S. N., Kompanets, G. G., & Tsygankov, V. Y. (2016). The experimental research (in vitro) of carrageenans and fucoidans to decrease activity of Hantavirus. Food Environ. Virol., 8, 120–124. https://doi.org/10.1007/s12560-016-9233-9. Peiser, L. S., & Gordon, S. (2001). The function of scavenger receptors expressed by macrophages and their role in the regulation of inflammation. Microbes and Infection, 3, 149–159. Potapnev, M. P. (2014). Autophagy, apoptosis, necrosis and immune recognition of self and nonself. Immunology, 2, 95–102 (In Russ.). Senthilkumar, K., Manivasagan, P., Venkatesan, J., & Kim, S. K. (2013). Brown seaweed fucoidan: Biological activity and apoptosis, growth signaling mechanism in cancer. International Journal of Biological Macromolecules, 60, 366–374. https://doi.org/ 10.1016/j.ijbiomac.2013.06.030. Stepanov, S. I., Konyshev, V. N., Kotlovanova, L. V., & Roganov, A. P. (1996). Karyotyping of individual cells with flow cytometry. Cytometry, 23, 279–283. https://doi.org/10.1002/(SICI)1097-0320(19960401)23:4<279::AID-CYTO3>3.0. CO;2-C. Tamura, Y., Adachi, H., Osuga, J., Ohashi, K., Yahagi, N., Sekiya, M., et al. (2003). FEEL1 and FEEL-2 are endocytic receptors for advanced glycation end products. Journal of Biological Chemistry, 278, 12613–12617. https://doi.org/10.1074/jbc.M210211200. Tolosa, L., Donato, M. T., P�erez-Cataldo, G., Castell, J. V., & G� omez-Lech� on, M. J. (2012). Upgrading cytochrome P450 activity in Hep G2 cells co-transfected with adenoviral vectors for drug hepatotoxicity assessment. Toxicology in Vitro, 26, 1272–1277. https://doi.org/10.1016/j.tiv.2011.11.008. Torres, M. A., Sousa, A. P., Silva Filho, E. A., Melo, D. F., Feitosa, J. P., de Paula, R. C., et al. (2007). Extraction and physicochemical characterization of Sargassum vulgare
Elena V. Zhurishkina: Investigation, Methodology, Formal analysis; Sergey I. Stepanov: Investigation; Olga N. Ayrapetyan: Visualization; Yury A. Skorik: Investigation, Methodology, Formal analysis; Elena N. Vlasova: Investigation; Igor V. Kruchina-Bogdanov: Investigation, Methodology, Formal analysis; Dmitry V. Lebedev: Investigation, Writing - Review & Editing; Anna A. Kulminskaya: Supervision, Re sources, Funding acquisition; Irina M. Lapina: Conceptualization, Investigation, Writing - Original Draft. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.bcdf.2019.100209. References Abudabbus, A., Badmus, J. A., Shalaweh, S., Bauer, R., & Hiss, D. (2017). Effects of fucoidan and chemotherapeutic agent combinations on malignant and nonmalignant breast cell lines. Current Pharmaceutical Biotechnology, 18, 748–757. https://doi.org/10.2174/1389201018666171115115112. Aisa, Y., Miyakawa, Y., Nakazato, T., Shibata, H., Saito, K., Ikeda, Y., et al. (2005). Fucoidan induces apoptosis of human HS-Sultan cells accompanied by activation of caspase-3 and down-regulation of ERK pathways. American Journal of Hematology, 78, 7–14. https://doi.org/10.1002/ajh.20182. Ale, M. T., Mikkelsen, J. D., & Meyer, A. S. (2011). Important determinants for fucoidan bioactivity: A critical review of structure-function relations and extraction methods for fucose-containing sulfated polysaccharides from brown seaweeds. Marine Drugs, 9, 2106–2130. https://doi.org/10.3390/md9102106. Anastyuk, S. D., Shevchenko, N. M., Ermakova, S. P., Vishchuk, O. S., Nazarenko, E. L., Dmitrenok, P. S., et al. (2012). Anticancer activity in vitro of a fucoidan from the brown alga Fucus evanescens and its low-molecular fragments, structurally characterized by tandem mass-spectrometry. Carbohydrate Polymers, 87, 186–194. Anastyuk, S. D., Shevchenko, N. M., Nazarenko, E. L., Imbs, T. I., Gorbach, V. I., Dmitrenok, P. S., et al. (2010). Structural analysis of a highly sulfated fucan from the brown alga Laminaria cichorioides by tandem MALDI and ESI mass spectrometry. Carbohydrate Research, 345, 2206–2212. https://doi.org/10.1016/j. carres.2010.07.043. Berwin, B., Delneste, Y., Lovingood, R. V., Post, S. R., & Pizzo, S. V. (2004). SREC-I, a type F scavenger receptor, is an endocytic receptor for calreticulin. Journal of Biological Chemistry, 279, 51250–51257. https://doi.org/10.1074/jbc.M406202200. Bilan, M. I., Grachev, A. A., Shashkov, A. S., Kelly, M., Sanderson, C. J., Nifantiev, N. E., et al. (2010). Further studies on the composition and structure of a fucoidan preparation from the brown alga Saccharina latissima. Carbohydrate Research, 345, 2038–2047. https://doi.org/10.1016/j.carres.2010.07.009. Cho, M. L., Lee, D. J., Kim, J. K., & You, S. (2014). Molecular characterization and immunomodulatory activity of sulfated fucans from Agarum cribrosum. Carbohydrate Polymers, 113, 507–514. https://doi.org/10.1016/j.carbpol.2014.07.055. Cho, M. L., Lee, B. Y., & You, S. (2011). Relationship between oversulfation and conformation of low and high molecular weight fucoidans and evaluation of their in vitro anticancer activity. Molecules, 16, 291–297. https://doi.org/10.3390/ molecules16010291. Cumashi, A., Ushakova, N. A., Preobrazhenskaya, M. E., D’Incecco, A., Piccoli, A., Totani, L., et al. (2007). A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic and antiadhesive activities of nine different fucoidans from brown seaweeds. Glycobiology, 17, 541–552. https://doi.org/10.1093/glycob/ cwm014. Deux, J. F., Meddahi-Pell� e, A., Le Blanche, A. F., Feldman, L. J., Colliec-Jouault, S., Br� ee, F., et al. (2002). Low molecular weight fucoidan prevents neointimal hyperplasia in rabbit iliac artery in-stent restenosis model. Arteriosclerosis, Thrombosis, and Vascular Biology, 22, 1604–1609. Dische, Z., Shettles, L. B., & Osnos, M. (1949). New specific color reactions of hexoses and spectrophotometric micro-metods for their determination. Archives of Biochemistry, 22, 169–184. Dodgson, K. S. (1961). Determination of inorganic sulphate in studies on the enzymatic and non-enzymatic hydrolysis of carbohydrate and other sulphate esters. Biochemical Journal, 78, 312–319. Durig, J., Bruhn, T., Zurborn, K. H., Gutensohn, K., Bruhn, H. D., & Beress, L. (1997). Anticoagulant fucoidan fraction from Fucus vesiculosus induce platelet activation in vitro. Thrombosis Research, 85, 479–491. Fitton, J. H., Stringer, D. N., & Karpiniec, S. S. (2015). Therapies from fucoidan: An update. Marine Drugs, 13, 5920–5946. https://doi.org/10.3390/md13095920.
10
E.V. Zhurishkina et al.
Bioactive Carbohydrates and Dietary Fibre xxx (xxxx) xxx cells. Journal of Agricultural and Food Chemistry, 57, 8677–8682. https://doi.org/ 10.1021/jf9010406. Yang, C., Chung, D., Shin, I., Lee, H., Kim, J., Lee, Y., et al. (2008). Effects of molecular weight and hydrolysis conditions on anticancer activity of fucoidans from sporophyll of Undaria pinnatifida. International Journal of Biological Macromolecules, 43, 433–437. https://doi.org/10.1016/j.ijbiomac.2008.08.006. Zayed, A., Muffler, K., Hahn, T., Rupp, S., Finkelmeier, D., Burger-Kentischer, A., et al. (2016). Physicochemical and biological characterization of fucoidan from Fucus vesiculosus purified by dye affinity chromatography. Marine Drugs, 14, E79. https:// doi.org/10.3390/md14040079. pii. Zhang, J., Riby, J. E., Conde, L., Grizzle, W. E., Cui, X., & Skibola, C. F. (2016). A Fucus vesiculosus extract inhibits estrogen receptor activation and induces cell death in female cancer cell lines. BMC Complementary and Alternative Medicine, 16, 151. https://doi.org/10.1186/s12906-016-1129-6. Zhang, Y., Yang, M., Portney, N. G., Cui, D., Budak, G., Ozbay, E., et al. (2008). Zeta potential: A surface electrical characteristic to probe the interaction of nanoparticles with normal and cancer human breast epithelial cells. Biomedical Microdevices, 10, 321–328. https://doi.org/10.1007/s10544-007-9139-2. Zhurishkina, E. V., Lapina, I. M., Ivanen, D. R., Stepanov, S. I., Shvetsova, S. V., Shavarda, A. L., et al. (2015). Effect of fucoidans isolated from seaweeds Laminaria digitata and Fucus vesiculosis on cell lines HeLa G-63, ECV 304 and PC 12. Tsitologiya, 57, 727–735. Zhurishkina, E. V., Stepanov, S. I., Shvetsova, S. V., Kulminskaya, A. A., & Lapina, I. M. (2017). A comparison of the effect of fucoidan from alga Fucus vesiculosus and its fractions obtained by anion-exchange chromatography on HeLa G-63, Hep G2, and Chang liver cells. Cell and Tissue Biology, 11, 242–249. Zhu, X. D., Zhuang, Y., Ben, J. J., Qian, L. L., Huang, H. P., Bai, H., et al. (2011). Caveolae-dependent endocytosis is required for class A macrophage scavenger receptor-mediated apoptosis in macrophages. Journal of Biological Chemistry, 286, 8231–8239. https://doi.org/10.1074/jbc.M110.145888.
alginate from Brazil. Carbohydrate Research, 342, 2067–2074. https://doi.org/ 10.1016/j.carres.2007.05.022. Trinchero, J., Ponce, N. M. A., Cordoba, O. L., Flores, M. L., Pampuro, S., Stortz, C. A., et al. (2009). Antiretroviral activity of fucoidans extracted from the brown seaweed Adenocystis utricularis. Phytotherapy Research, 23, 707–712. https://doi.org/10.1002/ ptr.2723. Usoltseva, R. V., Anastyuk, S. D., Ishina, I. A., Isakov, V. V., Zvyagintseva, T. N., Thinh, P. D., et al. (2018). Structural characteristics and anticancer activity in vitro of fucoidan from brown alga Padina boryana. Carbohydrate Polymers, 15, 260–268. https://doi.org/10.1016/j.carbpol.2017.12.071. Usoltseva, R. V., Shevchenko, N. M., Malyarenko, O. S., Ishina, I. A., Ivannikova, S. I., & Ermakova, S. P. (2018). Structure and anticancer activity of native and modified polysaccharides from brown alga Dictyota dichotoma. Carbohydrate Polymers, 180, 21–28. https://doi.org/10.1016/j.carbpol.2017.10.006. Usov, A. I., & Bilan, M. I. (2009). Fucoidans — sulfated polysaccharides of brown algae. Russian Chemical Reviews, 78, 785–799. https://doi.org/10.1070/ RC2009v078n08ABEH004063. Ustyuzhanina, N. E., Bilan, M. I., Ushakova, N. A., Usov, A. I., Kiselevskiy, M. V., & Nifantiev, N. E. (2014). Fucoidans: Pro- or antiangiogenic agents? Glycobiology, 24, 1265–1274. https://doi.org/10.1093/glycob/cwu063. Vishchuk, O. S., Tarbeeva, D. V., Ermakova, S. P., & Zvyagintseva, T. N. (2012). Structural characteristics and biological activity of fucoidans from the brown algae Alaria sp. and Saccharina japonica of different reproductive status. Chemistry and Biodiversity, 9, 817–828. https://doi.org/10.1002/cbdv.201100266. Wilson, E. N., Bristol, M. L., Di, X., Maltese, W. A., Koterba, K., Beckman, M. J., et al. (2011). A switch between cytoprotective and cytotoxic autophagy in the radio sensitization of breast tumor cells by chloroquine and vitamin D. Horm Cancer, 2, 272–285. https://doi.org/10.1007/s12672-011-0081-7. Yamasaki-Miyamoto, Y., Yamasaki, M., Tachibana, H., & Yamada, K. (2009). Fucoidan induces apoptosis through activation of caspase-8 on human breast cancer MCF-7
11