1.

Introduction

The primary cause of cancer-related deaths is metastasis of tumor cells to secondary tissues [1, 2]. Metastasis is one of the characteristics that distinguish malignant tumors from neoplasms [3] and preventing metastasis is an important goal of cancer treatment. Innate immunity plays a crucial role in inhibiting primary tumor metastasis in clinical trials [4]. Among immune cells, macrophages and NK cells are considered efficient effectors against tumors [5]. Thus, activation of immune system-related cells including macrophages and NK cells may contribute to inhibition of tumor metastasis. The root of Panax ginseng C.A. Meyer (ginseng) has been used as one of the most valuable traditional medicines for over 2000 years in China, Korea, and Japan. Pharmacological and clinical studies carried out over the past 50 years focused on the radioprotective, antitumor, 1

and antiviral activity of ginseng [6]. Ginsenosides are the main ingredients of ginseng and were shown to modulate the immune system and inhibit tumor metastasis in animal models [7]. Numerous data supporting the above results have indicated that the biological and pharmacological effects of ginseng are associated with its root [8, 9]. Processing ginseng roots is expensive because the roots can only be harvested every 4-6 years; however, the leaves can be harvested annually. If ginseng leaves are confirmed to exhibit biological activity comparable to that of ginseng roots, they can be introduced to clinical use. To the best of our knowledge, ginsenoside is one of the active ingredients responsible for the pharmacological effects of ginseng; however, not all the biological activities of ginseng are attributable to ginsenosides. Recently, the chemical properties of polysaccharides isolated from ginseng have been reviewed; 35 polysaccharides were identified from ginseng, including 18 from roots, 16 from leaves, and one from fruits [10]. Several studies have reported the chemical properties of polysaccharides isolated from the leaves of ginseng. For example, Gao et al. identified watersoluble and alkaline-soluble polysaccharide fractions for the first time in 1989 [11, 12]. Kiyohara et al. (1994) isolated an anti-ulcer polysaccharide fraction GL-BIII, which is composed of rhamnose (Rha), arabinose (Ara), mannose (Man), galactose (Gal), glucose (Glc), galacturonic acid (GalA), and glucuronic acid (GlcA) at molar ratios of 3:4:2:10:1:7:4 [13]. Subsequently, Shin et al. (1997) reported a complex pectic polysaccharide (GL-4IIb2) comprising 15 different monosaccharides including rarely observed sugars such as 2-Omethylfucose, 2-O-methylxylose, apiose, 3-C-carboxy-5-deoxy-l-xylose (acetic acid, AceA), 3-deoxy-d-manno-2-octulosonic acid (Kdo), and 3-deoxy-o-lyxo-2-heptulosatic acid (Dha) [14]. Polysaccharides are known to possess low toxicity for normal tissues, and can stimulate nonspecific immune cells to protect the host from foreign antigens [15]. In addition, activation of macrophages and NK cells by plant-derived extracts was reported to control tumor growth and metastasis [16, 17]. Recently, we have reported the biological activities of plant-derived polysaccharides such as activation of peritoneal macrophages [18] and inhibition of tumor angiogenesis [19] and metastasis [20]. Therefore, in this study, we focused on polysaccharides isolated from ginseng leaves. We extracted the polysaccharide fraction (GS-P) and analyzed its chemical properties. Further, we investigated the effect of GS-P on lung metastasis using colon 26-M3.1 carcinoma cells, and on activation of macrophages and NK cells.

2. Materials and methods

2.1. Extraction of the antimetastatic polysaccharide from ginseng leaves Ginseng leaves were collected from Keumsan, Chungnam Province, Korea (late fall, 2013). As shown in Fig. 1A, chopped ginseng leaves (1 kg) were decocted with distilled water (20 L) for 6 h. After centrifugation (1130 g, 30 min), the pH of supernatant (GS-W) was adjusted to pH 6.0 with 1 N HCl. To remove hydrophobic and low-molecular-weight materials, the supernatant was purified on a buffered Diaion HP-20 column (Mitsubishi Chemical Co. Ltd., Japan) and eluted with distilled water. The eluents (GS-C1) were adjusted to pH 8.0 and further fractionated via anion exchange chromatography on a Diaion PA312 column (Mitsubishi Chemical Co. Ltd., Japan). The Diaion PA312 column was washed with the designated buffer (20 mM Tris-HCl, pH 8.0), and the absorbed polysaccharide fraction (GSC2) was eluted with 0.55 M NaCl buffer (20 mM Tris-HCl, pH 8.0). GS-C2 fraction was concentrated using a vacuum evaporator, after which 4 volumes of ethanol (EtOH) were added. The precipitate was washed twice using 95 % EtOH and lyophilized after dialysis with a Spectrapor MWCO 1000 (Spectrum Medical Industries Inc., CA, USA) to obtain the purified polysaccharide fraction (GS-P). 2

2.2.

Analytical methods

Total carbohydrate content was estimated using the phenol-sulfuric acid method, with galactose (Gal) as the standard [21]. Uronic acid content was determined using the mhydroxybiphenyl procedure, with galacturonic acid (GalA) as the standard [22]. Protein content was quantified by the Bradford assay with bovine serum albumin (BSA) as the standard [23]. The contents of 2-keto-3-deoxy-d-manno-2-octulosonic acid (Kdo) and 2-keto3-deoxy-d-lyxo-2-heptulosaric acid (Dha) were determined using the thiobarbituric acid (TBA) method [24]. Monosaccharide composition of the GS-P was determined by gas chromatography (GC; Young-Lin Co., Anyang, Korea) analysis of their alditol acetates [25]. GS-P was hydrolyzed with 2 M trifluoroacetic acid (90 min, 121 °C) and converted to alditol acetates as previously described [20]. Kdo, Dha, and uronic acid contents were determined by GC analysis of alditol acetates, according to the modified methods of York et al. [26] and Stevenson, Darvill and Albersheim [27]. The alditol acetates were analyzed using an SP-2380 capillary column (Supelco, Bellefonte, PA, USA). The content of component sugars was calculated from the peak area of corresponding alditol acetate derivatives, their molecular weights, and the FID response factors. Molecular weight of the GS-P was determined by high-performance-size exclusion chromatography (HPSEC) using a high-performance liquid chromatography system (Agilent 1260, CA, USA) as previously described [18-20]. Relative MW was calculated from curves obtained using pullulan standards (Showa Denko Co. Ltd., Tokyo, Japan).

2.3. Mice and cell cultures BALB/c mice (5–6 weeks old, female) were purchased from Orient Bio (Seongnam, Korea) and maintained in s specific pathogen-free (SPF) room at Kyonggi University. Animal experiments were performed according to the guidelines of the Ethics Committee for Use of Experimental Animals at Kyonggi University (2015-0001). Colon 26-M3.1 carcinoma cells were maintained in Eagle's minimum essential medium (Gibco, MA, USA) supplemented with 10 % fetal bovine serum (FBS) (ATCC, VA, USA), vitamin solution, non-essential amino acids, and 1 % penicillin-streptomycin. Splenic lymphocytes and peritoneal macrophages from BALB/c mice were maintained in RPMI-1640 medium (Gibco, MA, USA) containing 10 % FBS and 1 % penicillin-streptomycin.

2.4. Cytotoxicity assay To analyze cytotoxicity of GS-P, colon 26-M3.1 carcinoma cells were incubated with various concentrations of GS-P for 48 h. Cytotoxicity was evaluated using the Cell Counting Kit-8 assay (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Similarly, splenic lymphocytes isolated from BALB/c mice were incubated with the indicated concentrations of GS-P in 96well plates for 72 h. Lymphocyte proliferation was evaluated with the CCK-8 assay and the absorbance was measured at 450 nm using a microplate reader (Molecular Device Co., CA, USA).

2.5. Cytokine production in peritoneal exudate macrophages (PEMs) Peritoneal exudate macrophages (PEMs) were collected from thioglycolate-treated BALB/c mice. The cells (2 × 105 cells/well) were seeded in 48-well plates. After 2 h, in order to remove non-adherent cells, PEMs were washed twice with warm phosphate-buffered saline (PBS). Afterwards, the cells were treated with various concentrations of GS-P or lipopolysaccharide (LPS) for 18 h. Production of cytokines tumor necrosis factor (TNF)-α and 3

interleukin (IL)-12 was determined using ELISA kits (BD Biosciences, NJ, USA) according to the manufacturer's protocols.

2.6. Assessment of lung metastasis Lung metastasis of colon 26-M3.1 cells was evaluated by intravenous (i.v.) injection of cells to BALB/c mice. In order to evaluate the antimetastatic activity of GS-P, the mice were injected with GS-P (4–500 μg/mouse; 5 mice/group; i.v.) 2 days before tumor cell injection. The mice were sacrificed 14 days after tumor cell injection and the lungs were fixed in Bouin's solution (Sigma, SL, USA). Tumor colonies were counted under a microscope.

2.7. Macrophage-mediated tumor cell cytotoxicity Macrophage-mediated cytotoxicity assay was performed as described previously [28] with some modifications. In brief, PEMs were harvested from mice 3 days after injection of GS-P (4–500 μg/mouse; i.v.). Single-cell suspensions of PEMs were added to colon 26-M3.1 cells (1 × 105 cells/well) to obtain effector (macrophages)-to-target cell (colon 26) ratios (E:T ratio) of 20:1 or 10:1 in a sterilized U-bottomed 96-well plate. After 18-h incubation, the culture supernatant was collected by centrifugation (200 g, 5 min). Following this, the supernatant (50 μL) was mixed with the same volume of lactate dehydrogenase (LDH) solution (Promega Co., WI, USA) in a new plate. After 20 min of incubation, the plate was analyzed at 490 nm using a microplate reader. Percentage of macrophage cytotoxicity was calculated using the following formula: Cytotoxicity (%) = [(absorbance value of experimental group – absorbance value of control group)/(absorbance value of untreated group – absorbance value of control group)]×100

2.8. NK cell-mediated tumor cytotoxicity To analyze the effect of GS-P on activation of NK-cells in mouse splenocyte samples, GS-P was intravenously injected into BALB/c mice. After 3 days, splenocytes were collected from mice and co-cultured with YAC-1 cells (1 × 105 cells/well) to obtain an E:T (Splenocytes: Yac-1) ratio of 100:1, 50:1, or 25:1 in sterilized, U-bottomed, 96-well plates. After 6 h, supernatant was collected by centrifugation (200 g, 5 min) and combined with LDH reagent in a new 96-well plate. The LDH assay was performed as described in section 2.7. Percentage of NK cell cytotoxicity was calculated using the following formula: Cytotoxicity (%) = [(absorbance value of experimental group – absorbance value of control group)/(absorbance value of untreated group – absorbance value of control group)]×100

2.9. Depletion of NK cell activity in vivo Inhibition of NK cells in vivo was performed as previously described [20]. Briefly, 50-fold diluted anti-asialo GM1 antibody (Wako Pure Chemicals Industries, Ltd., Osaka, Japan) was injected intraperitoneally into BALB/c mice (500 μL/mouse) at 1 day and 3 days before colon 26-M3.1 cell injection. The mice were intravenously administered GS-P 2 days before tumor inoculation, sacrificed 14 days after colon 26-M3.1 inoculation, and their lungs were fixed with Bouin’s solution. The tumor colonies were counted under a microscope. 2.10. Statistical analysis Statistical analysis was performed using the Student’s t-test, with p < 0.05 and p < 0.01 considered statistically significant. The analysis was performed using SPSS software version 12.0 (SPSS Inc., Chicago, IL, USA).

3. Results 4

3.1.

Extraction of the polysaccharide from ginseng leaves

A water extract (GS-W) containing high-MW polysaccharides and several low-MW materials were isolated from ginseng leaves by hot water extraction. GS-W showed a relatively high total sugar content (yield: 1.412 % w/w). When GS-W was purified on a Diaion HP-20 column, sugar-rich unabsorbed fraction (GS-C1) was obtained. GS-C1 fraction was re-loaded onto a Diaion PA312 column and the absorbed fraction was collected, yielding the acidic polysaccharide fraction, GS-C2. GS-C2 was further refined by EtOH precipitation, washing, and successive desalting to produce the purified fraction, GS-P (Fig. 1A). The yield of GS-P was 0.48 % w/w raw ginseng leaves. As shown in Fig. 1B, GS-P was eluted as a single peak with slight tailing in HPSEC analysis, with MW estimated at 10.2 kDa (Fig. 1B). The GS-P fraction was composed of 56.4 % neutral sugars, 39.8 % uronic acids, and 3.8 % Kdo-like materials, with no proteins detected. Moreover, the GS-P fraction comprised 15 kinds of monosaccharides, containing unusual sugars including 2-methylfucose, 2methylxylose, apiose, aceric acid, Kdo, and Dha (Table 1). These data suggest that GS-P has an RG-II polysaccharide component, as Kdo and Dha have only been found in rhamnogalacturonan-II (RG-II) in plant-derived polysaccharides [24].

3.2. Inhibitory effect of GS-P on lung metastasis using colon 26-M3.1 cancer cells To investigate the effect of GS-P on tumor metastasis, BALB/c mice were injected with various concentrations (4−500 μg/mouse) of GS-P 2 days before inoculation with colon 26M3.1 cells. As shown in Table 2, administration of GS-P (20−500 μg/mouse) significantly inhibited lung metastasis, and a low dose (20 μg/mouse) of GS-P caused slight inhibition. However, no inhibition of lung metastasis was observed with a GS-P dose of 4 μg/mouse. From this data, the optimal dose range of GS-P required to inhibit tumor metastasis is estimated to be 100-500 μg/mouse. Furthermore, no side effects of GS-P treatment, such as decreased body weight, were observed (data not shown). These findings indicate that GS-P reduced metastasis of colon 26-M3.1 cells.

3.3. Cytotoxicity assay We investigated GS-P cytotoxicity against tumor and normal cells. As shown in Fig. 2A, GSP did not affect the growth of colon 26-M3.1 tumor cells at concentrations up to 500 μg/mL (Fig. 2A) and induced proliferation of BALB/c mice splenocytes in a concentration-dependent manner (Fig. 2B). LPS was used as a positive control for proliferation of splenocytes. These results indicate that inhibition of tumor metastasis by GS-P is not caused by a cytotoxic effect on colon 26-M3.1 cells and that GS-P enhances the function of immune cells (Fig. 2).

3.4. GS-P-induced cytokine production in Peritoneal We analyzed the effects of GS-P treatment on macrophage activation and cytokine production. As shown in Table 3, GS-P treatment (10−100 μg/mL) induced the production of TNF-α and IL-12 in PEMs. LPS was used as a positive control for cytokine production in PEMs. These results suggest that GS-P-induced activation of macrophages and cytokine production may enhance macrophage-mediated cytotoxicity against tumor cells.

3.5. GS-P-induced macrophage-mediated cytotoxicity against tumor cells Activated macrophages are important effectors in innate immunity and can inhibit and disrupt tumor growth and metastasis [3, 4]. Macrophages are activated for tumoricidal activity by cytokines or various other stimuli, including herbal medicines and bacterial products [16, 28]. 5

Therefore, we investigated whether GS-P mediated macrophage activation to achieve tumoricidal activity. Therefore, we evaluated the level of lactate dehydrogenase (LDH), a cytosolic enzyme released from damaged cells, in the cell supernatant. A high level of LDH indicates a high tumoricidal activity by macrophages (effector cells) against colon 26-M3.1 (target cells). As shown in Fig 3, PEMs harvested from mice treated with GS-P (4−500 μg/mouse) showed increased cytotoxic activity against colon 26-M3.1 cells. Further, 100 μg/mouse of GS-P injected group exhibited the highest cytotoxicity against colon 26-M3.1 at different E/T ratios (PEMs:colon 26-M3.1). Collectively, these results indicate that GS-P activates macrophages, which in turn may inhibit lung metastasis of tumor cells.

3.6. Effects of GS-P on activity of mouse NK cells As NK cells participate in suppressing tumor growth and metastasis [30], we investigated the effects of GS-P on NK cell activity. As shown in Fig. 4A, splenocytes from GS-P-treated mice (20−500 μg/mouse) showed increased NK activity. An E/T ratio (splenocytes: Yac-1)dependent increase in NK activity was seen in the GS-P dose range of 20–500 μg/mice, whereas a decrease was observed at a dose of 4 μg/mouse. Next, we used the anti-asialo GM1 antibody to determine if NK cell activation was directly involved in GS-P-mediated inhibition of lung metastasis. An anti-asialo GM1 antibody is commercially used to inhibit NK cell function. As shown in Fig. 4B, lung metastasis was inhibited in GS-P-treated mice (3rd bar) compared to that in untreated control (1st bar). Pretreatment of mice with the anti-asialo GM1 antibody increased lung metastasis (2nd bar) compared to that reported for the control. However, GS-P treatment decreased lung metastasis even with the anti-asialo GM1 antibody pretreatment (4th bar). These data indicate that GS-P-induced NK cell activation partly contributed to antimetastatic activity.

4. Discussion Among polysaccharides commonly found in plants, pectic polysaccharides are the most structurally complex. These compounds comprise 1, 4-linked α-GalA residues (homogalacturonan, HG) glycosidically interlinked with substituted galacturonans (e.g., rhamnogalacturonan-I (RG-I) and RG-II) [26, 27]. RG-I is composed of a rhamnogalacturonan core with neutral carbohydrate side chains, such as arabinans, galactans, and arabinogalactans [28]. RG-II is a low molecular mass (5-10 kDa) polysaccharide that contains 11 different sugar residues linked together by more than 20 different glycosyl linkages [34]. For several decades, many studies reported that pectic substances isolated from plants exhibit pharmacological activities such as stimulating the immune system and activating the complement [15]. However, pharmacological activities have been associated with the RG-I and RG-II regions, not the HG region [35, 36]. It is well known that ginseng root and its purified constituents, such as ginsenosides or glycoproteins, enhance immune responses and reduce growth or metastasis of cancers in animal models [8-9, 17]. In contrast, there is little evidence of immune-stimulating and antitumor activity of ginseng leaves in vitro or in vivo. In this study, we demonstrated that the polysaccharides from ginseng leaves inhibit lung metastasis of colon 26-M3.1 carcinoma cells via activation of innate immune cells, macrophages, and NK cells. Our data from in vivo experiments showed that i.v. administration of GS-P inhibited lung metastasis of colon 26M3.1 carcinomas. As shown in Fig. 2A, GS-P did not significantly induce colon 26-M3.1 cell death at up to 500 μg/mL, suggesting that GS-P is not cytotoxic, and that its antitumor activity is not based on direct induction of tumor cell apoptosis. GS-P enhanced the proliferation of mouse splenocytes (Fig. 2B), suggesting that GS-P is a potent non-specific activator of immune cells. Therefore, we next focused on GS-P-induced stimulation of the innate immune system. 6

Activated macrophages and NK cells are important effector cells of innate immunity [4, 37]. GS-P treatment increased macrophage production of cytokines, including TNF-α, and IL-12. Inflammatory cytokines such as IL-1β and TNF-α produced by macrophages play a role in activating T cells and rejecting tumor cells [30]. In addition, at early stages of the immune response, macrophages and dendritic cells secrete IL-12, which is the most important cytokine in tumor-targeting immunity [38, 39]. IL-12 was shown to have potent antitumor and antimetastatic activity in animal models and is induced by activation of effector cells. The effects of IL-12 are most likely mediated by interferon-gamma (IFN-γ), mainly in NK cells [38]. Although many previous studies have reported rejection of tumors by activated macrophages in vivo, the underlying mechanisms remain unclear. Macrophages have also been reported to increase tumor growth or metastasis [40, 41]. These contradictory views of macrophages are widely debated and remain unreconciled [39]. In this study, peritoneal macrophages obtained from GS-P-treated mice showed higher cytotoxic activity against colon 26-M3.1 cells, than control (Fig. 3), suggesting that GS-P activates macrophages and induces cytokines in vivo, resulting in increased macrophage-mediated cytotoxicity towards tumor cells. Activation of NK cells by immunostimulants was reported to inhibit metastatic colonization of tumors. In this study, splenic NK cell activity in GS-P-treated mice revealed that GS-P markedly enhanced cytotoxic activity against YAC-1 cells (Fig. 4A). Furthermore, inhibition of NK cell activation by pretreatment with an anti-asialo GM1 antibody partly suppressed the antimetastatic effects of GS-P (Fig. 4B). These results suggest that cooperative activation of macrophages and NK cells is essential in inducing GS-P antitumor activity.

5. Conclusion Plant-derived polysaccharide is a good candidate for therapeutic development, because it exhibits relatively low toxicity and possesses pharmacological activities such as enhancement of immune response and reduction of tumor size and metastasis. However, many studies are focused on its efficacy, and not its chemical properties and underlying mechanisms. The biological activity of polysaccharides is related to its primary structure; monosaccharide composition, molecular weight, and type of glycosidic linkage [42, 43]. In this study, we describe the isolation of pectic polysaccharide from ginseng leaves and analyze its chemical composition and properties (molecular weight and sugar composition). In addition, we evaluated the antimetastatic activity of GS-P and showed that the antitumor effects were associated with the activation of macrophages and NK cells in vivo. The results of this study suggest that GS-P is a potential candidate for anticancer therapy. ACKNOWLEDGEMENT This work was supported by a grant from the Korean Food Research Institute (2013) and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2014R1A1A3050928). References [1] I.J. Fidler,;1; Cancer metastasis, Brit. Med. Bull. 47 (1991) 157-77. [2] L.A. Liotta, P.S. Steeg, W.G. Stetler-Stevenson,;1; Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation, Cell. 64 (1991) 327-336. [3] H. Rubin,;1; The significance of biological heterogeneity, Cancer Metastasis Rev. 9 (1990) 1-20. 7

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Fig. 1. Isolation and characterization of the polysaccharide fraction from ginseng leaves. A schematic representation of the isolation of polysaccharides from ginseng leaves, and their purification by two-successive column chromatographic separations (A). High-performance size-exclusion chromatography of the polysaccharide fraction, GS-P, isolated from ginseng leaves (B).

Fig. 2. Effect of GS-P on the growth of tumor cells and splenocytes. Colon 26M3.1 cells were co-cultured with or without the indicated concentrations of GS-P in 96-well plates for 48 h (A). Splenocytes from BALB/c mice were co-cultured with various concentrations of GS-P in 96-well plates for 72 h (B). Cytotoxicity against tumor cells or splenocyte proliferation was evaluated using a CCK-8 kit, as described in the materials and methods section. LPS (1 μg/mL) was used as a positive control for splenocyte proliferation activity.

Fig. 3. Effect of GS-P on the enhancement of peritoneal exudate macrophage (PEM) activity. PEM tumoricidal activity was determined by an LDH assay as described in the materials and methods section. *p<0.05, **p<0.01 as compared to control group.

Fig. 4. Effect of GS-P on the activation of mouse NK cells. NK cell activity was determined by an LDH assay as described in the materials and methods section (A). To deplete NK cells in vivo, mouse anti-asialo GM1 antibody was injected into mice, 1 and 3 days before inoculation of colon 26-M3.1 carcinoma cells. Mice were treated i.v. with GS-P (100 μg/mouse) 2 days before tumor inoculation. Mice were sacrificed 14 days after tumor inoculation for evaluation (B). *p<0.05, **p<0.01 as compared to each control group. Table 1. Chemical composition and monosaccharide constituents of the purified polysaccharides fraction (GS-P) from ginseng leaves. Chemical properties

GS-P

Molecular weight of HPSEC Yield from ginseng leaves

10.2 kDa 0.48% 11

Chemical composition

GS-P (%)

Neutral sugar Uronic acid Protein KDO-like material Component sugar1) 2-Methylfucose Rhamnose Fucose 2-Methylxylose Arabinose Xylose Apiose Aceric acid

56.4 39.8 0.0 3.8 GS-P (Mole%)2)

Component sugar1)

3.4 10.2 3.1 3.7 14.4 0.9 3.3 2.1

Mannose Galactose Glucose Galacturonic acid Glucuronic acid KDO3) DHA4)

GS-P (Mole%)2) 0.7 11.8 2.8 37.3 2.5 2.8 1.0

1)

Calculated from the peak areas and the molecular response factors of each alditol acetate in

GC. 2)

Mole% calculated from the detected total carbohydrate.

3)

KDO refers to 2-keto-3-deoxy-d-manno-2-octulosonic acid.

4)

DHA refers to 2-keto-3-deoxy-d-lyxo-2-heptulosaric acid.

Table 2. Inhibitory effect of GS-P on lung metastasis by i.v. inoculation of colon 26M3.1 carcinoma cells. GS-P (μg/mouse) Control

Number of lung metastases Mean ± SD (inhibition %) 59.4 ± 16.1

Range 42–85

500

2.4 ± 0.9 (96.0)

1-3

100

2.4 ± 2.5 (96.0)

0-5

20

18.4 ± 6.2 (69.0)

9–25

4

56.8 ± 15.5 (4.4)

42–78

Groups of five BALB/c mice were administered i.v. with the indicated doses of GS-P two days before i.v. inoculation of colon 26-M3.1 cells (3×104 cells/mouse). Mice were sacrificed 14 days after tumor inoculation for evaluation. Table 3. Effect of GS-P on cytokine production in peritoneal exudate macrophages (PEMs).

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Group Control LPS GS-P

Dose (μg/mL) 5 10 100

TNF-α (pg/mL) 103 ± 14 1148 ± 517 2459 ± 771 11058 ± 1316

IL-12 (pg/mL) 1.1 ± 0.4 243.3 ± 10.5 227.7 ± 6.6 300.9 ± 15.6

PEMs were harvested from 3% thioglycollate-treated BALB/c mice. The cells suspended in culture medium were plated onto 24-well culture plates and the non-adherent cells were removed after 2 h of incubation. The macrophages were co-incubated with the indicated concentration of GS-P or LPS for 24 h. The concentration of each cytokine in the cultured supernatants was determined by ELISA. TDENDOFDOCTD

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