Previous results showed that transfection of the α1, 2-FT gene into RMG-I cells resulted in a 20–30 fold increase in cellular α1, 2-FT activity [10]. We here verify the expression of LeY on cells. Laser confocal microscopy revealed that the expression of LeY on the surface of RMG-I-H cells was obviously higher than in RMG-I and RMG-I-C cells (Figure 1A). Immunohistochemical assays were performed to examine the expression of LeY in nude mouse xenograft tumors formed from subcutaneous injection with RMG-I, RMG-I-C and RMG-I-H cells. Similarly, the results showed that RMG-I-H cells had a higher expression of LeY than the two control groups (P < 0.05) (Figure 1B), indicating that the α1, 2-FT gene transfection increased the expression of LeY.

Since proliferation effectively reflects the malignancy of tumors, studying proliferation is an important way to understand the biological behavior of tumors. Using the MTT method, we detected absorbance values (490 nm) of cells at different time points after treatment with LeY mAb or the control IgM antibody. The results showed that the proliferation in LeY mAb-treated groups all decreased compared with that in the control IgM antibody-treated groups, among which the LeY mAb-treated RMG-I-H cells had the most obvious change (P < 0.05) (Figure 2). The inhibition rates of proliferation from day 1 through day 7 were 3.13%, 5.59%, 19.71%, 45.73%, 57.80%, 48.92% and 33.38%, respectively. The highest inhibition rate was on day 5, which was close to 60%. These results indicate that elevated expression of LeY led to an increase in the number of RMG-I cells when they were cultured in vitro.

This experiment was performed to determine the tumorigenic ability of the cell lines in nude mice. The results showed that the latent period of tumor growth was 8.8 ± 1.3 d and 8.6 ± 0.5 d in RMG-I cells and RMG-I-C cells, respectively, with no significant difference (P > 0.05), whereas the latent period of tumor growth was 5.2 ± 0.8 d in RMG-I-H cells, significantly less than that in the control groups (P < 0.01). The xenograft tumor volume was significantly larger in the RMG-I-H group compared to the control groups (P < 0.01) (Figure 3A). Similarly, tumor weight in the RMG-I-H group (2038.8 ± 241.9 mg) was also significantly higher than in the control RMG-I (696.8 ± 73.3 mg) and RMG-I-C (707.2 ± 21.8 mg) groups (P < 0.01) (Figure 3B). However, we found no difference between the RMG-I cells and RMG-I-C cells in growth potential in vivo. To further determine if tumor formation by RMG-I-H is dependent on LeY antigen, cells were inoculated after preincubation with LeY mAb. The results shown in Figures 3 (Figures 3C and 3D) illustrate that tumor formation was significantly inhibited by LeY mAb. These results demonstrate that LeY antigen enhances the tumorigenicity of RMG-I cells in vivo.

This experiment was performed to compare the adhesion of cells with or without LeY mAb treatment. Inverted microscopy revealed that cells in LeY mAb treated groups failed to spread or spread more slowly, with shorter and smaller cell bases according to morphological analysis. After the same culture time, the number of adhered cells in LeY mAb-treated RMG-I-H groups was decreased obviously compared with that in the control IgM antibody-treated groups (P < 0.05) (Figure 4), and the inhibition rates of adhesion were 53.17%, 61.50% and 50.93% at 10, 30 and 60 min, respectively. The results indicate that the LeY antigen is involved in the adhesion of ovarian cancer RMG-I cells.

RMG-I, RMG-I-C and RMG-I-H cells treated with different concentrations of LeY mAb (1 μg/mL, 5 μg/mL, 10 μg/mL and 20 μg/mL) were subjected to invasion assays using a transwell cell culture system. Matrigel were coated on the upper face of the transwell. The results suggested that there was no detectable difference in invasion between LeY mAb treated groups and control antibody treated groups (P > 0.05).

VEGF, b-FGF and TGF-β1 are growth factors with multiple biological functions that play important roles in enhancing tumor cell proliferation, angiogenesis and tumor development [17–19]. To gain insight into the mechanism for enhanced proliferation, tumorigenicity and adhesion by LeY antigen, we examined the expression of TGF-β1, VEGF and b-FGF in both nude mouse xenograft tumors and cells cultured in vitro by immunohistochemical staining and Western blot analysis, respectively. The results showed that the expression of TGF-β1, VEGF and b-FGF were all increased in RMG-I-H cells (P < 0.05) (Figure 5A and 5B), suggesting that the LeY is likely to enhance tumor cell proliferation and adhesion by promoting the secretion of these cell growth factors.

Mouse anti-human LeY mAb (clone A70-C/C8) [30] was purchased from Abcam (England). Rhodamine (TRITC)-conjugated AffiniPure goat anti-mouse IgG (H+L) was purchased from Zhongshan Biotechnology (China). Mouse anti-human IgM antibody, MTT reagents and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma (U.S.). The transwell (two vertical chambers separated by an 8 μm layer of polycarbonate membrane) was purchased from Corning (US). Matrigel was from BD Biosciences (US). Rabbit anti-human VEGF, rabbit anti-human b-FGF and rabbit anti-human TGF-β1 polyclonal antibodies were from Santa Cruz (US). The immunohistochemical SABC kit was purchased from Boster Biological Technology, Inc. (Wuhan, China).

The RMG-I cell line, which was originated from ovarian clear cell carcinoma, donated by Professor Iwamori Masao of Tokyo University of Japan. RMG-I-H is the cell line with a high expression of α1,2-FT and LeY antigen, and was established as previously reported [10,11]. RMG-I-C was the cell line transfected with the vector alone. The cells were cultured in DMEM (high-glucose) media (Invitrogen, Carlsbad, CA) containing 10% FBS (Clark, Australia) at 37 °C in a 5% CO₂ incubator.

The cells were seeded on a slide and fixed with 4% paraformaldehyde. The mouse anti-human LeY mAb was diluted at 1:100 as the working primary antibody; the goat anti-mouse TRITC fluorescence-conjugated secondary antibody was diluted at 1:200 as the working secondary antibody. After blocking with normal goat serum for 30 min, the cells were incubated with the working primary antibody at 37 °C for 1 h and then left at 4 °C overnight. After washing with PBS, the cells were incubated with the working secondary antibody at 37 °C for 100 min. DAPI was then used to stain the nuclei at room temperature for 5 min. The stained slide was observed under a laser confocal microscope (C1-SI; Nikon, Tokyo, Japan). Data were collected with a computer to generate digital images.

The cells were seeded in a 96-well plate at 2 × 10⁴ cells/well (three replicates), incubated at room temperature for 2 h, and then cultured in DMEM (with 10% FCS) containing LeY mAb (10 μg/mL) as previously described [31]. Mouse anti-human IgM antibody was added as control. Cells were cultured in 5% CO₂ at 37 °C for 72 h, and then 20 μL of 5 mg/mL MTT was added. After 5 h of subsequent culture, 150 μL of DMSO was added to terminate the reaction, and the absorbance value was detected at 490 nm using a microplate reader (Elx808; Bio-Tek Instruments, Vermont, USA).

Ethical approval was obtained from China Medical University animal ethics committee before the start of the study. Fifteen 5–6 week old healthy female nude mice (Balb/c nu/nu, purchased from Liaoning Experimental Animal Center) were randomized into three groups and given a dorsal subcutaneous injection with RMG-I, RMG-I-C or RMG-I-H single cell suspensions (10⁷ cells/0.3 mL). In the inhibition experiments, 10⁷ cells were first preincubated with LeY mAb or IgM antibody (10 μg/mL) as previously described [31]. Cells were then washed with PBS and resuspended in 0.3 mL of serum-free DMEM media. These cells were incubated subcutaneously as described above. Animals were raised under SPF conditions and their general status was periodically monitored. The length (a), width (b) and height (c) of the tumors were measured by Vernier caliper, and the approximate tumor volume (V) was calculated by the formula V = π/6(abc). For small tumors, the height (depth) was roughly equal to the smaller value between a and b. The nude mice were all sacrificed 50 days later, and the tumors were isolated and weighed. The tumor tissues were then fixed in 10% formaldehyde, embedded in paraffin and cut into sequential slices (4 μm).

LeY mAb (10 μg/mL) and the control IgM antibody were added to single-cell suspensions, respectively. The cells were incubated at 37 °C for 30 min and then seeded into Petri dishes (35 mm) at 3 × 10⁵ cells/dish. Three dishes were randomly placed into each group after 10, 30, and 60 min of culture, and were then washed with PBS to remove non-adherent cells. Adherent cells were digested off and counted. The inhibition rate (IR) was calculated by the following formula:

The bottom of the upper chamber was covered with a layer of matrigel to form a bottom barrier and dried overnight. 600 μL of 20% cell culture medium was added to the lower chamber, and 100 μL of cell suspension (1 × 10⁶/mL) was added to the upper chamber. After 2 h of incubation in 5% CO₂ at 37 °C, LeY mAb was added to both the upper and the lower chambers (at a final concentration of 1, 5, 10 or 20 μg/mL). Cells that did not migrate through the membrane were removed, and the cells that migrated to the lower face of the membrane were fixed with methanol followed by staining with Giemsa. The numbers of cells on the lower face were counted using a high-power field under microscope. There were three replicates at each concentration from each group, and the experiment was repeated three times.

The tissue sections were processed by the general deparaffinization and hydration procedure, and were then soaked in freshly prepared 3% hydrogen peroxide for 10 min. After antigen retrieval, the tissue sections were incubated in normal goat serum for 30 min. Primary antibodies (1:100) were then added and incubated at 4 °C overnight. Biotin-conjugated secondary antibodies were added, followed by 20 min incubation at 37 °C. The SABC reagent was then added, followed by 30 min incubation at 37 °C. The sections were developed by DAB and monitored under the microscope. After counterstaining with hematoxylin, the slides were dehydrated, cleared, mounted, and observed under a microscope.

Cells were rinsed with PBS, and 1% of Triton X-100 lysis buffer (20 mM Tris-HCl, pH 7.4, 10 mM EGTA, 10 mM MgCl₂, 1 mM benzamidine, 60 mM β-glycerophosphate, 1 mM Na₃VO₄, 20 mM NaF, 2 μg/mL aprotinin, 5 μg/mL leupeptin, 0.1 mM phenylmethylsulfonyl fluoride) was added. The suspension was centrifuged, and the supernatants were collected. Protein content was measured using the protein assay BCA kit (Beyotime biotechnology, China), and equal amounts of protein were loaded on SDS-PAGE gels. Subsequently, proteins were transferred to PVDF membranes (Millipore, Beaford, MA) and were probed with antibodies (1:1000). Immunoreactive bands were visualized by chemiluminescence (ECL; Pierce) using a secondary antibodies (1:8000).

The SPSS 12.0 statistical analysis software was used, and the analysis of One-Way ANOVA was employed. P < 0.05 was regarded as with statistical significance.