br P vs negative control group
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.)
considerable capacity for carrying biological information. Currently, extensive studies have indicated the important role of chemical com-position and structural characteristics of polysaccharides on their bio-logical activities [29,30]. Previous study demonstrated that the strong antioxidant and antitumor capacity of APS was highly dependent on the content of APS . 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 . Therefore, it is indispensable to under-stand the molecular structure of polysaccharides. In this study, che-mical 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 macro-phages, 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 in-teracting with cells, but also actively regulating the molecular process and cellular responses .
Macrophages yield up the defense line role against somatic FLAG tag Peptide 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 cy-totoxicity 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 ac-tivity. Besides, NO refers to short-lived, endogenously produced gas,
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 partici-pates in tumoricidal activity against diﬀerent tumor types . Fur-thermore, numerous studies have shown the macrophage-mediated cytotoxicity against many tumor cells was linked to TNF-α and NO . 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 cor-related with NO production in activated RAW264.7 cells . Another report demonstrated that APS induced RAW264.7 macrophage activa-tion 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 im-munoregulatory . 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 de-monstrated 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 macro-phages and activation of signal transduction pathway . At present, details to identify the polysaccharides-binding cellular receptors ex-pressed from immune cells are far from clear. However, some reports and experiments have introduced some cell membrane receptors di-rectly targeted by polysaccharides . 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 mac-rophages . As for APS, it has been demonstrated that APS was capable of activating mouse macrophages and B cells and enhanced cell
proliferation and cytokine production via the activation of TLR4 and NF-κB/Rel activation [17,42,43]. In our study, fluorescent images in-dicated 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.