Gamma‑irradiation degraded sulfated polysaccharide from a new red algal strain Pyropia yezoensis Sookwawon 104 with in vitro antiproliferative activity
- Authors:
- Published online on: August 6, 2020 https://doi.org/10.3892/ol.2020.11952
- Article Number: 91
Abstract
Introduction
Pyropia yezoensis (Porphyra yezoensis; P yezoensis) is an edible marine red alga containing various biological macromolecules, such as sulfated polysaccharides, which have antioxidant, anti-inflammatory, antitumor and immunomodulatory activity (1–5). The anticancer bioactivities and applications of natural polysaccharides are of considerable interest to researchers and have been investigated using in vivo and in vitro models. For example, Porphyra haitanensis polysaccharide exhibited an antiproliferative effect on the GC7901 human gastric cancer cell line via the induction of cell apoptosis, and demonstrated an in vivo antitumor effect on SGC7901 tumor-bearing mice (6). An ultrasound-degraded polysaccharide from P. yezoensis also demonstrated significant inhibitory activity in SGC7901 cells (2). In addition, an agar-type sulfated polysaccharide derived from Gracilaria dominguensis inhibited Ehrlich ascites carcinoma in mice (7). Furthermore, a sulfated polysaccharide from Champia feldmannii (Diaz-Pifferer) inhibited sarcoma 180 tumors in mice (8). These previous studies indicate that various sulfated polysaccharides isolated from red algae have the potential to be used as natural antitumor agents due to their effectiveness in inhibiting the proliferation of tumor cells in vitro and in vivo.
There is also evidence to suggest that oligosaccharides and polysaccharides derived from seaweed are beneficial for human health and may have a wide range of applications (9). In a review by Cheong et al (10), the notable biological activity of oligosaccharides from red seaweed was suggested to support their development for use in functional foods and the pharmaceutical industry. Therefore, polysaccharides, oligosaccharides and their derivates are of great interest to researchers. There are a number of reports concerning the use of polysaccharides with low molecular weight (Mw) to treat cancer in clinical trials, with these polysaccharides including glucan-based oligosaccharides, heparan sulfate mimetics and inulin/oligofructose (11–13). Low-Mw polysaccharides are generally prepared by methods including acid hydrolysis, ultrasonic degradation, an ascorbic acid/H2O2 redox system, enzymatic degradation, microwave-assisted acid hydrolysis and gamma-irradiation (2,14–17). The use of different degradation methods may help to broaden the scope of the polysaccharides.
The present study aimed to expand the antitumor applications of sulfated polysaccharides isolated from algae, and also to elucidate the characteristics and bioactivity of some degraded derivatives obtained using gamma-irradiation. Specifically, a sulfated polysaccharide was extracted from the newly cultivated strain P. yesoensis Sookwawon 104 by dilute hydrochloric acid extraction, and low-Mw polysaccharides were prepared from it by gamma-irradiation (20 and 100 kGy). The in vitro antiproliferative activity of the P. yezoensis sulfated polysaccharide (PYSP) and its derivatives on three tumor cell lines, namely the HeLa human cervical cancer cell line, MDA-MB-231 human breast carcinoma cell line and Hep3B human hepatic carcinoma cell line, and their potential mechanisms were also investigated.
Materials and methods
Materials and chemicals
The algal specimen P. yezoensis Sookwawon 104 was collected by the National Institute of Fisheries Science (South Korea). The specimen was identified by EJP and deposited at the Seaweed Research Center (South Korea) with the voucher specimen number Sookwawon 104. Thiazolyl blue tetrazolium bromide (MTT) were purchased from BBI Life Sciences Corporation. Mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose were obtained from the Sinopharm Chemical Reagent Co. Ltd for the monosaccharide composition analysis. TransScript All-in-One First-Strand cDNA Synthesis SuperMix and TransStart® Top Green qPCR SuperMix were purchased from Beijing TransGen Biotech Co., Ltd. Dulbecco's modified Eagle's medium (DMEM), Leibovitz's L-15 medium and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc.). Penicillin-streptomycin solution (100X) was obtained from Biosharp Life Sciences. All other chemical reagents used were of analytical grade.
Extraction of P. yezoensis polysaccharide
Dried and powdered P. yezoensis Sookwawon 104 (50 g) was passed through a 40-mesh sieve. Fat and pigment were then removed by refluxing with 250 ml 95% ethanol at 60°C for 6 h. The residue (45 g) was extracted twice with 1 mM HCl (1.3 l) at 80°C for 2 h. After filtration, the supernatant was concentrated to 0.65 l using a rotary evaporator at 50°C. Then, 95% ethanol (2.6 l) was added to the concentrate which was maintained at 4°C overnight. Following centrifugation at 2,000 × g for 10 min at room temperature, the precipitate, named PYSP, was collected and dried in a vacuum drying oven at 70°C (18).
Preparation of degradation derivatives by gamma-irradiation
PYSP (5% in water, w/v; pH 7.0) was degraded by gamma-irradiation at doses of 20 and 100 kGy, respectively, as previously described (15). The degradation derivatives were collected, lyophilized in a vacuum and freeze-dried. The derivatives obtained using 20 and 100 Gy were named as PYSP-20 and PYSP-100, respectively.
Component analysis
PYSP and its degradation derivatives were subjected to component analysis. The total sugar content was detected using the phenol-sulfuric acid method (19). The protein content was analyzed using the Bradford method (20). The sulfate group content was determined using a turbidimetric method (21).
Monosaccharide composition analysis
The monosaccharide compositions of PYSP, PYSP-20 and PYSP-100 were analyzed by high-performance liquid chromatography using a 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method (22). Briefly, the samples were hydrolyzed with 2 M trifluoroacetic acid at 100°C for 4 h. Excess acid was removed by adding ethanol at 60°C, and then NaOH (0.3 M, 300 µl) and PMP (0.5 M, 300 µl) were added to the reaction mixture, which was subsequently incubated at 70°C for another 1 h. Following neutralization by the addition of 0.3 M HCl, chloroform (1 ml) was added to the reaction mixture. The aqueous phase of three samples (20 µl) was analyzed by Waters 1525 HPLC system (Waters Corporation; http://www.waters.com/nextgen/us/en.html) on a Hypersil ODS-2 column (5 µm, 4.6×250 mm; Thermo Fisher Scientific Inc.) at a flow rate of 0.8 ml/min. The mobile phases were 0.05 M phosphate buffer solution (pH 6.8) and acetonitrile (83:17, v/v), and the detection wavelength was 254 nm at 25°C. Different monosaccharide standards (mannose, fucose, xylose, galactose, glucose, arabinose, rhamnose, galacturonic acid and glucuronic acid) were used to analyze the monosaccharide composition of PYSP and its derivatives.
Mw analysis
The Mw distributions of PYSP, PYSP-20 and PYSP-100 were measured by high-performance gel permeation chromatography. Dextran standards with different molecular weights (2,000, 150, 41.1, 21.4, 7.1 and 4.6 kDa, and 180 Da) were used to calibrate the column and establish a standard curve using linear regression (22). Each sample, dissolved in 0.1 M Na2SO4 solution, was analyzed using a TSK-GEL G5000 PWXL column (7.8×300 mm; Tosoh Corporation) and Waters 2424 Refractive Index Detector (Waters Corporation), which was eluted with 0.1 M Na2SO4 solution.
Fourier transform-infrared (FT-IR) analysis
Each sample (4 mg) was mixed with KBr powder (0.4 g), pressed into pellets and analyzed using an Infrared Spectrometer TENSOR 27 (Bruker Corporation) at the frequency range from 400 to 4,000 cm−1 (23).
Cell culture
HeLa, Hep3B and MDA-MB-231 cells were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology. The HeLa and Hep3B cells were maintained in DMEM, and the MDA-MB-231 cells were maintained in L-15 medium. All media were supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). The cell cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2.
MTT assay
Hep3B, HeLa and MDA-MB-231 cells were each seeded in 96-well plates at a density of 3×103 cells/well in 200 µl medium. The cells were treated with PYSP, PYSP-20 or PYSP-100 at concentrations of 200 or 500 µg/ml at 37°C for 48 h. Then, 20 µl MTT (5 mg/ml) was added to each well, and the cells were incubated for another 4 h. Finally, the cell viability was detected as previously described (24). The inhibition rate was calculated from the optical density (OD) at 490 nm using the following formula: Inhibition rate (%) = (1-ODtreatment/ODuntreated) ×100.
Crystal violet assay
Hep3B, HeLa and MDA-MB-231 cells were seeded in 24-well plates at a density of 5×104 cells/well in 1 ml medium overnight and then treated with PYSP-20 or PYSP-100 at 200 or 500 µg/ml at 37°C for 48 h. Untreated cells served as the control. The cells were then fixed with 4% paraformaldehyde at 25°C for 30 min, stained with 0.1% crystal violet for 30 min at room temperature, and then washed with distilled water. Finally, 10% acetic acid was added to each well and the absorbance at 595 nm was measured using a Cytation 3 microplate reader (BioTek Instruments, Inc.). The relative proliferation rate was calculated using the following formula: Relative proliferation rate = ODtreatment/ODuntreated.
Reverse transcription-quantitative PCR (RT-qPCR)
Hep3B, HeLa and MDA-MB-231 cells were seeded in 24-well plates at a density of 5×104 cells/well in 1 ml medium overnight and then treated with PYSP-20 or PYSP-100 at 200 µg/ml at 37°C for 48 h. The cells were then collected and total RNA was extracted from them using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA (2 µg/µl) was used for cDNA synthesis using TransScript All-in-One First-Strand cDNA Synthesis SuperMix. The cDNA samples were used as the template for the qPCR reaction using gene-specific primers. The final reaction volume of 10 µl contained 5 µl TransStart® Top Green qPCR SuperMix, 0.5 µl forward and reverse primers and 1 µl cDNA template. qPCR was conducted using a LightCycler 480 Instrument II (Roche Applied Science). The PCR thermocycling conditions were as follows: 1 cycle at 95°C for 30 sec followed by 40 cycles at 95°C for 30 sec, 58°C for 30 sec and 72°C for 20 sec. The relative amounts of mRNA were calculated using the 2−ΔΔCq method (25). The primer sequences were as follows: P53, forward: 5′-CCCCTCCTGGCCCCTGTCATCTTC-3′ and reverse: 5′-GCAGCGCCTCACAACCTCCGTCAT-3′; P21, forward: 5′-GCGGAACAAGGAGTCAGACA-3′ and reverse: 5′-GAACCAGGACACATGGGGAG-3′; Cyclin B1, forward: 5′-CTGCTGGGTGTAGGTCCTTG-3′ and reverse: 5′-TGCCATGTTGATCTTCGCCT-3′; Cdk1, forward: 5′-TTGAAACTGCTCGCACTTGG-3′ and reverse: 5′-TCCCGGCTTATTATTCCGCG-3′; GAPDH, forward: 5′-GCAGGGGGGAGCCAAAAGGGT-3′ and reverse: 5′-TGGGTGGCAGTGATGGCATGG-3′. GAPDH served as an internal reference.
Statistical analysis
Data are expressed as means ± standard deviation. GraphPad Prism 5.0 (GraphPad Software, Inc.) and Origin 8.5 (OriginLab Corporation) were used to prepare graphs and for analysis of the data using one-way and two-way ANOVA analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
Characterization of the polysaccharides
Extraction of 50 g dried P. yezoensis Sookwawon 104 using diluted hydrochloric acid extraction and ethanol precipitation yielded 5 g PYSP. The carbohydrate content, Mw and chemical composition of PYSP and its degradation products are shown in Table I. PYSP, PYSP-20 and PYSP-100 were composed of galactose, fucose and glucose in a molar ratio of 1.6:17.9:1.0, 2.0:17.6:1.0 and 1.7:13.5:1.0, respectively. The chemical compositions of PYSP, PYSP-20, and PYSP-100 were not markedly different; however, the Mw distribution was clearly reduced when the dose of gamma-irradiation was increased (Fig. 1). The FT-IR spectrum (Fig. 2) of each sample revealed a major broad stretching peak at ~3,430 cm−1 for the hydroxyl group, and a weak band at ~2,930 cm−1 for the C-H stretching vibration. The peak at 931 cm−1 indicated the existence of an ether bond (-C-O-C-), suggesting all samples contained 3,6-anhydro-α-L-galactose (14). The signals presented at ~1,250 and 890 cm−1 were respectively caused by the stretching vibrations of S=O and C-O-S groups (26,27), indicating that all samples contained sulfate groups. The peaks near 1,635 and 1,400 cm−1 observed for PYSP, PYSP-20 and PYSP-100 were the stretching vibrations of carboxyl and carbonyl groups (28). Together, the composition analysis and FT-IR spectra confirmed that PYSP and its derivatives did not exhibit any marked differences, with the exception of Mw distribution.
Antiproliferative activity
The in vitro antiproliferative effects of PYSP, PYSP-20 and PYSP-100 on Hep3B, HeLa and MDA-MB-231 cells were analyzed using MTT and crystal violet assays. As shown in Fig. 3, PYSP-20 and PYSP-100 exhibited marked antiproliferative effects on MDA-MB-231 cells, whereas PYSP had weaker antiproliferative activity. PYSP-20 and PYSP-100 displayed inhibition rates of 40–50% in MDA-MB-231 cells. Notably, the inhibition rate for PYSP-20 at a concentration of 500 µg/ml reached 50.6% (Fig. 3C). According to the results of the crystal violet assay, the relative proliferation rate of the cells decreased by almost half following treatment with PYSP-20 or PYSP-100 (Fig. 3A and B). The effects of PYSP, PYSP-20 and PYSP-100 on HeLa cells are shown in Fig. 4. PYSP-20 and PYSP-100 at concentrations of 200 and 500 µg/ml exhibited notable antiproliferative effects against HeLa cells, with a maximum inhibition rate of ~50% (Fig. 4C). However, PYSP exhibited an antiproliferative effect only at 200 µg/ml. Consistent with the results of the MTT assay, the relative proliferation rates of HeLa cells treated with PYSP-20 or PYSP-100 determined using the crystal violet assay (Fig. 4A and B) exhibited a similar inhibitory trend as those of the MTT assay in Fig. 4C. In Hep3B cells, PYSP, PYSP-20 and PYSP-100 exhibited antiproliferative effects at concentrations of 200 and 500 µg/ml. and their inhibition rate reached ~50%. The inhibition rate of PYSP-100 was significantly higher than that of PYSP or PYSP-20 at the concentration of 500 µg/ml (Fig. 5C). Also, the relative proliferation rates of PYSP-20 and PYSP-100 were been reduced by almost half compared with those in the control group (Fig. 5A and B).
Due to the greater in vitro antiproliferative activity of PYSP-20 and PYSP-100 when compared with PYSP, their potential antiproliferative mechanism was further explored through measuring the expression of genes regulating the cell cycle, namely Cyclin B1, Cdk1, P53 and P21 (Fig. 6). The treatment of Hep3B cells with PYSP-20 or PYSP-100 appeared to reduce the mRNA levels of cyclin B1 and Cdk1 compared with those in the control group, while the expression levels of P53 and P21 significantly increased. In HeLa cells, Cdk1 was significantly decreased after PYSP-20 treatment. Furthermore, P53 and P21 in HeLa cells appeared to be upregulated following PYSP-20 or PYSP-100 treatment, with P21 exhibiting a significant increase in response to treatment with PYSP-20. In the MDA-MB-231 cells, Cyclin B1 and Cdk1 appeared to be slightly downregulated after PYSP-20 or PYSP-100 treatment. Moreover, PYSP-20 exposure significantly increased the mRNA levels of P21, while PYSP-100 significantly increased the mRNA levels of P53 expression in MDA-MB-231 cells.
Discussion
Red algae is an abundant marine resource that comprises various species, including Gracilaria gracili and P. yezoensis. P. yezoensis contains multiple bioactive macromolecules, including polysaccharides, proteins and polyunsaturated fatty acids (29). In the present study, the polysaccharide PYSP was extracted from a new red alga strain P. yezoensis Sookwawon 104 and the degradation derivatives PYSP-20 and PYSP-100 were prepared by gamma-irradiation. The antiproliferative activities of these polysaccharides were investigated in vitro against Hep3B, HeLa and MDA-MB-231 cells. In the Mw analysis, the elution curve indicated that PYSP mainly comprises high-Mw polysaccharide. Since the available evidence shows that the reduction of Mw may improve the bioavailability of polysaccharides (30), a degradation method using gamma-irradiation was used to prepare low-Mw polysaccharides from PYSP using the irradiation doses 20 and 100 kGy, according to our previous studies (15,31,32). In the present study, PYSP-20 and PYSP-100 exhibited a significant reduction in Mw compared with PYSP, but the monosaccharide composition and sulfate group content did not change markedly, consistent with our previous study (32). In addition, in the FT-IR spectra, there was also no clear difference in the characteristic absorption bands among these polysaccharides, the only exception being that PYSP-20 and PYSP-100 exhibited a slight difference in the stretching vibrations of carboxyl and carbonyl groups, possibly due to the breaking of those chemical bonds by the gamma-irradiation (32).
Polysaccharides have been shown to exhibit lower inhibition rates on tumor cells when used at low concentrations. For example, a polysaccharide from Cordyceps gunnii mycelia demonstrated only weak inhibitory activity against tumor cells when used at a low concentration, such as 50 or 100 µg/ml (33). Zhang et al (34) also demonstrated that low concentrations of polysaccharide, ranging from 25 to 100 µg/ml, had only a weak inhibitory effect on tumor cell viability. Therefore, with consideration of these previous studies, the sample concentrations used in the present study were selected as 200 and 500 µg/ml. PYSP and its derivatives exhibited different Mw distribution ranges, and PYSP with a higher Mw exhibit weaker antitumor capability compared with its low-Mw derivatives (PYSP-20 and PYSP-100). Therefore, we speculate that Mw is a key factor affecting the distinct antiproliferative activity of PYSP and its derivatives. This is consistent with a previous study (35), in which the Mw of sulfated Artemisia sphaerocephala polysaccharides was highly associated with their antitumor activity, and low-Mw polysaccharide demonstrated a greater inhibitory ability against A549, HepG2 and HeLa cells in vitro. However, for the HeLa cells in the present study, the high-Mw polysaccharide PYSP exhibited antiproliferative activity only at the lower concentration, indicating that the antiproliferative activity of high-Mw polysaccharide might also be affected by the dosage. Choromanska et al (36) demonstrated that high-Mw β-glucan had stronger growth inhibitory activity against A549 and H69AR cells at a low concentration (200 µg/ml) compared with other higher concentrations. Although these results indicate that the low-Mw polysaccharides in the present study have a promising in vitro antiproliferative effect on cancer cell lines, validation of their antitumor effect and evaluation of toxicity are required in further studies. Also, previous studies have demonstrated that the upregulation of P53 and P21, together with the downregulation of Cyclin B1 and Cdk1, serve important roles in blocking the cell cycle, which is the potential antitumor mechanism of a variety of clinical anticancer medicines (37–40). The present data demonstrate that PYSP-20 and PYSP-100 are able to regulate the expression of P53, P21, Cyclin B1 and Cdk1 and so may induce cell cycle arrest. As was shown above the studies have shown that polysaccharides have antitumor activity. Meanwhile, it was reported that polysaccharide was also a kind chemotherapeutic assistant drug. For example, one study reported that when low-Mw polysaccharide was used as a carrier for 5-fluorouracil (5-FU), the antitumor activity of 5-FU against transplanted S180 tumors in mice was enhanced (41). Thus, the synergistic effects of polysaccharide with conventional chemotherapeutic drugs, as a combination therapy against cancer, are of considerable interest.
In summary, a sulfated polysaccharide from P. yezoensis Sookwawon 104 and its low-Mw derivatives obtained by gamma irradiation were investigated in the present study. Gamma irradiation did not cause significant changes in the sulfate group content and monosaccharide composition, although changes in the Mw distribution were observed. The in vitro antiproliferation assays indicated that Mw had a significant influence on the antitumor activity of the sulfated polysaccharides. The low-Mw polysaccharides exhibited stronger antiproliferative effects than PYSP, and the potential mechanisms may involve cell cycle arrest. Prior to the further research and development of PYSP and its degradation derivatives, strong supporting data from in vivo antitumor assays are urgently required. However, the current findings promote the exploitation and utilization of polysaccharide from P. yezoensis Sookwawon 104 as a promising candidate for cancer adjuvant therapy.
Acknowledgements
The authors would like to thank Dr Alan K Chang (Wenzhou University; Wenzhou, China) for helpful discussion and for revising the language of the manuscript.
Funding
This study was financially supported by the National Natural Science Foundation of China (grant nos. 41876197 and 81872952), the National Key Research and Development Project (grant no. 2018YFD0901503), the Natural Science Foundation of Zhejiang Province (grant nos. LY18C020006 and LGN18C020004), the Scientific Foundation of Education Department of Zhejiang Province (grant no. Y201737374), the National Research Foundation of Korea (grant no. NRF-2018R1D1A1B07049359) and a Golden Seed Project Grant funded by Ministry of Oceans and Fisheries (grant no. 213008-05-4-SB910).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
DH, LY, YC, XM, SW, JZ and EJP performed the experiments. JL designed the experiments used to evaluate the physicochemical properties. DH, HT, MW and JIC designed the study; DH and HT analyzed the data. DH wrote the original draft of the manuscript, and DH, LY and HT revised it. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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