Anti-tumor potential of astragalus polysaccharides on breast cancer cell line mediated by macrophage activation

Materials Science & Engineering C 98 (2019) 685–695

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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec

Anti-tumor potential of astragalus polysaccharides on breast cancer cell line mediated by macrophage activation

T

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Wenfang Lia, Kedong Songa, , Shuping Wanga, Chenghong Zhangb, Meiling Zhuanga, Yiwei Wangc, Tianqing Liua a

State Key Laboratory of Fine Chemicals, Dalian R&D Center for Stem Cell and Tissue Engineering, Dalian University of Technology, Dalian 116024, China Department of Basic Medicine, Dalian Medical University, Dalian 116011, China c Burns Research Group, ANZAC Research Institute, University of Sydney, Concord, NSW 2139, Australia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Astragalus polysaccharides Macrophage activation Breast cancer Apoptosis TNF-α

Adverse effects are pressing challenges produced by chemotherapy and radiotherapy for the treatment of breast cancer. Nontoxic herbal medicines are therefore considered as a favorable alternative. Astragalus membranaceus has attracted growing interest in the field of biomedicine thanks to its various biological activities, among which the anticancer activity is considered to be closely associated with its active component-astragalus polysaccharide (APS). Currently, direct anti-tumor activity and the activation of immune response of the host have been widely acknowledged as the mechanism by which APS exerts its anti-cancer activity. In this study, we aimed to investigate whether APS could inhibit the growth of MCF-7 cells and activate macrophages to further kill cancer cells. The results indicated that the obtained APS was a pyran-type polysaccharide, containing 89.75% total carbohydrate and a minor amount of uronic acid (9.3%). Although APS did not significantly inhibit the growth of MCF-7 cells growth, encouragingly, APS-activated RAW264.7 macrophages present anti-cancer activity as evidenced by (a) cell proliferation inhibition (with an inhibitory rate of 41%), (b) G1-phase cell cycle arrest, as well as (c) the regulation of apoptosis-related genes (Bax/Bcl-2, 13.26-fold increase than untreated cells). In addition, APS could upregulate the level of nitric oxide (NO) and tumor necrosis factor-α (TNF-α), which acted as inducers of tumor cell apoptosis. Collectively, our findings suggest that APS can activate macrophages to release NO and TNF-α, which directly blocks cancer cell growth. The anti-breast cancer effect of APS and the in vivo mechanism will be further elucidated with a review to provide a therapeutic strategy for breast cancer.

1. Introduction Breast cancer is the most common cancer and the second leading cause of cancer-related death among women following lung cancer [1]. Although considerable progress against cancer has been achieved, multi-faceted challenges still remain including the severe adverse effects caused by chemotherapy and radiotherapy and the drug resistance developed over time [2]. Therefore, more attentions have been paid on new anti-cancer strategies, such as herbal medicines or natural products which are considered nontoxic, multi-targeted and do not lead to the development of drug resistance. Currently, polysaccharides have attracted considerable attention in biomedical field due to their various pharmacological activities [3,4]. Among the polysaccharides identified, astragalus polysaccharide (APS), the primary active component extracted from Chinese medicinal herb

Astragalus membranaceus, has been shown to exhibit broad-range inhibitory effect on various types of solid tumors [5]. Previous report demonstrated APS exhibited favorable anti-melanoma potency by down-regulating CD40 expression [6]. Besides, APS exerted a synergistic anti-tumor effect in combination with other chemotherapy agents with increased sensitivity and decreased side-effects. Tian et al. reported that APS combined with adriamycin yielded an enhanced antitumor activity in H22 tumor-bearing mice by regulating immune cytokines [7]. Despite these progresses, the anti-tumorigenic activity and mechanism of APS against breast carcinogenesis has rarely been reported. The anti-tumor activity of polysaccharides is generally mediated via two major pathways: direct inhibitory/eradicative effect against malignant cells, and activated innate and/or adaptive immune system, while the latter contributes to the activation of various immune cells

⁎ Corresponding author at: State Key Laboratory of Fine Chemicals, Dalian R&D Center for Stem Cell and Tissue Engineering, Dalian University of Technology, 116024, China. E-mail address: [email protected] (K. Song).

https://doi.org/10.1016/j.msec.2019.01.025 Received 8 December 2017; Received in revised form 30 October 2018; Accepted 7 January 2019 Available online 08 January 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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purchased from Abcam Inc. (Cambridge, MA, USA). Human TNF-α ELISA kit was obtained from Boster Biological Technology Co. (Wuhan, China). Cell cycle analysis, Annexin V-FITC apoptosis and nitric oxide (NO) detection kits were purchased from Beyotime Institute of Biotechnology (Haimen, China). All the other reagents were of analytical grade.

(e.g. macrophage, T and B lymphocytes, NK cells etc.) and augmented production of a range of important immunoregulatory cytokines [8–10]. Among the available immune cells, macrophages are commonly chosen as the research model for the anti-tumor activity of polysaccharides in vitro since tumor necrosis factor-α (TNF-α) and nitric oxide (NO) generated by activated macrophages are two important cytotoxic mediators that are targeted towards destroying cancer cells [11,12]. Currently, many polysaccharides have been shown to be macrophage activators and further mediate cytotoxicity against tumors [13]. Fucogalactan at 50 μg/mL markedly augmented the release of TNF-α and NO in macrophages of mice in vitro and contributed to antitumor activity in tumor-bearing hosts as well as immunomodulating effects [14]. β-glucan-related polysaccharides of the higher fungus activated macrophages and released NO which mediated the destruction of leukemia L1210 cells [15]. At present, numerous evidence have demonstrated that APS can activate mouse peritoneal macrophages and RAW264.7 macrophages accompanied by the up-regulation of TNF-α, NO production and the transcription of inducible NO synthase (iNOS) [16,17]. However, until now, no sufficient information is available for the determination of whether macrophages activated by APS are capable to mediate the destruction of MCF-7 cells in vitro and the involved mechanism of anti-tumor action. In view of this, the ability of APS to stimulate macrophages and further attenuated activity towards MCF-7 cells growth were therefore investigated. In this study, we aimed to investigate the effect of APS and APSmediated macrophages on the growth of MCF-7 cells, and to further elucidate the mechanism of growth-inhibition to cancer cells. We found that APS could be an ideal activator of RAW264.7 murine macrophages and the secreted TNF-α and NO acted as a killing mechanism to induce the apoptosis of MCF-7 cells (Fig. 1).

2.2. Physical properties of APS 2.2.1. Appearance and chemical composition of APS A scanning electron microscope (SEM, SU1510, Hitachi High Technologies, Japan) coupled to an energy dispersive X-Ray spectrometer (EDS) was used to investigate the morphology and elemental compositions of APS. The samples were mounted onto a copper grid and sputtered with gold, followed by observation with multiple magnifications. Carbohydrate content was determined by phenol‑sulfuric acid method using glucose as the standard [18]. The content of total uronic acid was quantified according by carbazole‑sulfuric acid method at 530 nm using glucuronic acid as standard [19]. 2.2.2. Ultraviolet and infrared spectrum analysis APS was dissolved in deionized water to final concentrations of 0.5–2 mg/mL. The samples were scanned with a UV–Vis spectrophotometer (Lambda 750 s, PerkinElmer, USA) in the range of 190 to 800 nm. The structural characteristics of APS were determined by Fourier transformed IR spectrophotometer (EQUINOX55, Bruker, Germany). 2.3. Macrophage activation 2.3.1. Nitrite determination The generation of nitric oxide (NO) was determined by Griess reaction. Briefly, RAW 264.7 cells (1 × 105 cells/mL) were seeded in 24well plates and incubated for 24 h. Afterwards, cells were subjected to different concentrations of APS. Here, LPS (1 μg/mL) and the complete medium was used as positive and negative control groups, respectively. Besides, cells were separately treated with TLR4 antibody (20 μg/mL) for 2 h following the addition of APS (1000 μg/mL) and LPS. After 24 h stimulation, 100 μL culture supernatant was mixed with an equal volume of Griess reagent at room temperature for 10 min. The absorbance of the mixture was measured by a microplate reader at 540 nm. All of the experiments were performed in triplicates.

2. Materials and methods 2.1. Materials and reagents APS was purchased from Pharmagenesis Inc. (Beijing, China). 5fluorouracil (5-FU) and lipopolysaccharides (LPS) were purchased from Sigma Aldrich Co. (St Louis, MO, USA). MCF-7 and RAW264.7 murine macrophage-like cells were purchased from Zhong Qiao Xin Zhou Biotechnology Co. Ltd. (Shanghai, China). Dulbecco's modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Co. (Carlsbad, CA, USA). Calcein-AM, propidium iodide (PI) and Hoechst 33342 were obtained from Calbiochem (San Diego, CA). Cell counting kit-8 (CCK-8) was obtained from Selleck.cn (Shanghai, China). Anti-Bcl-2 antibody, anti-Bax antibody, and anti-TLR4 antibody were

2.3.2. TNF-α expression by ELISA ELISA was performed to evaluate TNF-α expression in the presence

Fig. 1. Schematic diagram of the apoptosis of MCF-7 cells induced by APS-activated macrophages. 686

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of APS. In brief, RAW 264.7 cells (1 × 105 cells/mL) were seeded into 24-well plates and incubated overnight, followed by the addition of serial dilutions of APS and LPS. Similarly, cells were subjected to TLR4 antibody as described above. After stimulation for 24 h, the level of TNF-α in cell-free supernatant (i.e. CM) was assessed by ELISA according to the manufacturer's instructions.

Japan). 2.4.4. Colony formation assay Colony formation assay was conducted to evaluate the inhibitory effect of CM on MCF-7 cells. MCF-7 cells in logarithmic phase were seeded at a density of 800 cells/well in 6-well culture plates. After cell adhesion, CM with different concentrations was added and the cells were incubated for 2 weeks. Afterwards, cells were stained with Giemsa's stain for the colony count.

2.3.3. FITC-labeling of APS and interaction with macrophages APS was labeled with fluorescein isothiocyanate (FITC) as previously described [20]. APS was dissolved in 50 mL formamide and 50 mL methylsulfoxide containing 0.05 g dibutyline-dilaurate. FITC (0.25 g) and NaHCO3 (0.1 g) were added and the mixture was heattreated at 100 °C for 1 h. After several precipitations in ethanol, the FITC-labeled APS was dissolved in PBS and dialyzed against distilled water and lyophilized. FITC-labeled polysaccharides were added to the macrophage cells and incubated for 20 min, 1 h, and 6 h, respectively. Similarly, unlabeled APS was treated with RAW264.7 cells for 1 h followed by exposure to FITC-labeled APS for 5 h. After being washed in PBS for three times, macrophages were imaged with a fluorescence microscopy at 488-nm.

2.4.5. Cell cycle analysis MCF-7 cells (1 × 106 cells/mL) were seeded in 6-well culture plates and treated with various concentrations of APS and CM for 48 h. Cells were collected according to the routine digestion by trypsin. Then they were washed with cold PBS and fixation with 70% ethanol at 4 °C for 12 h. Subsequently, cells were treated with 100 μg/mL RNase solution and PI staining at 37 °C for 30 min. The samples were analyzed by a flow cytometer (FACS Canto, BD Biosciences, USA). The relative proportions of cells in the G0/G1, S and G2/M phases were analyzed with Modfit software (Verity Software House Inc., Topsham, ME, USA). 2.4.6. Detection of cell apoptosis MCF-7 cells (3 × 103 cells/well) were seeded in 96-well plates and were treated by various concentrations of APS and CM for 48 h. DAPI (5 μg/mL in PBS) staining was conducted to evaluate the nuclear morphology. Cell apoptosis was evaluated by dual (AO/EB) fluorescent staining [21]. Apoptotic rate of MCF-7 cells was also assessed using an Annexin V–FITC/PI (Sigma, St. Louis, MO) staining assay after exposure to CM for 48 h as previously described [22].

2.4. Antineoplastic activities of APS 2.4.1. Cell culture and conditioned medium collection MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS at 37 °C and 5% CO2 in a humidified incubator. APS was dissolved in DMEM at different concentrations (50, 100, 200, 500, and 1000 μg/mL, respectively) and filter-sterilized with a membrane filter (0.22-μm pore size). RAW264.7 cells at 1 × 105 cells/mL were seeded in 24-well culture plates, followed by the addition of different concentrations of APS. Besides, untreated RAW264.7 cells and cells treated by LPS (1 μg/mL) were used as the negative control and positive control, respectively. At 24 h, the supernatants from the aforementioned groups were collected and centrifuged at 1000 rpm for 10 min. The supernatants with various concentrations of APS (hereafter simply referred to as conditioned medium) were collected and stored at −20 °C for later use.

2.4.7. SEM analysis A total of 20 μL MCF-7 cells (1.5 × 105 cells/mL) were seeded on cover slips and treated with various concentrations of APS and CM for 48 h. Subsequently, cells on the cover slips were fixed with 2.5% glutaraldehyde at 4 °C for 3 h, post-dehydrated with progressively increasing concentrations of ethanol (50%, 70%, 90% and 100%) and dried at room temperature. The specimens were coated with gold and examined with SEM with accelerating voltage of 30.0 kV and exposure time of 6 μs.

2.4.2. Inhibitory effect of APS and conditioned medium CCK-8 assay was conducted to investigate the effect of APS and conditioned medium (CM) on the viability of MCF-7 cells. Briefly, 3 × 103 cells were seeded in each well of 96-well culture plates containing 100 μL complete medium. Following overnight incubation, serial concentrations of APS (50, 100, 200, 500 and 1000 μg/mL) and CM were added and maintained in a humidified incubator for 24, 48 and 72 h, respectively. MCF-7 cells incubated with complete medium and 5FU (50 μg/mL) were used as negative and positive control, respectively. Subsequently, APS-containing medium and CM in each well were replaced with 100 μL fresh medium and 10 μL CCK-8 solution, and the cells were then incubated at 37 °C for 3 h. Afterwards, the absorbance of each well was detected at 450 nm by a microplate reader (Varioskan Flash, Thermo Fisher Scientific, San Jose, CA, USA). All experiments were performed in triplicates. The absorbance of the negative control group was considered as 100% and the inhibitory rate was calculated according to the equation: ζ = (1 − A1/A0) × 100%, where A1 and A0 were the absorbance of the sample and negative control group, respectively.

2.4.8. Bcl-2/Bax expression Immunofluorescence and flow cytometry assays were conducted to evaluate CM effects on Bcl-2/Bax expression. After 48 h of incubation, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Subsequently, cells were blocked with 10% normal goat serum for 1 h at room temperature and exposed to human anti-Bcl-2 antibody (rabbit monoclonal antibody, 1:100), anti-Bax antibody (rabbit monoclonal antibody, 1:100) overnight at 4 °C. Following the removal of primary antibodies, cells were then incubated with Dylight 549 conjugated goat anti-rabbit antibody (Beyotime, 1:50) for 1 h and the nuclei were counterstained with DAPI. Furthermore, Bcl2/Bax protein expression was evaluated by flow cytometry as previously described [23]. 2.5. Cell migration and invasion assay The effects of APS and CM on cell migration and invasion were evaluated in a 24-Transwell cell culture chamber (8 μm, Corning Incorporated, Acton, MA). A total of 300 μL cells (2 × 105 cells/mL) were seeded in the upper chamber in serum-free DMEM culture medium and different concentration of APS and CM, while DMEM medium with 10% FBS (600 μL) was added to the bottom well as a chemoattractant. After 24 h incubation, cells on the upper surface of membranes were removed with cotton swabs and the migrated cells on the lower surface were fixed with 4% paraformaldehyde for 10 min and stained with eosin for 20 min. Migrated cells were visualized and

2.4.3. Calcein-AM/PI viability assay Cell viability was also qualitatively evaluated by live/dead staining. A total of 2 × 103 cells were seeded in 96-well plates with 100 μL complete medium. After exposure to various APS and CM, all cells were treated with calcein-AM, PI and Hoechst 33342 staining solution at each time point and incubated at 37 °C for 30 min. Imaging was performed by a confocal laser scanning microscope (FV1000, Olympus, 687

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Fig. 2. The morphology and chemical properties of APS. (a) Macroscopic morphology of APS. (b–c) SEM observation of APS at 40,000× and 160,000× magnification. (d) EDS analysis of APS. (e) UV absorption spectra of APS. (f) IR spectrum of APS.

photographed with an inverted phase contrast microscope (Olympus, Japan). For the invasion assays, the experimental procedures were similar to that of migration assays except that the Transwell invasion chambers were coated with Matrigel (50 μL per filter) (BD Biosciences, Franklin Lakes, NJ, USA) and the incubation was extended to 48 h. After eosin staining, images of three random fields from three replicate wells were selected and the numbers of cells were calculated.

vibration and CeH bending vibration, respectively. The four bands above were characteristic absorption bands of polysaccharide. The three bands at 1010–1155 cm−1 indicated the pyran configurations of polysaccharides. The absorption peaks at 1081 and 1022 cm−1 were attributed to the glycosidic linkage CeOeH and CeOeC stretching vibration. The bands at 848 cm−1 and 930 cm−1 were characteristic of α-1,6 glucan [24].

2.6. Statistical analysis

3.2. Macrophage activation of APS

All data were expressed as the mean ± standard deviation (SD). All experiments were performed at least three times. Significances were analyzed by one-way analysis of variance (ANOVA) and Student's t-test. P < 0.05 was considered as significant difference. All statistical analyses were performed using Origin 8.0 software (Origin Lab, MA, USA).

Following 24 h incubation with APS, the production of NO was gradually up-regulated with the increasing doses of APS (Fig. 3a). Significant enhancement of NO generation was identified after exposure to APS at the concentration of 200–1000 μg/mL compared with that of untreated cells (P < 0.01). However, NO production stimulated by APS was markedly lower than that in the presence of LPS (P < 0.05). For the expression of TNF-α, ELISA results indicated that all the APS-treated groups presented a significant increase of TNF-α levels in a dose-dependent manner as compared with that of control group (P < 0.001, Fig. 3b). Nevertheless, significant superiority of TNF-α secretion was noticed in LPS group compared with that of APS at the concentration of 1000 μg/mL (P < 0.05). On this basis, we speculated that APS may indirectly exert cytotoxicity against cancer cells though the macrophage activation with the release of TNF-α and NO. To preliminarily elucidate the action mechanism of APS to macrophages, we localized the site of their interaction by labeling APS with FITC. The obtained FITC-labeled APS was light yellow powder, and exhibited strong green fluorescence at 488 nm excitation. The major site of interaction could be noticed on cell membrane rather than penetrate into cells. FITC-APS specifically bound to RAW264.7 macrophages in a time-dependent manner (20 min, 1 h, and 6 h), and the binding was blocked by unlabeled APS, which suggested that membrane receptors in macrophages appeared to saturate (Fig. 3c). It explained the opposite decrease of NO and TNF-α levels over 1000 μg/mL. Dramatic changes of cellular morphology with long protrusions and

3. Results 3.1. Chemical properties of APS APS was white powder in extrinsic feature as shown in Fig. 2a. SEM observation found that most of the APS presented spherical-like shape with the mean size of 191.4 ± 51.84 nm, and the size of smallest particle of APS was 58.3 nm (Fig. 2b). At a higher magnification, the surface of APS was relatively rough with small protrusions (Fig. 2c). Meanwhile, the EDS analysis indicated the main elements of APS were carbon (72.56%) and oxygen (27.44%), and the undetected nitrogen and phosphorus also explained the preferable purity without mixing protein and nucleic acid (Fig. 2d). Chemical composition analysis showed that APS contained 89.75% total carbohydrate and a small proportion of uronic acid (9.3%). As shown in Fig. 2e, the UV absorption spectra of APS indicated no absorption at 280 nm or 260 nm, which implied the absence of protein or nucleic acid. The IR spectrum of APS (Fig. 2f) exhibited absorption bands at 3400, 2925, 1647 and 1371 cm−1, attributing to OeH stretching vibration, CeH stretching vibration, C]O stretching 688

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Fig. 3. APS activated macrophages. (a) and (b) indicated the effects of APS on NO and TNF-α production in RAW264.7 murine macrophage cells. The data represent the means ± standard deviation; n = 3; *P < 0.05, **P < 0.01; # P < 0.05, APS (1000 μg/mL) vs. the LPS group. (c) Fluorescence microphotographs of RAW264.7 cells after incubation with FITC-labeled APS for 20 min, 1 h, and 6 h, as well as incubation with APS (1000 μg/mL) for 1 h followed by exposure to FITClabeled APS for 5 h. Scale bar = 30 μm. (a–b) Macroscopic morphology of FITC-labeled APS. (d) Cellular morphology of RAW264.7 at P5 passage with/without the intervention of APS under phase contrast microscopy and SEM. The blue circle and arrows indicated the long protrusions. Scale bar = 3 μm. (e–f) The effect of antiTLR4 antibody on the production of APS-induced NO and TNF-α. *** P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

those in the experimental group. Conversely, 5-FU presented significant toxicity effects on MCF-7 cells. The cell viability was obviously decreased after exposure to 5-FU in a time-dependent manner compared with those in experimental and negative control group (P < 0.001) (Fig. 4b). To investigate the possible anti-tumor activity from the increased production of NO and TNF-α in CM mediated by APS on macrophages, MCF-7 cells were also subjected to CM for 24, 48, and 72 h, respectively. After exposure to CM for 2 days, the number of living cells gradually decreased and that of dead cells increased in a dose-dependent manner. However, the cytotoxicity effect of CM was weaker than that in 5-FU group (Fig. 4c). The results of CCK indicated that CM exerted growth inhibitory effects on MCF-7 cells in a time- and dose-dependent manner, with the most remarkable toxicity effect (41%) at the concentration of 1000 μg/mL on day 3. Significant inhibitory effects of CM were identified at the concentration of 1000 μg/mL as compared to those at the concentration of 50–200 μg/mL at 24, 48, and 72 h (P < 0.001). However, the toxicity effect of CM at the highest concentration was markedly inferior compared than that in 5-FU group at 24 h (P < 0.01). Furthermore, the difference between two groups became even more pronounced over time (P < 0.001, Fig. 4d). The 6-well plate-based colony formation assays were carried out to uncover the effect of CM on the long-term growth of MCF-7 cells. Upon 14-d incubation with CM, the numbers of Giemsa-stained colonies were remarkably decreased with the increase of APS concentration in CM. Notably, no colony was noticed in the positive control group since the 5-FU dose on each cell was enhanced with the lower cell-seeding density (Fig. 4e). Compared to untreated cells, CM significantly inhibited the colony formation at the concentration of 200–1000 μg/mL of APS

pseudopodia were identified after exposure to APS compared with those of untreated RAW 264.7 cells (Fig. 3d). Moreover, treatment of RAW264.7 cells withTLR4 antibodies for 2 h before adding APS to the culture resulted in the down-regulation rather than complete suppression of NO and TNF-a production than that in corresponding samples treated with APS alone (Fig. 3e–f), which demonstrated that binding of TLR4 receptor with APS was partially associated with the activation of macrophages and following cytokine production. 3.3. Antineoplastic activity 3.3.1. Growth-inhibitory effects of APS and CM on MCF-7 Live/dead staining and CCK were conducted to qualitatively and quantitatively evaluate the effects of APS on the growth of MCF-7 cells, respectively (Fig. 4a and b). The results of live/dead staining indicated that APS groups failed to inhibit cell growth as an overwhelming proportion of living cells were positively stained by calcein-AM with strong green fluorescence, and sporadic dead cells exhibited red fluorescence with PI staining compared with those of untreated cells. Meanwhile, a great many living cells also exhibit slight blue fluorescence in nuclei stained by Hoechst. Instead, a small number of living cells were identified in positive control group accompanied by numerous dead cells. In addition, the results of CCK also suggested that no cytotoxicity on MCF7 cells was observed after the treatment with different doses of APS for 3 days, particularly on day 1 with the higher cell viabilities in the experimental group than that in the negative control group. Although better viabilities were identified in the negative control group on day 1 and day 3, there were no significant differences in comparison with 689

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Fig. 4. Inhibitory effect of CM on proliferative activity of MCF-7 cells. (a,c) Live/dead staining to evaluate the effect of APS and CM on cell viability after incubation for 72 h, respectively. Cells were stained with calcein-AM, PI and Hoechst 33342 after exposure to APS and APS mediated CM (100–1000 μg/mL), and 50 μg/mL 5-FU (scale bar = 100 μm). (b, d) The viability and inhibitory of MCF-7 cell cultured with different dosages of APS and APS-mediated CM were evaluated by CCK-8 assay after incubation for 24, 48, and 72 h, respectively, (n = 4). (e) Colony formation of MCF-7 cells in the presence of APS mediated CM with different concentration and 5-FU. (f) Colony number of MCF-7 cells after incubation with CM and 5-FU. *** P < 0.001 compared between CM groups and negative control group; ## P < 0.01, ### P < 0.001 compared between 5-FU and APS mediated CM at the concentration of 1000 μg/mL.

addition, consistent with the above results that APS alone failed to inhibited cell proliferation, no significant difference of cell cycle phase distribution was identified among APS groups (Fig. S1).

(P < 0.001). Nevertheless, the colony number in the presence of 5-FU was markedly lower than that of highest concentration of APS in CM (Fig. 4f, P < 0.001). These findings indicated that APS was capable to active macrophages to inhibit MCF-7 cells growth, even if the cytotoxicity was inferior in comparison with 5-FU.

3.3.3. CM induced cell apoptosis The formation of apoptotic bodies is a typical characteristic of cells undergoing apoptosis. After treatment with CM for 48 h, DAPI staining indicated that CM led to a strong chromatin condensation and nuclear fragmentation with the increasing concentration of APS in CM, while the cells presented normal nuclear morphology and slight blue stain in nuclei in the negative control group (Fig. 6a), and also in those groups after treatment with APS at the different concentrations (Fig. S2). Increased nuclear fragmentation and dot-like apoptotic bodies were identified in the presence of 5-FU compared with that in CM (1000 μg/ mL APS) group (Fig. 6a). MCF-7 cells were labeled by AO/EB and observed with a fluorescent microscope after exposure to CM for 48 h. No significant apoptosis was detected in the negative control group. As is presented in Fig. 6b, we found that the number of apoptotic cells, especially the late-stage proportion, gradually increased with growing concentrations. Moreover, it was notable that the overall number of cells tended to decrease in a dose-dependent manner. Basically, all cells were apoptotic at different stages and appeared to disintegrate in 5-FU group.

3.3.2. CM induced G1 phase arrest and sub-G1 DNA fraction To examine the mechanism responsible for CM-mediated cell proliferation inhibition, cell cycle phase distribution and proliferation index were analyzed. CM treatment induced cell cycle arrest in G1 phase in a dose-dependent manner (Fig. 5a and b). Compared to the negative control group, significant elevation was identified in the CM group mediated by APS at the concentration of 500 μg/mL (P < 0.05) and 1000 μg/mL (P < 0.01). The formed sub-G1 peak appeared in the cell cycle histogram as a result of apoptosis with the characteristic of karyopyknosis and DNA cleavage in apoptotic cells. As is presented in Fig. 5a, an apoptotic peak with the sub-G1 fraction of 3.19% appeared after exposure to CM with APS (1000 μg/mL), while the fraction was 9.01% after the treatment with 5-FU. Meanwhile, APS mediated CM down-regulated proliferation index of MCF-7 cells in particular at the concentration of 500 μg/mL (35.94%, P < 0.05) and 1000 μg/mL (24.52%, P < 0.01) compared to those of untreated cells (44.02%, Fig. 5c), which further appeared to inhibit cell proliferation. In 690

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Fig. 5. DNA cell cycle analyses of MCF-7 cells after treatment with CM. (a) Cell cycle distribution by flow cytometry of MCF-7 cells in the presence of CM mediated by various APS and 5-FU for 48 h. (b) Cell cycle profile of MCF-7 cells after exposure to CM for 48 h. The fraction of cells from apoptosis, G1, S and G2 phases analyzed from PI-A vs. cell accounts of each phase were shown in as percentages. (c) Proliferation index of MCF-7 cells. Data expressed as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 vs. negative control group (untreated cells).

Instead, CM incubation led to cell deformation with decreased cell size, and loss of microvilli and pseudopods with smooth surface in the majority of cells. Meanwhile, CM treatment resulted in apoptotic body formation with different sizes (Fig. 6e). Cell morphological changes with apoptotic bodies were also identified after exposure to 5-FU. Here, the reduction in microvilli in CM group may potentially inhibit MCF-7 cell attachment and invasion.

To further evaluate the role of APS in the apoptosis of MCF-7, we used flow cytometry to quantitatively analyze the apoptotic cells. As is shown in Fig. 6c and d, the apoptotic rates of MCF-7 cells were gradually increased in a dose-dependent manner. Significant elevation was noticed in the experimental group with the concentration of 1000 μg/ mL APS compared with that of untreated cells (P < 0.001). However, the apoptotic rate (29.7%) was markedly inferior as compared to the rate of 57.1% in 5-FU group (P < 0.001). SEM examination revealed that MCF-7 cells are polymorphic and triangular in shape with elongated pseudopods and microvilli on cell surface. No significant change of cell morphology was noticed after treatment with APS at various concentrations, as shown in Fig. S3.

3.3.4. CM down-regulated Bcl-2 and upregulated Bax expression To further explore the apoptotic mechanism of APS mediated CM on MCF-7 cells, the expression levels of apoptosis-related genes and proteins (Bcl-2 and Bax) were examined. It was found that CM inhibited the

Fig. 6. APS mediated CM induced the apoptosis in MCF-7 cells. (a) Nuclear condensation of MCF-7 cells was visualized under fluorescent microscope after exposure to CM and 5-FU. Condensed nuclei and the apoptotic bodies are indicated by arrows. (b) Apoptosis cells were detected by AO/EB staining in the presence of APS mediated CM and 5-FU. Green nuclei of living cells, green–yellow nuclei of cells at early stages of apoptosis with different forms of condensed, bright orange nuclei of cells at the late stage of apoptosis with condensed chromatin, and uneven orange-red or bright red nuclei of necrotic cells. Scale bar = 50 μm. (c) Flow cytometry analysis of apoptosis induced by CM in MCF-7 cells using Annexin V-FITC staining. (d) The percentage of apoptotic MCF-7 with the intervention of CM. Significant differences between the CM-treated groups and negative control group were indicated as *** (P < 0.001), while significant difference between APS-mediated CM (1000 μg/mL) and 5-FU was indicated as ###(P < 0.001). (e) SEM observation of MCF-7 apoptosis after exposure to CM and 5-FU. Magnification of the images was 3000× and 10,000× (green border) on electron microscope. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 691

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Fig. 7. Bcl-2 and Bax expression in MCF-7 cells in different groups. (a) Immunofluorescent pictures of FITC-Bcl-2 and DAPI staining and their merge of MCF-7cells after exposure to APS mediated CM and 5-FU, respectively. (b) Immunofluorescent pictures of Bax protein after treatment with APS mediated CM and 5-FU, respectively. Scale bar = 30 μm. (c) Bcl-2 and Bax protein expression by flow cytometry intracellular staining. (d) The ratio of Bax and Bcl-2 protein expression. * P < 0.05, ** P < 0.01, vs. negative control group (untreated cells). # P < 0.05, compared between 5-FU and APS mediated CM at the concentration of 1000 μg/mL.

S4–S5). Here, Transwell assay further verified that CM possessed highly strong inhibitory efficacy on cell migration and invasion, and the underlying mechanism still needs to be determined in our subsequent research.

mitochondrial anti-apoptotic protein Bcl-2 in a dose-dependent manner (Fig. 7a). Compared with the experimental group, the protein level of Bcl-2 was significantly down-regulated after the exposure to 5-FU. In addition, the protein level of Bax was gradually up-regulated with the intervention of CM. Following 5-FU treatment, the protein level of Bax was markedly enhanced in the MCF-7 cells (Fig. 7b). The results of flow cytometry intracellular staining showed that Bcl-2 protein levels were markedly decreased and Bax protein levels were significantly increased in MCF-7 cells after treatment with CM and 5-FU (Fig. 7c). Significant up-regulation of Bax/Bcl-2 ratio was identified in CM group (200–1000 μg/mL) compared with that of negative control group (Fig. 7d). Notably, the Bax/Bcl-2 ratio increased from 0.227 in negative control to 3.01 in the CM group (1000 μg/mL).

4. Discussion Nowadays, traditional herbal medicine and natural products are becoming increasingly popular among cancer patients responsible for their enhanced immunity potential and quality of life. Particularly, numerous polysaccharides isolated from microorganisms, plants and animals have attracted growing interests in biomedical field and appear to be a favorable source for oncotherapy, as they possess anti-tumor activities or augment the efficacy of chemotherapy agents [25–27]. Among these polysaccharides, APS has been considered as an ideal candidate for oncotherapy, particularly being used in combination with chemotherapy and radiotherapy [28]. As an adjuvant in cancer treatment, APS can enhance therapeutic effects and reduce adverse effects, contributing to the improvement of host immune response. However, few reports are available concerning whether APS can activate the immune potential in vitro. In the present study, we initially examined the effect of APS on MCF-7 cells viability and received unsatisfactory outcome. Considering the antitumor mechanism of APS in vivo, the macrophage-like RAW264.7 cells was chosen as model in vitro to evaluate whether APS was able to stimulate immune cells and further inhibited cancer cells growth. We found the obtained APS could activate RAW264.7 cells accompanied by up-regulation of NO and TNF-α production, which may act as cell apoptosis inducer to inhibit MCF-7 cells viability. Based on the encouraging observation, APS may provide an adjuvant strategy for the treatment of breast cancer. The corresponding anti-breast cancer effect of APS and its mechanism in vivo will be further elucidated. The immune regulation and antitumor activity of polysaccharides are closely associated with their structure variability which presents a

3.4. CM inhibited MCF-7 cells migration and invasion The previous SEM results indicated that CM led to the loss of microvilli and pseudopods of MCF-7 cells which may contribute to the inhibition of cell migration and invasion. On this basis, we examined the effect of CM on the migration and invasion activities of MCF-7 cells by Transwell migration assays. As is shown in Fig. 8a, cells migrated to the lower chamber by shrink motion, and penetrated Matrigel by secreting hydrolases and further invaded to the lower chamber. The Transwell assays indicated that CM depressed the migration and invasion of MCF-7 cells in a dose-dependent manner (Fig. 8b). The selected random fields of cells number suggested that the migration of MCF-7 cells were significantly decreased when exposed to CM mediated by APS with the concentration of 200–1000 μg/mL (Fig. 8c) as compared to that of untreated cells (P < 0.001). Similarly, APS mediated CM made the invasive potential of MCF-7 cells markedly decreased in comparison with that of negative control group (Fig. 8d). Expectedly, APS had no inhibitory effect either on cell migration or invasion. No significant difference was observed of the migrated or invasive numbers of MCF-7 cells after treatment with various concentrations of APS (Fig. 692

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Fig. 8. APS mediated CM suppressed MCF-7 cells migration and invasion. (a) Representative SEM images of cells that passed through the Transwell membrane with/ without Matrigel at 1000× and 2000× magnification. Green arrows indicated that the secreted hydrolases digested Matrigel, and blue arrows indicated that cells migrated to low chamber. (b) Representative images of migrating or invading MCF-7 cells after treatment with APS mediated CM, respectively. Scale bar = 100 μm. (c–d) MCF-7 cell numbers that passed through the Transwell chambers without Matrigel (motile cells/field) or with Matrigel (invasive cells/field). ** P < 0.01, *** P < 0.001, vs. negative control group (untreated cells). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

has become a molecule of interest in carcinogenesis and tumor growth progression [34,35]. Accumulating evidence suggests that NO derived from macrophages, endothelial cells, and natural killer cells participates in tumoricidal activity against different tumor types [36]. Furthermore, numerous studies have shown the macrophage-mediated cytotoxicity against many tumor cells was linked to TNF-α and NO [37]. Takeda et al. found that fucoidan polysaccharide alone failed to inhibit S-180 cell growth in vitro, but fucoidan-treated RAW264.7 cells depressed S-180 cell growth which was considered to be partially correlated with NO production in activated RAW264.7 cells [38]. Another report demonstrated that APS induced RAW264.7 macrophage activation and up-regulated the concentrations of NO, TNF-α, IL-1β and IL-6 in vitro, and further exhibited anti-tumor activity in vivo by immunoregulatory [39]. Based on these considerations, we evaluated the ability of APS to induce TNF-α and NO production by RAW264.7 cells and the related anti-tumor effect towards MCF-7 cells. Our results demonstrated that APS stimulated the production of NO and TNF-α from RAW264.7 cells in a dose-dependent manner, which further acted as the priming agents to mediate the growth inhibition of MCF-7 cells. For the activation mechanism of macrophage, the first step of action is to recognize them by certain receptors/proteins located on macrophages and activation of signal transduction pathway [40]. At present, details to identify the polysaccharides-binding cellular receptors expressed from immune cells are far from clear. However, some reports and experiments have introduced some cell membrane receptors directly targeted by polysaccharides [39]. For instance, previous study showed that polysaccharide fraction from P. umbellatus enhanced the production of TNF-α and IL-1β by TLR4-depedent activation of macrophages [41]. As for APS, it has been demonstrated that APS was capable of activating mouse macrophages and B cells and enhanced cell

considerable capacity for carrying biological information. Currently, extensive studies have indicated the important role of chemical composition and structural characteristics of polysaccharides on their biological activities [29,30]. Previous study demonstrated that the strong antioxidant and antitumor capacity of APS was highly dependent on the content of APS [6]. Zhu et al. reported that the diverse anti-hepatoma activities of APS-I (55.47%) and APS-II (47.72%) were considered to be most closely related to their chemical composition, configuration and their physical properties [5]. Therefore, it is indispensable to understand the molecular structure of polysaccharides. In this study, chemical properties results indicated that the obtained APS were highly purified, without protein and nucleic acid. IR analysis showed that APS had a backbone composed of (1,6)-α-D-glucopyranosyl (Glcp) residues. In addition, SEM was used to evaluate the morphology and mean size of APS. According to the anti-tumor activity of APS-activated macrophages, the activated effect of APS might be attributed to its relative smaller sizes and spherical shape with high specific surface area, which favored the full exposure of the active site of APS to macrophages. For the dominant nanoscale of APS, it is not only capable of passively interacting with cells, but also actively regulating the molecular process and cellular responses [31]. Macrophages yield up the defense line role against somatic cells infected by parasite or fungi and tumor cells in host defense system. It is well known that macrophages can secrete 70 different soluble factors such as many proteases and a variety of cytolytic factors, some of which have been proven to play a crucial role in macrophage mediated cytotoxicity against tumors [32,33]. Among these secreted cytokines, TNF-α, derived from monocytes/macrophages, has been considered as an important cytotoxic mediator contributing to the tumoricidal activity. Besides, NO refers to short-lived, endogenously produced gas, 693

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dose- and time-dependent manner, and blocked cell cycle at G1 stage. Meanwhile, CM was capable of inducing cell apoptosis by increasing the Bax/Bcl-2 ratio. Overall, apoptosis induction and cell cycle arrest may be the anti-tumor mechanisms of APS mediated CM. Currently, the efforts in the study of APS are gradually shifting to prevention and clinical intervention in neoplastic disease because of its capability to enhance host immune function and its role as an adjuvant to chemotherapeutic agents. Our findings indicated that APS appeared to activate the immune response in vitro and then inhibit breast cancer cells growth, further research to evaluate the antitumor effect in vivo was underway in our lab. The molecular mechanisms for macrophages activation by APS and inhibitory activity on cancer cell migration are still required for the investigations in the near future.

proliferation and cytokine production via the activation of TLR4 and NF-κB/Rel activation [17,42,43]. In our study, fluorescent images indicated the major sites of APS and macrophages located at the outer surface of cell membrane. The down-regulated production of NO and TNF-α in RAW 264.7 cells after treatment with TLR4 antibodies prior to APS further demonstrated the TLR4 receptor on the cell membrane is involved in APS binding and the subsequent production of cytotoxic factors. Apoptosis refers to a form of highly regulated process of physiological cell death, which evokes cell death through extrinsic (via death receptors) or intrinsic (via mitochondria) pathways. The defective apoptosis and the uncontrolled cell proliferation appear to disrupt the balance between cell division and cell death, finally leading to the development of cancer [44]. Therefore, targeting cell apoptosis and dysfunctional cell proliferation have become a great interest for the treatment of cancer [45,46]. Apoptotic cell death is characterized by cell shrinkage, plasma membrane blebbing, nuclear condensation and ultimately DNA fragmentation [47]. Currently, cancer cell apoptosis and cell proliferation inhibition have been shown to be the most pervasive anti-tumor mechanism induced by many kinds of chemotherapeutic agents or antitumor drugs [48], and the same is true for some polysaccharides [49,50]. A previous report indicated that Angelica sinensis polysaccharide could inhibit cell proliferation and promote the apoptosis of HeLa cells, which primarily involved the activation of the intrinsic mitochondrial pathway [51]. Shen et al. found that APS was capable of inhibiting MKN45 cells proliferation by blocking cell cycle at G1 stage and inducing cells apoptosis in a dose- and time-dependent manner [52]. In this study, APS failed to induce cell apoptosis and inhibited cell proliferation, migration and invasion. Encouragingly, APS mediated CM markedly repressed the proliferation of MCF-7 cells in a time- and dose-dependent manner and induced cell cycle arrest in G1phase. Simultaneously, we further demonstrated that CM decreased the survival of MCF-7 cells through an apoptotic mechanism, as was verified by cell shrinkage, loss of microvilli, apoptotic bodies formation and nuclear fragmentation. Meanwhile, to obtain further information regarding apoptotic modulation, we examined the expression of Bax and Bcl-2 proteins in MCF-7 cells. Bcl-2 and Bax function as tumor antiapoptotic and pro-apoptotic factors, respectively, which have been demonstrated to play crucial roles in the regulation of apoptosis, and the balance of Bax and Bcl-2, i.e. the ratio of Bax/Bcl-2, is a decisive factor for cell survival or death [53]. Our results revealed that apoptosis was significantly stimulated as indicated by increasing the Bax/Bcl-2 ratio upon CM treatment. On this basis, APS mediated CM induced apoptosis in MCF-7 cells by modulating Bcl-2 family protein. Cancer metastasis is a major underlying event for cancer-related morbidity and mortality [54]. It begins with the migration and invasion of cancer cells into surrounding tissues and lymphatics followed by targeting organs. Nowadays, great attention has been paid on searching for new strategies against tumor progression, of which some polysaccharides have shown the potential to intervene tumor cell migration and invasion [55–57]. In our study, it was reasonable to speculate that CM could suppress the migration and invasion of MCF-7 cells based on their loss of microvilli and pseudopods as one of the crucial steps in directed migration and invasion is lamellipodia formation at the leading edge of cells. Transwell assay further verified that CM possessed highly strong inhibitory efficacy on cell migration and invasion, and the underlying mechanism is still to be determined in our subsequent research.

Acknowledgments This work was supported by the National Natural Science Foundation of China (31670978/31370991/21676041), the Fok Ying Tung Education Foundation (132027), the State Key Laboratory of Fine Chemicals (KF1111), the Natural Science Foundation of Liaoning (20180510028) and the Fundamental Research Funds for the Central Universities (2016ZD210) and SRF for ROCS, SEM (42). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.01.025. References [1] C.E. DeSantis, S.A. Fedewa, A. Goding Sauer, J.L. Kramer, R.A. Smith, A. Jemal, Breast cancer statistics, 2015: convergence of incidence rates between black and white women, Cancer J. Clin. 66 (2016) 31–42. [2] A.I. Neugut, G.C. Hillyer, L.H. Kushi, L. Lamerato, D.L. Buono, S.D. Nathanson, D.H. Bovbjerg, J.S. Mandelblatt, W.Y. Tsai, J.S. Jacobson, A prospective cohort study of early discontinuation of adjuvant chemotherapy in women with breast cancer: the breast cancer quality of care study (BQUAL), Breast Cancer Res. Treat. 158 (2016) 127–138. [3] Y. Liu, B. Zhang, S. Ibrahim, S.S. Gao, H. Yang, W. Huang, Purification, characterization and antioxidant activity of polysaccharides from Flammulina velutipes residue, Carbohydr. Polym. 145 (2016) 71–77. [4] G.J. Wu, S.M. Shiu, M.C. Hsieh, G.J. Tsai, Anti-inflammatory activity of a sulfated polysaccharide from the brown alga Sargassum cristaefolium, Food Hydrocoll. 53 (2016) 16–23. [5] Z.Y. Zhu, R.Q. Liu, C.L. Si, F. Zhou, Y.X. Wang, L.N. Ding, C. Jing, A.J. Liu, Y.M. Zhang, Structural analysis and anti-tumor activity comparison of polysaccharides from Astragalus, Carbohydr. Polym. 85 (2011) 895–902. [6] R. Li, W.C. Chen, W.P. Wang, W.Y. Tian, X.G. Zhang, Antioxidant activity of Astragalus polysaccharides and antitumour activity of the polysaccharides and siRNA, Carbohydr. Polym. 82 (2010) 240–244. [7] Q.E. Tian, H.D. Li, M. Yan, H.L. Cai, Q.Y. Tan, W.Y. Zhang, Astragalus polysaccharides can regulate cytokine and P-glycoprotein expression in H22 tumorbearing mice, World J. Gastroenterol. 18 (2012) 7079–7086. [8] L. Meng, S. Sun, R. Li, Z. Shen, P. Wang, X. Jiang, Antioxidant activity of polysaccharides produced by Hirsutella sp. and relation with their chemical characteristics, Carbohydr. Polym. 117 (2015) 452–457. [9] C. Xun, N. Shen, B. Li, Y. Zhang, F. Wang, Y. Yang, X. Shi, K. Schafermyer, S.A. Brown, J.S. Thompson, Radiation mitigation effect of cultured mushroom fungus Hirsutella sinensis (CorImmune) isolated from a Chinese/Tibetan herbal preparation–Cordyceps sinensis, Int. J. Radiat. Biol. 84 (2008) 139–149. [10] Z.Y. Zhu, X.C. Liu, X.N. Fang, H.Q. Sun, X.Y. Yang, Y.M. Zhang, Structural characterization and anti-tumor activity of polysaccharide produced by Hirsutella sinensis, Int. J. Biol. Macromol. 82 (2016) 959–966. [11] C. Bogdan, Nitric oxide and the immune response, Nat. Immunol. 2 (2001) 907–916. [12] F.R. Balkwill, M.S. Naylor, S. Malik, Tumour necrosis factor as an anticancer agent, Eur. J. Cancer Clin. Oncol. 26 (1990) 641–644. [13] M.F. Moradali, H. Mostafavi, S. Ghods, G.A. Hedjaroude, Immunomodulating and anticancer agents in the realm of macromycetes fungi (macrofungi), Int. Immunopharmacol. 7 (2007) 701–724. [14] M. Mizuno, Y. Shiomi, K.I. Minato, S. Kawakami, H. Ashida, H. Tsuchida, Fucogalactan isolated from Sarcodon aspratus elicits release of tumor necrosis factor-α and nitric oxide from murine macrophages, Immunopharmacology 46 (2000) 113–121. [15] S.P. Wasser, Reishi or Ling Zhi (Ganoderma lucidum), Encycl. Diet. Suppl. 1 (2005) 603–622.

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