Degalactosylated/Desialylated Human Serum Containing GcMAF Induces Macrophage Phagocytic Activity and In Vivo Antitumor Activity

Materials and Methods

Preparation of GcMAF-containing human serum. Gc protein and GcMAF were prepared as previously reported by Uto et al. (17, 18). Human serum containing 1f1f-subtype of Gc protein (100 μl, 100 μg/μl) was obtained from human volunteers and each serum sample was incubated with 65 mU of β-D-galactosidase (from Escherichia coli; Wako, Osaka, Japan) and neuraminidase (sialidase, from Clostridium perfringens; Sigma-Aldrich Japan Co. Ltd., Tokyo, Japan) in 100 mM sodium phosphate buffer (pH 7.0) at 37°C for 3 h. The reaction mixture was heated at 60°C for 10 min and the solution was concentrated using a microcon concentration unit (10,000 MWCO; Nihon Millipore Co. Ltd., Tokyo, Japan). The protein concentrations were determined using a BCA protein assay kit (Pierce Chemical Co., Rockford, IL, USA).

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Figure 1.

Analysis of group-specific component protein-derived macrophage-activating factor (GcMAF) generated by the enzymatic treatment of human serum. A: Western blots probed with anti-human Gc globulin and Helix pomatia agglutinin (HPA) lectin. B: Quantification of the signal intensity of the detected protein bands was performed using JustTLC image analysis software.

Western blotting of GcMAF-containing human serum. The GcMAF-containing human serum was subjected to sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and subsequently electroblotted onto a nitrocellulose membrane. Non-specific binding was blocked by overnight incubation in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 and 1% bovine serum albumin (BSA) at 4°C. The membranes were then probed with anti-human Gc globulin (DakoCytomation Co. Ltd., Kyoto, Japan) and with biotin-conjugated Helix pomatia agglutinin (HPA) lectin (Sigma-Aldrich, St. Louis, MO, USA) specific for GcMAF with N-acetylgalactosamine (GalNAc) moiety. After membrane washing, the blots were incubated with horseradish peroxidase (HRP)-labeled anti-rabbit IgG (GE Healthcare Life Sciences, Uppsala, Sweden) as a secondary antibody. The blots were developed using an ECL western blotting detection system (GE Healthcare Life Sciences, Uppsala, Sweden) and visualization and quantification of the western blot bands was carried out with a LumiCube chemiluminescence analyzer and JustTLC image analysis software (Liponics, Tokyo, Japan).

Isolation and culture of mouse peritoneal macrophages. Mouse peritoneal adherent cells containing macrophages were collected from 8-week-old female ICR mice (Japan SLC, Hamamatsu, Japan), as previously reported by Uto et al. (17, 18) and cultured in 24-well plates at a density of 5×10⁵ cells/well in serum-free RPMI-1640 (Life Technologies, Carlsbad, CA, USA) for 1 h. The cultured cells were then washed three times with serum-free RPMI-1640 to separate adherent macrophages from non-adherent cells such as T- and B-cells. The cultured macrophages were treated with different concentrations of serum proteins for 15 h and used for phagocytosis assay as described below.

Phagocytosis assay. Mouse peritoneal macrophages were layered onto coverslips in a 24-well plate. After 3 h of human serum treatment, the cultures were assayed for phagocytic activity. Sheep red blood cells (SRBCs; Nippon Bio-Supp. Center, Tokyo, Japan) were opsonized by rabbit hemolytic serum (anti-sheep red blood cells; Cosmo Bio Co., Tokyo, Japan). Opsonized SRBCs (0.5%) in serum-free RPMI-1640 were overlaid onto each macrophage-coated coverslip and cultured for 1.5 h. The non-internalized erythrocytes were lysed by immersing the coverslip into a hypotonic solution [1/5-diluted phosphate-buffered saline (PBS)]. The macrophages were fixed with methanol, air-dried, and stained with Giemsa stain. The number of phagocytosed erythrocytes per cell was determined microscopically; 250 macrophages were counted for each data point. The data were expressed in terms of the phagocytosis index (PI), which was defined as the percentage of macrophages with ingested erythrocytes multiplied by the mean number of erythrocytes ingested per macrophage.

Animals. ICR female mice weighing 20-25 g were housed in polycarbonate cages under standard laboratory conditions (24±1°C, 12 h light/dark cycle) with food and water ad libitum. All procedures used for animal experimentation were approved by the Animal Research Committee of the University of Tokushima (TokuDobutsu12025). Ehrlich ascites carcinoma (EAC) cells were obtained from the Cancer Research Institute of Kanazawa University and were maintained in the peritoneal cavity of mice by injecting 0.1 ml of ascitic fluid every seven days. Ascitic tumor cell counts were carried out in a Neubauer hemocytometer using the trypan blue dye exclusion method. Tumor cell suspensions were prepared in PBS.

In vivo antitumor activity assay. Ten-week-old female ICR mice were inoculated with 1×10⁷ EAC cells (0.2 ml/mouse) intraperitoneally (i.p.). The day of tumor implantation was assigned as day ‘0’. On day 1, the animals were randomized and divided into five groups (n=5) and administered i.p. with PBS, GcMAF (40 ng/kg/d), or GcMAF-containing human serum (172, 517, and 1,552 μg protein/kg/d) for seven days. The animals with ascitic tumor were weighed every day. Kaplan-Meier survival curves were analyzed by the log-rank test.

Statistical analysis. Data are expressed as the mean and standard deviation. The statistical significance of the differences between the results of the independent experiments was analyzed using Student's t-test. A p-value of <0.05 was considered statistically significant.