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  • br Astragalus membranaceus has been


    Astragalus membranaceus, has been shown to exhibit broad-range in-hibitory 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 sy-nergistic 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 anti-tumor activity in H22 tumor-bearing mice by regulating immune cy-tokines [7]. Despite these progresses, the anti-tumorigenic activity and mechanism of APS against breast carcinogenesis has rarely been re-ported.
    The anti-tumor activity of polysaccharides is generally mediated via two major pathways: direct inhibitory/eradicative effect against ma-lignant 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).
    (e.g. macrophage, T and B lymphocytes, NK 4μ8C 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 poly-saccharides 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 anti-tumor activity in tumor-bearing hosts as well as immunomodulating effects [14]. β-glucan-related polysaccharides of the higher fungus ac-tivated 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 cap-able 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 APS-mediated 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. Materials and methods
    2.1. Materials and reagents
    APS was purchased from Pharmagenesis Inc. (Beijing, China). 5-fluorouracil (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 (Shanghai, China). Anti-Bcl-2 antibody, anti-Bax antibody, and anti-TLR4 antibody were  Materials Science & Engineering C 98 (2019) 685–695
    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 analy-tical grade.
    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 spectro-meter (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 magnifi-cations. 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].