M1 Polarization but Anti-LPS-Induced Inflammation and Anti-MCF-7 Breast Cancer Cell Growth Effects of Five Selected Polysaccharides

Five potential polysaccharides from guava seed (GSPS), common buckwheat (CBPS), bitter buckwheat (BBPS), red Formosa lambsquarters (RFLPS), and yellow Formosa lambsquarters (YFLPS) were selected to measure their effects on mouse peritoneal macrophages in the absence or presence of lipopolysaccharide (LPS). Macrophage-conditioned media (MCM) in the absence or presence of 5 selected polysaccharides were prepared to treat MCF-7 cells. The cell viability was determined using 3-(4,5-dimethylthiazol-2-diphenyl)-2,5-tetrazolium bromide (MTT) assay. Proinflammatory (also known as M1 type) (interleukin- (IL-) 1β, IL-6 and tumor necrosis factor- (TNF-) α) and anti-inflammatory (also known as M2 type) (IL-10) cytokines secreted by macrophages were determined using ELISA. The relationship between MCF-7 cell growth and M1/M2 cytokine secretion profiles in the corresponding MCM were delineated. The results showed that 5 selected polysaccharides, except BBPS, significantly (P < 0.05) and dose-dependently increased M1 (IL-1β + IL-6 + TNF-α)/M2 (IL-10) cytokine secretion ratios by macrophages in the absence of LPS, suggesting that four selected polysaccharides have M1 polarization property. However, all of 5 selected polysaccharides significantly (P < 0.05) decreased proinflammatory (IL-1β + IL-6 + TNF-α)/anti-inflammatory (IL-10) cytokine secretion ratios by LPS-stimulated macrophages, exhibiting that all of the 5 selected polysaccharides, particularly GSPS, have anti-inflammatory potential. All MCM prepared with these selected polysaccharides (except YFLPS) significantly enhanced their inhibitory effects on MCF-7 cell growth. A negative correlation was noted between MCF-7 cell viabilities and M1/M2 cytokine secretion ratios ((IL-6 + TNF-α)/IL-10) in the corresponding MCM, suggesting that increases in M1 macrophages in the tumor microenvironment might inhibit MCF-7 cell growth. Particular polysaccharides including RFLPS, GSPS, YFLPS, and CBPS may increase the percentage of M1 macrophages in the tumor environment and further inhibit MCF-7 cell growth via immunotherapy.


Introduction
Macrophages that are inflammatory cells can be activated during inflammation process resulting from bacteria or endotoxin invasion to combat pathogens, thereby reducing the damage to the body [1]. Activated macrophages produce proinflammatory cytokines (also known as M1-type cytokines), such as interleukin-(IL-) 1β, IL-6 and tumor necrosis factor-(TNF-) α, to recruit more white blood cells to the inflammation site and enhance the inflammation status [2].
Mild or acute controlled inflammation is a normal and essential process for protection; however, chronic or repeatedly uncontrolled inflammation might cause inflammation-related diseases such as cancers. Beyond enhancing inflammation and activating the immune system, macrophages via cytokines release are also found to play an important antiinflammatory role. A balanced mechanism to regulate excess inflammation exists in the body via producing anti-inflammatory cytokines, such as IL-10 [3]. Consequently, the secretion profile of proinflammatory (M1) and anti-inflammatory (M2) cytokines by macrophages may reflect the inflammation status in vitro or in vivo [4,5]. Active phytochemicals with antiinflammatory potential such as polysaccharides have been proven anti-inflammatory effects through regulating pro-and anti-inflammatory cytokine profiles [6]. e cell differentiation of macrophages has been related to the development of chronic diseases such as cancers. Macrophages can be classified into two main groups designated as M1 and M2, although this dichotomy has been challenged and further complexity has been reported. M1type macrophages encourage inflammation that help to clear infection and may inhibit tumor cell growth, whereas M2type macrophages decrease inflammation through producing an anti-inflammatory cytokine IL-10 and improve tissue repair [7]. Macrophages may infiltrate a number of tumors; however, tumor-associated macrophages (TAMs) are mainly M2-type macrophages that seem to actively promote tumor growth and progression [8]. In contrast, M1 phenotype macrophages in the tumor environment may help to control cancer development in the early stage.
Among cancers, breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females in the United States [9]. During tumorigenesis, macrophages, neutrophils, mast cells, myeloidderived suppressor cells, dendritic cells (DCs), natural killer (NK) cells, and lymphocytes may be recruited due to recurrent inflammation in the tumor microenvironment [10]. NK cells and cytotoxic T cells may destroy tumor cell itself if they can detect the variation of tumor cells [11]. Unfortunately, about 5-10% of all cancer cases result from escaping from surveillance by NK and cytotoxic T cells [12]. Immune stimulation by biological response modifiers (BRMs) may enhance the ability of NK or cytotoxic Tcells to remove cancer cells in vivo. Moreover, a decrease of inflammation in the tumor microenvironment may be another possible target to pharmaceutically reduce tumorigenesis and angiogenesis in the later stage. Recently, new and possible effective anticancer compounds from natural sources with anti-inflammatory potential such as polysaccharides have been introduced in the treatment of human breast cancer cells via immunotherapy of splenocytes [13]. Potent anti-inflammatory polysaccharides have been a promising agent for anticancer diseases due to regulating M1/M2 or inflammation status in the tumor microenvironment via influencing cytokine secretion profiles by macrophages but not inhibiting the intact immune system.

Determination of Noncytotoxic Optimal Concentrations of Five Selected Polysaccharides to Mouse Primary
Macrophages Using Cell Viability (MTT Assay). To determine the noncytotoxic optimal concentrations for the five selected polysaccharide fractions, the cell viabilities of macrophages treated with individual polysaccharides at different concentrations were determined using 3-(4,5-dimethylthiazol-2-diphenyl)-2,5-tetrazolium bromide (MTT, Sigma, MO, USA) assay. All polysaccharide stock solutions were aseptically diluted into working solutions using TCM medium before use. e macrophages (50 μl/well) in the absence or presence of polysaccharide samples (50 μl/well) at the indicated final concentrations of 0, 1.6, 8, 40, 200, 500, and 1000 μg/ml were cultured in 96-well plates and incubated at 37°C in a humidified incubator with 5% CO 2 and 100. e noncytotoxic optimal doses of individual polysaccharides were selected to conduct cytokine secretion assessments. Based on the results of MTT assay, noncytotoxic optimal administration concentrations for the five selected polysaccharides were determined.

Effects of Five Selected Polysaccharides at eir Optimal Administration Concentrations on M1 (Pro-) and M2 (Anti-Inflammatory) Cytokine Secretions by Macrophages in the
Absence or Presence of LPS. GSPS, CBPS, BBPS, RFLPS, and YFLPS polysaccharides at their optimal administration concentrations were cultured with peritoneal macrophages in the absence or presence of LPS to assess possible M1/M2 polarization and anti-inflammatory effects using the following two experimental models: Model A: mouse peritoneal macrophages (0.5 ml/well) were cocultured with GSPS, CBPS, BBPS, RFLPS, and YFLPS (0.5 ml/well) at their noncytotoxic optimal administration concentrations in 24-well plates and incubated at 37°C in a humidified incubator with 5% CO 2 and 95% air for 48 h. Endotoxin LPS (L-2654, Sigma-Aldrich Co., St. Louis, MO, USA) was selected as a positive control at 2.5 μg/ml in the culture in each experiment. e supernatants in the cell cultures were collected and stored at −80°C for following M1/M2 cytokine assays. Model B: an inflammation-concurrent cell culture model was designed using LPS addition to the test samples. An endotoxin LPS was selected to stimulate inflammation in macrophages. e peritoneal macrophages were cultured in the presence of LPS at 2.5 μg/ml and polysaccharide samples at their noncytotoxic optimal administration concentrations. e plates were incubated in a humidified incubator with 5% CO 2 and 95% air at 37°C for 48 h. e supernatants in the cell cultures were collected and stored at −80°C for following pro-and anti-inflammatory cytokine assays.

Preparation of Macrophage-Conditioned Media (MCM) in the Absence or Presence of Five Selected Polysaccharides.
To prepare MCM, isolated peritoneal macrophages (2 × 10 6 cells/ml TCM medium, 0.5 ml/well) were cocultured with GSPS, CBPS, BBPS, RFLPS, or YFLPS at the indicated noncytotoxic concentrations of 0, 8, 40, and 200 μg/ml TCM medium (0.5 ml/well) in 24 well plates [24]. e plates were incubated at 37°C in a humidified incubator with 5% CO 2 and 95% air for 48 h. e cultured plate was centrifuged at 400 ×g for 10 min to collect the supernatant (ca. 1.0 ml/well) in the cell cultures, which comprised the macrophageconditioned medium (MCM). e supernatant of cell cultures was collected and lyophilized. e lyophilized MCM was dissolved in 0.5 ml MEM/EBSS medium. e two-fold concentrated MCM was stored at −80°C until use. e MCF-7 cells were quickly defrosted at 37°C and maintained in the medium of MEM/ Earle's balanced salts solution (MEM/EBSS) supplemented with 10% fetal bovine serum (FBS), 0.1 mM nonessential amino acid, 1.0 mM sodium pyruvate, penicillin 100 units/ ml, streptomycin 100 μg/ml, and amphotericin B 0.25 μg/ml, at 37°C in a humidified incubator with 95% air and 5% CO 2 . After the cells had grown to 90% confluence in a 75 T tissue culture flask (TPP Biochrom AG, Trasadingen, Switzerland), they were plated at a density of 2 × 10 5 cells/ml in the 96-well plates to perform the following bio-assay.

Effect of MCM Using Five Different Polysaccharides on the Cell Viability of MCF-7 Cells.
To evaluate effects of MCM administration on the cell viability of MCF-7 cells, MCF-7 cells (50 μl/well) were treated with MCM (50 μl/well) or paclitaxel at 2.5 μΜ as a positive control. e plates were incubated in a humidified incubator with 5% CO 2 and 95% air at 37°C for 24 or 48 h. e live cells were measured by MTT assay. e cell viability (%) in each biological determination was calculated using the equation: cell viability (% of control) � ((A sample − A blank )/(A control − A blank )) × 100 [26].

Statistical Analysis.
Results are expressed as the mean ± standard deviation (SD). Differences among Evidence-Based Complementary and Alternative Medicine treatments were analyzed using one-way analysis of variance (ANOVA), followed by Duncan's multiple range test using the SPSS system with 19.0. e relationship between cytokine levels in MCM and cell viabilities of MCF-7 cells was described as Pearson product-moment correlation coefficient (r). P < 0.05 considered a significant difference.

Noncytotoxic Optimal Concentrations of Five Selected
Polysaccharides for Primary Peritoneal Macrophages. To avoid cytotoxicity from excess concentration administration for the five selected polysaccharides, GSPS, CBPS, BBPS, RFLPS, and YFLPS at indicated concentrations of 0, 1.6, 8, 40, 200, 500, and 1000 μg/ml, were cocultured with primary peritoneal macrophages for 48 h, respectively. e LPS at 2.5 μg/ml was selected as a positive control. e cell viability was assayed using MTT assay. e results showed that all GSPS, CBPS, BBPS, RFLPS, and YFLPS administrations at the indicated concentrations did not significantly (P > 0.05) exhibit any cytotoxicity to macrophages compared to that of the control, respectively ( Figure 1). Interestingly, GSPS and BBPS treated at appropriate concentrations significantly (P < 0.05) increased the macrophage cell viability, suggesting that some of these selected polysaccharides have immunostimulatory potential (Figures 1(a) and 1(c)). In contrast, GSPS and RFLPS treated at the indicated concentrations of 500 and 1000 μg/ml slightly decreased (P > 0.05) the macrophage cell viability (Figures 1(a) and 1(d)). To avoid unpredictable cytotoxicities at the higher doses, GSPS, CBPS, BBPS, RFLPS, and YFLPS at the same concentrations of 1.6, 8, 40, and 200 μg/ml were selected as optimal concentrations for treating macrophages and used for the following cytokine secretion assays and MCM preparation.

Effects of Five Selected Polysaccharides on Cytokine Secretion Profiles by Primary Peritoneal Macrophages in the Absence or Presence of LPS.
To evaluate possible M1/M2 polarization and anti-inflammatory potential of the five selected polysaccharides, GSPS, CBPS, BBPS, RFLPS, and YFLPS at the indicated noncytotoxic optimal concentrations of 1.6, 8, 40, and 200 μg/ml were selected to treat macrophages for 48 h in the absence or presence of LPS. Cytokine secretion profiles including M1 (pro-) (IL-1β, IL-6, and TNF-α) and M2 (anti-inflammatory) (IL-10) cytokines from primary peritoneal macrophages were measured, respectively. Table 1 shows the effect of five different polysaccharides on M1 and M2 cytokine secretions by peritoneal macrophages from female BALB/c mice. LPS treatment (2.5 μg/ml) alone was selected as a positive control. e results showed that IL-1β, IL-6, TNF-α, and IL-10 secretions by the LPS-stimulated macrophages significantly (P < 0.05) increased as compared to those of negative controls, indicating that the cell culture model used in this study is suitable and effective for M1/M2 bioassay (Table 1). Interestingly, both M1 and M2 cytokine secretion levels by the primary peritoneal macrophages treated with five selected polysaccharides dose-dependently and significantly (P < 0.05) increased, suggesting that all five selected polysaccharides have immunostimulatory potential, particularly GSPS, RFLPS, and CBPS in order (Table 1). In traditional Chinese herbal medicine, some active ingredients are proven polysaccharides [6]. Parts of the polysaccharides are considered BRMs for enhancing immune activity [27]. In the present study, we evidence that GSPS, CBPS, and RFLPS among the five selected polysaccharides presented substantial immunostimulatory effects on macrophages. Our study suggests that GSPS, CBPS, and RFLPS showed substantial immunostimulatory effects on macrophages that may be applicable to BRMs in the future. Further analysis on M1/M2 cytokine secretion ratios showed that RFLPS, GSPS, YFLPS, and CBPS in order significantly (P < 0.05) increased M1 (IL-1β + IL-6 + TNF-α)/M2 (IL-10) secretion ratios as compared to that of the control, suggesting that these four selected polysaccharides treated alone might result in macrophage activation and reveal a M1 polarization property ( Figure 2).

Effects of MCM Prepared in the Absence or Presence of Five Selected Polysaccharides on MCF-7 Cell Growth.
We found that direct administrations of the five selected polysaccharides could not markedly influence the growth of MCF-7 cells [13]. To further evaluate possible effects of the five selected polysaccharides on breast cancer via tumor immunotherapy, MCF-7 cells were treated with MCM prepared with the five selected polysaccharides at the indicated noncytotoxic concentrations of 0, 8, 40, and 200 μg/ml (Figure 4). e results showed that there was no significant difference in the viabilities of MCF-7 cells between cultured with MEM/EBSS (medium for the cancer cell line) and TCM medium (medium for mouse primary macrophages), indicating that TCM medium in the MCM did not influence MCF-7 cell growth. Paclitaxel (a treatment control) significantly (P < 0.05) inhibited MCF-7 cell growth as compared to that of negative control (cells alone either in MEM/EBSS or TCM medium) through 24 or 48 h incubation, implying that paclitaxel might be applied for breast cancer treatment. Importantly, the control group (MCM without polysaccharides) significantly (P < 0.05) inhibited the MCF-7 cell viability as compared to that of negative control through 24 or 48 h incubation, implying that secretions by macrophages, e.g., cytokines, might influence MCF-7 cell growth. Individually, MCM prepared with GSPS at 40 and 200 μg/ml significantly (P < 0.05) and dose-dependently enhanced their inhibitory effects on MCF-7 cell viabilities through 48 h incubation (Figure 4(a)). Incubation with MCM prepared with CBPS at 8 μg/ml for 24 h significantly (P < 0.05) enhanced its inhibitory effect on MCF-7 cell viabilities (Figure 4(b)). MCM prepared with BBPS at the indicated concentrations for 48 h significantly (P < 0.05) enhanced their inhibitory effects on MCF-7 cell viabilities (Figure 4(c)). MCM prepared with RFLPS at the indicated concentrations either through 24 or 48 h incubation significantly (P < 0.05) enhanced its inhibitory effects on MCF-7 cell viability ( Figure 4(d)). However, MCM prepared with YFLPS at the indicated concentrations could not enhance its inhibitory effects on MCF-7 cell viability either through 24 or 48 h incubation (Figure 4(e)). Among five selected polysaccharides, GSPS had the highest potential for anti-breast cancer via cancer immunotherapy by modulating macrophages secretion.
Recently, certain plant polysaccharides have been found to enhance or activate the immune response of macrophages. DDP1-1 obtained from Dendrobium denneanum polysaccharides enhances a curative effect on cancer and may be served as a novel anticancer drug [29]. SCP-60, a water-extracted crude polysaccharide from Sarcodia ceylonensis, exhibits a strong antitumor activity in S180 mice [30]. In the present study, our results suggest that MCM cultured with selected polysaccharides (except YFLPS) markedly enhanced their inhibitory effects on MCF-7 cell growth, indicating that these four polysaccharides, particularly GSPS, may be served as a novel potential anti-breast-cancer drug via tumor immunotherapy.

Association between MCF-7 Cell Viabilities and Cytokine
Levels in the Corresponding MCM. We presumed that cytokine secretions by macrophages in MCM might influence MCF-7 cell growth. To clarify association between  (Figure 5(j)). Even though low mutuality, our results evidence that increased M1/M2 cytokine ratios in the tumor microenvironment significantly inhibited the growth of human breast MCF-7 cancer cells. Increased M1/M2 cytokine ratios revealed that M1 macrophages might dominate in the MCM that enhanced inflammation via producing proinflammatory cytokines [7]. Increased inflammation status, namely, M1 macrophages dominate, may help to control cancer development in the early stage. We found that RFLPS, GSPS, YFLPS, and CBPS in order significantly (P < 0.05) increased M1/M2 cytokine secretion ratios, suggesting that these four selected polysaccharides treated alone enhanced macrophage activation and inflammation ( Figure 2). Consequently, particular polysaccharides including RFLPS, GSPS, YFLPS, and CBPS may increase the cell number of M1 macrophages in the tumor microenvironment and inhibit MCF-7 cell growth via increasing M1/M2 cytokine secretion ratios. Currently, cancer immunotherapy is becoming a viable alternative to traditional cancer treatment options [31,32]. Our results exhibited that cytokine immunotherapy, particularly M1-type cytokines, might be effective to inhibit human breast cancers (Figures 4 and 5). Numerous animal tumor model studies have demonstrated that cytokines have broad antitumor activity [33]. Intervention of polysaccharides to immune cells, particularly macrophages, may modulate their cytokine secretion profiles. Polysaccharides from Cymbopogon citratus (CCPS) have been found to inhibit the growth of transplanted S180 tumors, improve the immunity of tumor-bearing mice and increase IL-2, IL-6, IL-12, and TNF-α cytokine secretions, suggesting that CCPS may have an antitumor activity via modulating cytokine secretion [34]. In addition, traditional oriental medicine such as Portulaca oleracea L. has known for its immunestimulatory effect through upregulating inflammatory cytokines (IL-2, IL-12, TNF-α, and IFN-c) and NK cell activity in vitro [35]. In the present study, we demonstrated that MCM prepared with four selected polysaccharides including GSPS, CBPS, BBPS, and RFLPS enhanced their inhibitory effects on MCF-7 cell growth, possibly via cytokine immunotherapy. We hypothesized that particular polysaccharides might modulate cytokine secretion profiles of macrophages and subsequently change a microenvironment to inhibit tumor growth. Importantly, RFLPS and GSPS had the highest antitumor potential among the five selected      polysaccharides, via regulating macrophages cytokine secretion. Differentiated macrophages (M1 or M2) can be easily discriminated by their unique cytokine secretion profiles [36]. In the present study, we investigated the main functions of polarized macrophages (M1 or M2) in cancer diseases through correlating cytokine secretion profiles and MCF-7 cell growth. Our results provide the evidence to infer that increased M1 in the tumor microenvironment may inhibit breast cancer MCF-7 cell growth. Moreover, the polarization of different macrophages can be stimulated by adding certain stimuli such as polysaccharides.
e five selected polysaccharides have been partially characterized [13]. We presumed that these selected polysaccharides, particularly RFLPS and GSPS, are a mixture of nonstarch galactan, possibly proteopolysaccharides [13]. Macrophages in Peyer's patches may be activated by particular polysaccharides via certain receptors, such as glucan receptors on macrophages. Once immune cells are firstly activated by potent polysaccharides in the gut, indirect prevention or treatment of cancer by the activated immune cells may happen, namely, tumor immunotherapy.

Conclusions
All of the selected polysaccharides, except BBPS, significantly and dose-dependently increased (IL-1β + IL-6 + TNFα)/(IL-10) cytokine secretion ratios by macrophages, suggesting that these four selected polysaccharides have M1 polarization property and activate slight inflammation in macrophages. Treatments using the five selected polysaccharides in the presence of LPS significantly decreased (IL-1β + IL-6 + TNF-α)/(IL-10) cytokine secretion ratios in macrophages, suggesting that all five selected polysaccharide fractions, particularly GSPS, have anti-inflammatory potential. All MCM prepared with selected polysaccharides (except YFLPS) significantly (P < 0.05) enhanced their inhibitory effects on MCF-7 cell growth. A negative correlation was noted between MCF-7 cell viabilities and M1/M2 cytokine secretion ratios ((IL-6 + TNF-α)/IL-10) in the corresponding MCM, suggesting that increases in M1 macrophages in the tumor microenvironment might inhibit MCF-7 cell growth. Particular polysaccharides, including RFLPS, GSPS, YFLPS, and CBPS, may increase the percentage of M1 macrophages in the tumor environment to inhibit MCF-7 cell growth.

Data Availability
e data used to support the findings of this study available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Jin-Yuarn Lin took part in the experiment design, interpretation of the data, and the writing and review of the manuscript. Hsiao-Chien Lin conducted the experiments and analyzed the data. All authors read and approved the final manuscript.