Fucoidans Stimulate Immune Reaction and Suppress Cancer Growth

Materials and Methods

Materials. Individual samples were purchased from the manufacturers or distributors as shown in Table I. RPMI 1640 medium, sodium citrate, 4-(2-hydroxyethyl)-1-piperazine-ethanol-sulfonic acid (HEPES), antibiotics, Wright stain, Freund's adjuvant, ovalbumin, Concanavalin A (Con A) and lipopolysaccharide (LPS) were purchased from Sigma (St. Louis, MO, USA). Fetal calf serum (FCS) was from Hyclone Laboratories (Logan, UT, USA).

Cells. J774A.1 mouse macrophages and YAC cells were obtained from the ATCC (Manassas, VA, USA). The BALB/c mouse-derived mammary tumor cell line Ptac64 was generously provided by Dr. Wei-Zen Wei of the Michigan Cancer Foundation, Wayne State University, Detroit, MI, USA. The cell lines were maintained in RPMI 1640 medium containing HEPES buffer supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, in plastic disposable tissue culture flasks at 37°C in a 5% CO₂/95% air incubator.

Animals. Female, 8-week-old BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All animal work was performed according to the University of Louisville Institutional Animal Care and Use Committee (IACUC) protocol. Animals were sacrificed by cervical dislocation.

Phagocytosis. The technique employing phagocytosis of synthetic polymeric microspheres was described in Vetvicka et al. (16, 17). Briefly, peripheral blood cells were incubated in vitro with 0.05 ml of 2-hydroxyethyl methacrylate particles (HEMA; 5×10⁸/ml). The test tubes were incubated at 37°C for 60 min, with intermittent shaking. Smears were stained with Wright stain. Cells with three or more HEMA particles were considered positive. Mice were injected intraperitoneally with individual samples or phosphate-buffered saline (PBS; control). All experiments were performed in triplicate. At least 200 cells in 60 high-power fields were examined in each experiment. In in vitro experiments, cells were incubated with 0.01 ml of HEMA particles/well. Incubation and staining was identical to in vivo experiments.

Mitogenic response assay. For the mitogenic response assay, splenic lymphocytes (5×10⁵ per well) were co-cultured with tested samples (10 μg) and either 10 μg of Con A or 1 μg of LPS in 200 μl of RMPI 1640 plus 10% FCS for 72 h. Proliferation was evaluated using the Biotrak cell proliferation ELISA system (GE Healthcare Bio-Sciences, Pittsburg, PA, USA).

In vitro cytotoxicity assay. Spleen cells were isolated from mice by standard methods. Cell suspension was generated by pressing minced spleen against the bottom of a petri dish containing PBS. After erythrocyte elimination by 10-second incubation in distilled water and five washes in cold PBS, cells were re-suspended in PBS and counted. Viability was determined by Trypan blue exclusion. Only cells with viability better than 95% were used in subsequent experiments. Splenocytes (10⁶/ml; 0.1 ml/well) in V-shaped 96-well microplates were incubated with individual samples (2 μg/ml) for 30 min at 37°C and then washed three times with RPMI 1640 medium. After washing, 50 μl of target cell line YAC-1 (two different concentrations of target cells were used so the final effector-target ratio was 32:1 and 64:1, respectively). After spinning the plates at 250 × g for 5 min, the plates were incubated for 4 h at 37°C. The cytotoxic activity of cells was determined by the use of CytoTox 96 Non-Radioactive Cytotoxicity Assay from Promega (Promega, Madison, WI, USA), according to the manufacturer's instructions. Briefly, 10 μl of lysis solution was added into the appropriate control wells 45 min before the end of incubation. The next step was to spin the plates at 250 × g for 5 min, followed by transferring 50 μl of supernatant into flat-bottomed, 96-well microplates. After 50 μl of reconstituted substrate was added into each well, plates were covered and incubated for 30 min at room temperature at dark. The optical density was determined by using a STL ELISA reader (Tecan U.S., Research Triangle Park, NC, USA) at 492 nm. Specific cell-mediated cytotoxicity was calculated using the formula: Percent-specific killing (% cytotoxicity)=100×[(OD492 experimental - OD₄₉₂ spontaneous) divided (OD₄₉₂ maximum - OD₄₉₂ spontaneous)] as described in manufacturer's instructions, where spontaneous release was target cells incubated with medium alone and maximum release was obtained from target cells lysed with the solution provided in the kit.

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Figure 1.

Effects on phagocytosis by peripheral blood monocytes by orally administered individual samples against phosphate-buffered saline (PBS). Each value represents the mean±SD. *Represents significant differences between control (PBS) and tested samples at p≤0.05 level. All experiments were performed in triplicate.

Macrophage inflammatory assay. One hundred thousand of J774A.1 mouse macrophages were incubated in RPMI 1640 medium supplemented with 10% FCS in 12-well tissue culture plates. The cells were incubated with either 5 μg/ml of LPS or with LPS plus tested samples for 18 h. The supernatant was collected, filtered and stored at −80°C. Interleukin (IL)-10 and tumor necrosis factor (TNF)-α levels were determined using an ELISA and Quantikine mouse IL-10 or TNF-α kit (R&D Systems, Minneapolis, MN, USA), respectively.

Antibody formation. Formation of antibodies was evaluated using ovalbumin (OVA) as an antigen. Mice were injected twice (two weeks apart) with 0.1 mg of OVA and the serum was collected 7 days after last injection. Experimental groups received daily intraperitoneal injections of tested material. ELISA detected the level of specific antibodies against OVA. As a positive control, a combination of OVA and Freund's adjuvant was used.

Breast cancer model. Mice were injected directly into the mammary fat pads with 1×10⁶/mouse of Ptas64 cells in PBS. The experimental treatment begun after palpable tumors were found (usually 14 days after injection of cells) and after mice were assigned to experimental groups. Experimental treatment was achieved by intraperitoneal injections of tested samples diluted in PBS (once/day for 14 days). After treatment, the mice were sacrificed, tumors removed and weighed (18).

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Figure 2.

Effects on formation of antibodies against ovalbumin (OVA) by orally administered individual samples. Animals were injected twice (two weeks apart) with antigen. The serum was collected 7 days after last injection. Level of specific antibodies against ovalbumin was detected by ELISA. As positive control, Freund's adjuvant (FA) was used. *Represents significant differences between control (OVA alone) and samples at p≤0.05 level.