Running Title: Antitumor Activity of Orally Administered Ascophyllan Inhibitory Effect of Orally Administered Sulfated Polysaccharide Ascophyllan Isolated from Ascophyllum nodosum on the Growth of Sarcoma-180 Solid Tumor in Mice ZEDONG JIANG1, RYOGO ABU1, SHOGO ISAKA1, SATORU NAKAZONO1, MIKINORI UENO1, TAKASI OKIMURA2, KENICHI YAMAGUCHI1 and TATSUYA ODA1
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Graduate School of Fisheries Science and Environmental Studies, Nagasaki University,
Nagasaki, Japan; 2
Research and Development Division, Hayashikane Sangyo Co., Ltd., Shimonoseki,
Yamaguchi, Japan
Correspondence to: Tatsuya Oda, Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan. Tel./Fax: +81 958192831, e-mail:
[email protected]
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Abstract. We evaluated antitumor activity of crude extract and ascophyllan prepared from Ascophyllum nodosum in sarcoma-180 solid tumor-bearing mice with continuous intraperitoneal (i.p.) administration at a dose of 50 mg/kg body weight/day or oral administration at a dose of 500 mg/kg body weight/day. Ascophyllan and crude extract administered via oral route showed greater antitumor effects than via i.p. route, and the tumor sizes in mice treated with ascopyllan and crude extract were reduced by a mean of 68.7±6.8% and 42.4±24.8% by oral route, and 41.4±16.1% and 13.6±20.6% by i.p. route compared to control mice. Splenic natural killer cell activity in the mice treated with ascophyllan and crude extract by i.p. route was significantly enhanced, while only a slight increase in this activity was observed in orally treated mice. Furthermore, increase in spleen weight of tumor-bearing mice was slightly suppressed by oral administration of ascophyllan, whereas i.p. administration resulted in further enlargement. Analysis of serum cytokines revealed that oral treatment with ascophyllan resulted in significant increase intumor necrosis factor- and interleukin-12 levels. Since ascophyllan showed no direct cytotoxic effect on sarcoma-180 cells, orally administered ascophyllan is suggested to exhibit its antitumor activity through the activation of the host immune system. Key Words: Ascophyllan, sarcoma-180 cells, antitumor activity, oral administration, intraperitoneal administration, host immune system.
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Cancer is one of the most serious diseases for human beings. Although the efficacy of modern medical treatments for cancer has been improved in recent years, the associated side-effects are still serious problems limiting the rehabilitation and the survival rate of patients (1, 2). Therefore, further efforts are required to discover new anticancer strategies such as utilization of natural products as medicines for a safe and feasible approach to cancer treatment. Certain seaweeds have long been used in traditional Chinese medicine for the cure of malignant tumor (3), and consumption of brown algae and their extracts may contribute to reducing the incidence of cancer, especially breast cancer in Asian countries (4). Hence, much attention has been focused on natural compounds isolated from brown algae. Yamamoto et al. reported that the non-dialyzable fractions consisting of polysaccharides from four brown seaweeds markedly inhibited sarcoma-180 solid tumor growth in mice (5, 6). They further reported that the main antitumor components against L-1210 leukemia in a mouse model were sulfated polysaccharides (7, 8). Similarly, crude fucoidan from Eisenia bicyclis has been found to exert immune-potentiating effects in sarcoma-180 tumor-bearing mice, leading to its antitumor effect (9). Furthermore, fucoidan purified from brown seaweed Sargassum thunbergii was reported to inhibit the growth of Ehrlich ascites carcinoma and the metastasis of Lewis lung carcinoma in mice through the enhancement of host-immune responses (10, 11). It has recently been reported that fucoidan isolated from Fucus vesiculosus showed antitumor activities against sarcoma-180, Lewis lung carcinoma and B16 melanoma in mouse models. It was proposed that the antiangiogenic activity of the fucoidan is partly responsible for the antitumor mechanisms, and the sulfation levels in the fucoidan molecule plays an important role in the antiangiogenic and antitumor activities (12). Ascophyllan, a sulfated polysaccharide isolated from brown alga Ascophyllum nodosum, is distinguishable from fucoidan in terms of its characteristic monosaccharide composition (13) and biological activity (14, 15). Interestingly, ascophyllan induced production of much higher levels of nitric oxide (NO) and cytokines, such as tumor necrosis factor- (TNF-) and granulocyte colony-stimulating factor (G-CSF), from RAW264.7 cells than those induced by fucoidans isolated from F. vesiculosus and A. nodosum (15). More recent study found that ascophyllan was capable of stimulating a respiratory burst in RAW264.7 cells (16). Furthermore, our previous studies found that 3
intraperitoneally administered ascophyllan showed antitumor activity in sarcoma-180 ascites tumor model (17). Similar to fucoidan (18), we have recently found that ascophyllan administered by intraperitoneal (i.p.) injection increased the natural killer (NK) activity of splenic lymphocytes (19). Thus, ascophyllan is considered to be a promising candidate as a cancer chemotherapeutic agent. In this study, we evaluated the antitumor activities of purified ascophyllan together with crude extract of A. nodosum in sarcoma-180 solid tumor-bearing mice with i.p. and oral administration routes. Materials and Methods Materials. Calcein-AM was obtained from Molecular Probes, Inc. (Eugene, OR, USA). Mouse TNF- enzyme-linked immunosorbent assay (ELISA) kit
mouse
interleukin-12 (IL-12) total ELISA kit were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Quantikine ELISA kit to mouse interferon- (IFN-) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Other chemicals were of the highest grade commercially available.
Preparation of crude extract and ascophyllan. Brown seaweed A. nodosum collected on the coast of Norway was obtained from KAISEI (Shimonoseki, Japan). Ascophyllan was prepared from A. nodosum as described previously (13). Crude extract was prepared by the following procedures. Milled A. nodosum was suspended in water and stirred at 100ºC for 1 h. The water extraction was repeated twice. After filtration, activated charcoal was added to the filtrate and stirred at 100ºC for 30 min. After removal of activated charcoal by filtration, the solution was subjected to spray drying, and the obtained powder was used as crude extract. The crude extract is currently commercially available as Ascophyllan HS from Hayashikane Sangyo Co. Yamaguchi, Japan. Ascophyllan and crude extract solutions were passed through endotoxin-removal filters (Zetapor Dispo: Wako Pure Chemical industries, Ltd, Osaka, Japan) before use. Molecular mass and composition analysis. Molecular mass of ascophyllan was estimated by a gel-filitration chromatography with a Sepharose 4B column (1.5 cm100 4
cm) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) as described previously (20). Composition of neutral monosaccharide in ascophyllan was analyzed by Alliance® High performance liquid chromatography (HPLC) system (Milford, MA, USA) equipped with GlyScope Honenpak C18 column (75 mm 4.6 mm I.D.) (J-OIL MILLS, INC., Tokyo, Japan) as previously reported (20). Uronic acids in ascophyllan were estimated by a modified carbazole-sulfuric acid method using galacuronic acid as a standard (20). The sulfate level of ascophyllan was measured with Dodgson-Price method, using K2SO4 as a standard, as reported previously (20). Animals and cell lines. Specific pathogen-free (SPF) male ddY mice (4 weeks old, 17.0-26.0 g) were obtained from Texam, Nagasaki, Japan. These mice were housed at a constant room temperature of 24℃ under controlled conditions of 12 h light/dark cycle photoperiod and provided with free access to standard laboratory food CE-2 (Texam, Nagasaki, Japan). All mice were treated according to the Guidelines of the Japanese Association for Laboratory Animal Science and the Guidelines for Animal Experiments of Nagasaki University, Japan. Murine sarcoma-180 cells obtained from the Institute of Development, Aging, and Cancer of Tohoku University were maintained in the abdominal cavity of 4-week-old SPF male ddY mice. A murine T-lymphoma cell line YAC-1, which is sensitive to the cytotoxic activity of naturally occurring killer cells in mice, obtained from the Institute of Development, Aging, and Cancer of Tohoku University, were cultured at 37°C in RPMI-1640 medium supplemented with 10% Fetal bovine serum (FBS), penicillin (100 IU/ml), and streptomycin (100 g/ml) in a humidified atmosphere with 5% CO2 and 95% air. Cytotoxicity assay. Cytotoxic effects of ascophyllan and crude extract on sarcoma-180 cells were measured by Alamar blue assay as described previously (14). Briefly, 10-day-old sarcoma-180 ascites cells, obtained from the abdominal cavity of ascites tumor-bearing mice, were washed twice with phosphate buffered saline (PBS) by centrifugation (270g) for 5 min at 4°C, and then seeded into 96-well plates (2104 cells/well) with different concentrations of ascophyllan or crude extract (0-1,000 g/ml by two-fold dilution) in RPMI-1640 medium supplemented with 20% FBS. After 48 h at 37°C, Alamar blue reagent was added to each well at a final concentration of 10%. 5
After 3 h incubation at 37°C, the absorbance of each well was measured at 535 nm with reference wavelength at 600 nm using a Multiskan GO microplate reader (Thermo Fisher Scientific K.K., Yokohama, Japan). In vivo antitumor assay. In vivo antitumor activities of ascophyllan and crude extract with different routes of continuous administration (i.p. or oral route) were evaluated in a sarcoma-180 solid tumor model as previously reported, with some modifications (21, 22). Ascophyllan and crude extract were dissolved in saline for i.p. or distilled water for oral administration. Administration of ascophyllan or crude extract i.p. (dose: 50 mg/kg b.w./day; n=10) and orally (dose: 500 mg/kg b.w./day; n=8) to mice were initiated two days and four days prior to tumor implantation, respectively. Twelve hours after final administration of each sample, 50 l of 10-day-old sarcoma-180 ascites tumor cells (1108 cells/ml) was intradermally inoculated into the dorsal area of test mice. Subsequently, ascophyllan or crude extract solution was consecutively administered to tumor-bearing mice via i.p. or oral route once daily for 10 consecutive days. Control tumor-bearing mice and normal mice were administered saline (for i.p. route; n=10) or distilled water (for oral route; n=8) on the same schedule. Mortalities and body weights of mice in each group were recorded every day from the beginning of ascophyllan and crude extract administration to sacrifice day. On day 11 after tumor transplantation, all mice were sacrificed, and then the solid tumor tissues were excised and weighed for evaluating the in vivo antitumor activity. The tumor volume was estimated according to the following formula: tumor volume (cm3) = xyz, where x, y, and z are the three perpendicular diameters of the tumor. Tumor growth inhibition ratio was calculated by following formula: inhibition ratio (%) = [(A-B)/A]100, where A is average tumor weight (WC) or volume (VC) of control tumor-bearing mice, and B is tumor weight (WT) or volume (VT) of mice treated with test sample (ascophyllan or crude extract). The major organs (liver, spleen, and kidney) were also excised and weighed at the same time. Splenic NK cell activity analysis. Three mice were selected randomly from each group, and splenic NK cell activity was measured by a method described previously (19). Briefly, 15 M of calcein-AM was added to the YAC-1 cells (1.5 106 cells/ml) in RPMI-1640 medium supplemented with 10% FBS, and the cells were incubated for 30 min at 37℃
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with gentle stirring. The calcein-loaded YAC-1 cells were washed twice with the medium and resuspended in the medium containing 2.5 mM of probenecid, an anion transporter inhibitor. Two hundred microliters of the calcein-loaded YAC-1 cells (106 cell/ml) was added to 200 l of splenic lymphocytes (5107 cells/ml) prepared from mice of each group at effector/target ratio of 50:1. After incubation at 37℃ for 4 h, the cells were centrifuged, then 40 l of supernatant was harvested and mixed with 500 l of 40 mM Tris-HCl (pH 8.0) buffer. The fluorescence intensity at 530 nm excited at 485 nm was then measured with a F-2500 fluorescence spectrophotomer (Hitachi High-Technologies Co., Tokyo, Japan) Analysis of cytokines in the serum. Blood was collected from the portal vein of each mouse, and serum was obtained by centrifugation. The levels of TNF-, IL-12, and IFN- in the serum of tumor-bearing mice and control mice were determined by sandwich ELISA with two antibodies to two different epitopes on TNF-, IL-12, or IFN- molecule according to the instructions of the manufacturers. The concentration of each cytokine was estimated from a reference to a standard curve for serial three-fold dilution of murine recombinant TNF-, IL-12, or IFN-. According to the manufacturers’ instructions, the detection limit of ELISA kit for TNF-, IL-12, and IFN- pg/ml, 12 pg/ml, and 2 pg/ml, respectively. Statistical analysis. All the experiments were repeated at least three times. The results are expressed as means±SE, and the data were analyzed using one-way ANOVA followed by t-test to determine significant differences. A value of p
