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Keywords:

  • sialyl Lewis X;
  • prostate cancer;
  • prostatic stromal cell;
  • prostate cell adhesion

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Elevated expression of sialyl Lewis X has been postulated to be a prognostic indicator of prostate cancer. However, direct evidence for the relationship between increased expression of sialyl Lewis X and malignancy of prostate cancer is still lacking. To determine whether increased levels of sialyl Lewis X leads to malignancy in prostate tumor, we transfected the human prostate cancer cell line PC-3 with α1,3-fucosyltransferase III (FTIII) to obtain stable transfectants, PC-3-FTIII lines, that highly express sialyl Lewis X. When inoculated in the prostate of nude mice, PC-3-FTIII cells produced large prostate tumors, while mock-transfected PC-3 cells, which are negative for sialyl Lewis X antigen, produced small prostate tumors. The aggressive tumor formation by PC-3-FTIII cells was inhibited by preincubation of the tumor cells with anti-sialyl Lewis X antibody, by the presence of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide but not by control peptide. PC-3-FTIII cells and mock-transfected PC-3 cells exhibited no significant difference in cell numbers when cultured in vitro. Remarkably, PC-3-FTIII adhered to prostatic stromal cells in vitro with higher affinity than mock-transfected PC-3. Such adhesion was inhibited by preincubation of PC-3-FTIII cells with antisialyl Lewis X antibody, by the addition of sialyl Lewis X oligosaccharide or by selectin ligand mimic peptide. However, anti-E-selectin, anti-P-selectin or anti-L-selectin antibodies did not inhibit the adhesion of PC-3-FTIII cells to the stromal cells. These results suggest that prostate cancer cells gain aggressiveness through adhesive interaction with prostatic stromal cells by a novel mechanism involving sialyl Lewis X. © 2003 Wiley-Liss, Inc.

Prostate cancer is the most common cancer in men and the second highest cause of cancer death in Western society.1 Although intensely studied, the molecular and biochemical determinants of prostate cancer development and progression remain unknown. Measurement of prostate-specific antigen so far has been the best method for detection of the disease, but it is limited in its ability to distinguish benign prostate hypertrophy from prostate cancer.2, 3 Moreover, current markers and methodologies cannot distinguish a tumor that will remain benign and not impinge on patient survival versus a tumor with aggressive traits culminating in metastatic spread and death.

Progression of cancer in primary organs and development of metastasis are complex processes involving genetic and epigenetic mechanisms.4, 5 Carbohydrates expressed on the cell surface are altered during carcinogenesis and apparently play important roles in cancer metastasis.6, 7 Among various carbohydrate molecules, sialyl Lewis X, NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ-R, has been associated with breast, colon and lung carcinomas,8, 9, 10, 11, 12 while sialyl Lewis A, NeuNAcα2-3Galβ1-3(Fucα1-4)GlcNAcβ-R, is associated with colorectal and pancreatic carcinomas.13, 14 In colon and lung cancer patients, the expression of sialyl Lewis X correlates positively with poor prognosis.9, 10, 11, 12

It has been shown that many human adenocarcinoma cells adhere to E-selection in vitro in a sialyl Lewis X- and sialyl Lewis A-dependent manner.15, 16, 17, 18, 19 However, it is not clear whether E- and P-selectins play a role in tumor cell adhesion. E- and P-selectins are not expressed on endothelial cells in vivo unless endothelial cells are stimulated by an inflammatory cytokine.20 It has been demonstrated that experimental inoculation of cancer cells can induce expression of cytokines in the hepatocytes, leading to expression of E-selectin on endothelial cells.21, 22 On the other hand, it has been demonstrated recently that B16-α1,3-fucosyltransferase III (FTIII) cells expressing sialyl Lewis X can form lung tumor foci in mice deficient in E- and P-selectins.23 These results indicate that sialyl Lewis X expressed on tumor cells can be recognized by an adhesion molecule distinct from selectins on endothelial cells.

Previous studies have shown that sialyl Lewis X is a reliable indicator of malignant potential and prognosis in prostate cancer.24, 25 In tumor formation of prostate cancer cells, it has been demonstrated that adhesion of epithelial prostate cells to stromal cells plays a critical role.26, 27, 28 However, the roles of sialyl Lewis X in adhesion of prostate cells to stromal cells have not been evaluated. In the present study, we used gene transfer of α1,3-fucosyltransferase to provide direct evidence for roles of sialyl Lewis X in prostate tumor growth and metastasis through remodeling of the membrane carbohydrate moiety. We found that acquisition of sialyl Lewis X by the PC-3 prostate cancer cell line results in prostate tumors of large size while mock-transfected PC-3 cells produced relatively smaller tumors. Our results suggest strongly that sialyl Lewis X-positive prostate cancer cells acquire aggressiveness, probably due to sialyl Lewis X-dependent adhesive interaction with prostatic stromal cells.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Human prostate cancer biopsy specimens

Prostate biopsy specimens from 118 randomly selected patients at various clinical stages of prostate cancer were used for immunohistochemical staining. Routine prostate biopsy for pathologic diagnosis was performed at the Department of Urology, Tohoku University School of Medicine, Sendai, Japan. Informed consent was obtained from all patients whose biopsy specimens were used in this study.

Pathologic diagnosis and Gleason scoring

Pathologic diagnosis for prostatic adenocarcinoma and Gleason scoring29 were performed by a single pathologist (M.E.) to obtain a uniform diagnosis.

Immunohistochemistry

Formalin-fixed, paraffin-embedded prostate biopsy specimens were deparaffinized and stained with anti-sialyl Lewis X monoclonal antibody (CSLEX1; Becton Dickinson, Mountain View, CA) as described previously.10 Tissue specimens containing more than 10% positively stained cancer cells were defined as CSLEX-1-positive.

Plasmid preparation

Plasmid pcDNA3-Fuc-TIII was constructed by first isolating a 1.7 kilobase HindIII-XbaI fragment corresponding to the Fuc-TIII cDNA from pcDNAI-Fuc-TIII.30 This fragment cDNA was then cloned into the pcDNA3(neo) plasmid vector.

Stable transfectants

The human prostate cancer cell line PC-3 was purchased from the American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 medium containing 10% fetal calf serum. PC-3 cells were transfected with pcDNA3(neo)-FTIII using LipfectAMINE (Life Technologies, Bethesda, MD) as described previously.31, 32 After 2 weeks in G418 selection (400 μg/ml; Gibco-BRL, Gaithersburg, MD), 20 single colonies were examined for immunochemical detection of sialyl Lewis X using CSLEX1 and fluorescein isothiocyanate (FITC)-conjugated goat affinity-purified (Fab′)2 fragment specific to mouse IgM (Cappel). Eight stable transfectants expressing sialyl Lewis X were established (PC-3-FTIII). Among the stable transfectants, 2 clones (PC-3-FTIII-1 and -2) were subjected to tumor assays. Since PC-3-FTIII-1 and PC-3-FTIII-2 yielded identical results in the following experiments, we designated PC-3-FTIII-1 cell as PC-3-FTIII and the results obtained by PC-3-FTIII-1 cells are shown. As a control cell line, mock-transfectant (vector only) PC-3-pcDNA3 was used.

Immunocytochemistry of human prostate stromal cells

Human prostate stromal cells were cultured on glass coverslips and stained with each mouse monoclonal antibody for E-, L- and P-selectin (Pharmingen, San Diego, CA) followed by biotinylated anti-mouse IgG antibody and peroxidase-conjugated avidin. A peroxidase substrate, AEC, was used to develop the color reaction.

Flow cytometric analysis

PC-3-FTIII cells and mock-transfectant cells were assessed by FACS analysis after incubation with CSLEX1, anti-sialyl Lewis A (CSLEA-1, UCLA Tissue Typing Laboratory, Los Angeles, CA) or anti-Lewis X (CD15) monoclonal antibody followed by incubation with FITC-conjugated secondary antibodies. Prostatic stromal cells were incubated with anti-E-selectin antibody, anti-P-selectin antibody or anti-L-selectin antibody (Sigma, St. Lois, MO) followed by incubation with FITC-conjugated secondary antibody. Positive control cells for E-selectin and P-selectin were prepared by stimulating human lung microvascular endothelial cells (HMVAC-L; Clonetics, East Rutherford, NJ) with IL-1β at 25 μg/ml for 6 hr. Human peripheral lymphocytes were used for positive control for L-selectin. Analyses were carried out by FACSort flow cytometry using the CellQuest program (Becton Dickinson) as described previously.32

Orthotopic tumor cell inoculation

Balb/c nude (nu/nu) mice, 6- to 8-week-old males obtained from Tacomic (Germantown, NY), were used for orthotopic tumor cell injection. Mice were anesthetized with avertin and laparotomy was performed; 2 × 106 of PC-3-FTIII cells and mock-transfected PC-3 cells were suspended in 20 μl of serum-free RPMI 1640 medium and inoculated into the posterior lobe of prostate. After tumor cell inoculation, the wound was closed with surgical clips. Four weeks after injection, mice were sacrificed and prostates were removed and fixed with a buffered formalin solution.

To determine the roles of various carbohydrates in tumor formation, PC-3-FTIII (2 × 106) cells were first preincubated with anti-sialyl Lewis X (CSLEX-1, 10 μg/ml) or anti-Lewis X antibody (CD15, 10 μg/ml; Immunetech, Marseille, France). Cells were then washed twice with PBS and resuspended in 20 μl of serum-free RPMI 1640 media. These tumor cells were then inoculated into the posterior lobe of the prostate as described above. Similarly, 2 × 106 of PC-3-FTIII cells were suspended in 20 μl of serum-free RPMI 1640 medium containing 250 μg/ml of sialyl Lewis X oligosaccharides attached to linear polyacrylamide33, 34 or 5 mg/ml of synthetic selectin ligand mimic IELLQAR or control FAQLDWH peptide35 and inoculated into the mouse prostate as described above.

Growth rate of cell lines

PC-3-pcDNA3 (mock-transfected) cells and PC-3-FTIII cells were seeded in 96-well plates at 105 cells/ml in RPMI 1640 containing 10% fetal calf serum and 400 μg/ml of G418 and cultured for various times. The number of living cells was measured each day using the Cell Counting Kit (Wako Pure Chemical Industries, Tokyo, Japan). Triplicate cultures were used for each sample.

Motility and invasion assays

A transwell cell culture chamber (Costar, Cambridge, MA) was used for in vitro motility and invasion assays with some modifications.36 The bottom of the upper chamber was sealed with a polyvinylpyrrolidone-free polycarbonate filter with a pore size 8 μm. The lower face was covered with 50 μg/ml of fibronectin (Wako Pure Chemical Industries). Cells (1 × 105) were plated in the upper chamber and incubated for 2 hr. The lower chamber was filled with serum-free RPMI 1640 medium. 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. The mean number of 10 different fields were plotted. For the invasion assay, the upper face of the filter was covered with 1 mg/ml of Matrigel (Collaborative Research, Bedford, MA), and the number of cells on the lower face was counted. These assays were carried out in triplicate. The standard deviation of these values was always within 5%.

Adhesion assay

The prostatic stromal cell line27, 37 was kindly provided by Dr. Kenji Nishimura, Department of Urology, Osaka University School of Medicine, Osaka, Japan. Prostate stromal cells were cultured to confluence in a 24-well culture plate as described previously.37 The wells were washed 3 times with PBS. PC-3-pcDNA3 and PC-3-FTIII cells were harvested and resuspended in RPMI 1640 containing 1% fetal calf serum and seeded on prostate stromal cells at 105/ml. For antibody treatment, PC-3-FTIII cells were incubated with CSLEX-1 (10 μg/ml) or anti-Lewis X antibody (10 μg/ml) on ice for 30 min before seeding. Antibody-treated cells were washed with PBS and seeded on the stromal cells. The cells were incubated for 5 min at room temperature with continuous rotation at 1g. After washing away unbound cells with cold PBS, bound cells were counted under a light microscope. The mean cell numbers in 10 different fields were plotted. The adhesion assays were carried out in triplicate. Similarly, sialyl Lewis X oligosaccharide (250 μg/ml) or selectin ligand mimic peptide (500 μg/ml) was added in the adhesion assay mixture. Effect of anti-selectin antibodies was tested by treating the stromal cells with monoclonal antibodies for E-selectin, P-selectin or L-selectin (Sigma) at the final concentration of 40 μg/ml dissolved in RPMI medium containing 1% fetal calf serum at 4°C for 30 min followed by washing with PBS. Under these conditions, the antibodies specific to E-, P- or L-selectin inhibited the adhesion of sialyl Lewis X expressing tumor cells to E-, P- or L-selectin chimeric protein coated on plates, as described previously.38, 39

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Sialyl Lewis X expression in prostate

To explore a potential correlation between sialyl Lewis X expression and tumorigenicity, we used immunohistochemistry with an anti-sialyl Lewis X antibody to investigate the relationship between sialyl Lewis X expression and the Gleason sum in prostate biopsy specimens from prostate cancer patients. The Gleason score/sum is a widely accepted histopathologic grading system that often predicts the prognosis of patients with prostate cancer.29 Figure 1 shows representative micrographs of human prostate cancer specimens after staining with anti-sialyl Lewis X (CSLEX-1) antibody. While prostate cancer with a low Gleason sum is negative for CSLEX-1 staining (Fig. 1a), specimens with Gleason sum of 7 (Fig. 1b) and 9 (Fig. 1c) were strongly positive for sialyl Lewis X. We divided the prostate cancer specimens into 3 groups according to Gleason sum: Gleason sum of lower than 7, a score of 7 and sum higher than 7. Positive CSLEX-1 staining showed significant correlation with the Gleason sum group, indicating that a sialyl Lewis X-positive tumor has more aggressive properties and predicts a poor prognosis (Table I). Thus, the higher the Gleason sum, the more positive the staining is, suggesting strongly that expression of sialyl Lewis X positively correlates with the malignancy of prostate tumor.

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Figure 1. Immunohistochemistry of the human prostate cancer biopsy specimens. Prostate biopsy specimens were stained with antisialyl Lewis X antibody. Each specimen with Gleason sum 6 (a), 7 (b) or 9 (c) was stained with antisialyl Lewis X antibody, CSLEX-1. Counterstaining is performed by hematoxylin.

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Table I. Pathological Grade and Sialyl Lewis X Expression
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Establishment of sialyl Lewis X expressing prostate cancer cell line, PC-3-FTIII

To determine directly the role of sialyl Lewis X in prostate cancer malignancy, the human prostate cell line PC-3, which is negative for sialyl Lewis X expression, was transfected with FTIII, a fucosyltransferase that leads the synthesis of sialyl Lewis X, to obtain sialyl Lewis X-positive 2 PC-3-FTIII cell lines, PC-3-FTIII-1 and PC-3-FTIII-2 cells. Virtually identical results were obtained in the following experiments using both PC-3-FTIII-1 and PC-3-FTIII-2 lines; therefore, we did not specify PC-3-FTIII-1 and PC-3-FTIII-2 and designated these cell lines collectively as PC-3-FTIII. PC-3-FTIII cells and controls transfected with PC-3-pcDNA3 (mock-transfected) were stained with antisialyl Lewis X monoclonal antibody (CSLEX-1) followed by FITC-conjugated anti-mouse IgM. Mock-transfectants were negative for CSLEX-1, while PC-3-FTIII cells were positive (Fig. 2). PC-3-FTIII cells were characterized by enhanced expression of sialyl Lewis X, sialyl Lewis A and Lewis X. Since treatment with antisialyl Lewis A antibody did not inhibit tumorigenecity, we focused our efforts in determining the roles of sialyl Lewis X in the following experiments.

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Figure 2. Flow cytometric analysis of PC-3 cells transfected with fucosyltransferase. Flow cytometry for sialyl Lewis X antigen, sialyl Lewis A antigen or Lewis X antigen. Closed histogram denotes the incubation with antisialyl Lewis X (a), sialyl Lewis A (b) or Lewis X (c) antibody followed by FITC-conjugated antibody. Open histograms indicate control experiments omitting the primary antibodies. Note that PC-3-FTIII cells showed elevation of all sialyl Lewis X, sialyl Lewis A and Lewis X antigens.

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In vitro growth kinetics and motility

Because malignancy is closely associated with cell proliferation activity, we examined the cell numbers of PC-3-FTIII cells and mock-transfected PC-3 cells cultured in vitro. The results show no significant difference in cell numbers obtained during in vitro culture between PC-3-FTIII cells and mock-transfectants (Fig. 3a), indicating that acquisition of sialyl Lewis X did not lead to increase in the cell numbers of PC-3 cells when they were cultured in vitro.

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Figure 3. In vitro growth kinetics and motility assays. (a) Cell numbers exhibited by 3 PC-3 transfectants under normal tissue culture conditions are shown. Note that there are no significant differences in in vitro growth between the mock-transfectants and the PC-3-FTIII cells. (b) Motility activities exhibited by PC-3-pcDNA3 (mock) and PC-3-FTIII cells assayed by transmigration chamber are shown. Note that there are no significant differences in motility between mock-transfectants and PC-3-FTIII cells. The results are shown for triplicate experiments including the standard deviation.

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PC-3-pcDNA3 and PC-3-FTIII cells were also subjected to motility/invasion assays using a transwell cell culture system. Fibronectin and Matrigel were coated on the lower and upper face of the transwell for motility and invasion assays, respectively. However, there was no detectable difference in either motility or invasion between PC-3-FTIII cells and mock-transfectants in these in vitro assays (Fig. 3b).

Tumorigenecity of PC-3-FTIII cells upon orthotopic innoculation into prostate of nude mice

To determine the role of sialyl Lewis X in tumor progression of PC-3 cells in situ, PC-3-FTIII cells and mock-transfectants were inoculated in the mouse prostate. Four weeks after inoculation, mice were sacrificed and the prostates examined. As shown in Figure 4(a) and (b), PC-3-FTIII cells produced larger tumors than did mock-transfectants.

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Figure 4. Tumor formation by PC-3 cells in the nude mice. PC-3-pcDNA3 (mock-transfectant) and PC-3-FTIII cells were inoculated in the prostate of the nude mice. (a) Extirpated prostate specimens obtained after inoculation of PC-3-pcDNA3 (mock) and PC-3-FTIII cells. Scale bar = 10 mm. (b) Wet weight of prostates derived from PC-3-pcDNA3 and PC-3-FTIII. (c) Prostate specimens obtained after inoculation of PC-3-FTIII cells preincubated with various antibodies, sialyl Lewis X oligosaccharide or selectin ligand mimic peptide. (d) Wet weight of prostates produced by PC-3-FTIII before and after preincubation with anti-Lewis X (CD15) or antisialyl Lewis X (CSLEX-1) antibody or in the presence of sialyl Lewis X oligosaccharide, selectin ligand mimic peptide (IELLQAR) or control peptide (FAQLDWH). PC-3-FTIII cells produced much larger tumors than mock-transfectant PC-3 cells. The tumor formation of PC-3-FTIII cells was inhibited by antisialyl Lewis X antibody, selectin ligand mimic peptide, sialyl Lewis X oligosaccharide, but not by anti-Lewis X antibody or control peptide.

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Microscopic examination showed that PC-3-FTIII produced a large tumor in prostates (Fig. 5a and b). Figure 5(b) is a higher magnification of the region denoted by rectangular in Figure 5(a), showing invasion of PC-3-FTIII into the muscle layer around the prostate capsule (shown by arrows in Fig. 5b). By contrast, the prostate tumors produced by mock-transfectant were confined within seminal vesicles (Fig. 5d), and the seminal vesicles were intact (arrows in Fig. 5e, which is a higher magnification of the region denoted in Fig. 5d). The expression of sialyl Lewis X was maintained in PC-3-FTIII-derived tumor cells throughout the experimental period in host mice (Fig. 5c), and most of those tumor cells at the invasive front, which runs from the upper left to the lower right of Figure 5(c), are positive for sialyl Lewis X. Interestingly, some cancer cells lost sialyl Lewis X after formation of tumors. By contrast, control PC-3-derived tumor cells remained negative for sialyl Lewis X expression (Fig. 5f). These results indicate that expression of sialyl Lewis X on PC-3 cells resulted in aggressive tumor formation.

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Figure 5. Microscopic examination of prostate tumors in mice. (a) and (b): prostate tumors produced by PC-3-FTIIII cells stained by hematoxylin and eosin. Tumor cells have invaded beyond the prostate capsule, and disrupted the seminal vesicles (arrows in a). (b) is a higher magnification of a region shown in the rectangular in (a). Tumor cells have invaded the muscle layer around the prostate capsule (arrows in b). (d) and (e): The prostate tumors produced by mock-transfectants were confined within the prostatic capsule (arrowheads in d), and the seminal vesicles were intact (arrows in d). Higher magnification shows the intact prostatic capsule (arrows in e). (c) and (f): Immunohistochemistry for sialyl Lewis X in the prostate tumor inoculated in mice. The prostate tumor produced by mock-transfectants was negative for sialyl Lewis X antigen (f), while the tumor produced by PC-3-FTIII cells was positive (c).

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Inhibition of tumor formation by sialyl Lewis X oligosaccharides and selectin ligand mimic peptides

To determine if tumor formation by PC-3-FTIII is dependent on sialyl Lewis X antigen, PC-3-FTIII cells were inoculated after preincubation of these cells with antisialyl Lewis X antibody. The results shown in Figure 4(c) and (d) illustrate that tumor formation was inhibited by anti-sialyl Lewis X antibody (CSLEX-1) but not by anti-Lewis X (CDI5) antibody. Furthermore, the tumor weight was significantly reduced in the presence of sialyl Lewis X oligosaccharide, selectin ligand mimicking IELLQAR peptides but was not affected by the presence of control FAQLDWH peptide (Fig. 4c and d). These results indicate that the aggressiveness of PC-3-FTIII depends on sialyl Lewis X carbohydrate antigen expressed on PC-3 cells.

Adhesion of PC-3-FTIII cells to human prostate stromal cells

To gain insight into the mechanisms for aggressive tumors produced by PC-3-FTIII cells, we compared the adhesion of PC-3-FTIII and mock-transfected PC-3 cells to prostate stromal cells by cell adhesion assay in vitro. As shown in Figure 6, PC-3-FTIII cells adhered efficiently to stromal cells, while mock-transfected PC-3 cells barely adhered to the same stromal cells. Remarkably, adhesion of PC-3-FTIII cells to stromal cells could be inhibited by preincubation of PC-3-FTIII cells with antisialyl Lewis X antibody (Fig. 6a, panel c). Moreover, adhesion of PC-3-FTIII cells was also inhibited by sialyl Lewis X oligosaccharides and selectin ligand mimicking IELLQAR peptide but not by the control peptide (Fig. 6b). These results suggest strongly that adhesion of PC-3-FTIII cells to stromal cells depends on sialyl Lewis X expressed on PC-3 FTIII.

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Figure 6. Adhesion of PC-3-pcDNA3 and PC-3-FTIII cells to stromal cells. (a) Representative photomicrographs of PC3-FTIII cells adhered to stromal cells. Each shows (panel a) PC-3-FTIII cells adhered to the stromal cells, (panel b) PC-3-mock (mock-transfectant) to stromal cells and (panel c) PC-3-FTIII cells after preincubation with PC-3-FTIII cells of anti-sialyl Lewis X antibody, CSLEX-1. Scale bar = 100 μm. (b) Numbers of PC-3-FTIII cells adhered to the stromal cells are shown. Note that antisialyl Lewis X antibody (CSLEX-1) but not anti-Lewis X antibody (CD15) blocked the adhesion of PC-3-FTIII cells. Similarly, adhesion was inhibited by sialyl Lewis X (sLeX) oligosaccharides (250 μg/ml) and selectin ligand mimic peptide (IELLQAR; 500 μg/ml) but not by a control peptide (FAQLDWH; 500 μg/ml).

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Since sialyl Lewis X is a specific carbohydrate ligand for selectins, we tested if stromal cells express E-, P- and L-selectin. Flow cytometric analysis showed no signals for selectin expression on the stromal cells while control IL-1β-stimulated human lung microvascular endothelial cells for E- and P-selectins and peripheral blood lymphocytes (for L-selectin) showed a strong staining (Fig. 7a). No inhibition for adhesion of PC-3-FTIII cells to stromal cells was observed upon preincubation of stromal cells with anti-E-, P- or L- selectin antibody (Fig. 7b). The adhesion of tumor cells expressing sialyl Lewis X to E-, P- or L-selectin chimeric proteins, assayed as shown in previous reports,38, 39 was significantly inhibited by using the same neutralizing antibodies under the same condition. These results exclude the possibility that sialyl Lewis X expressing PC-3-FTIII cells is recognized by a selectin expressed on stromal cells.

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Figure 7. Detection of selectins on stromal cells and effect of antiselectin antibodies on the adhesion of PC-3 cells to stromal cells. (a) Flow cytometric analysis of prostatic stromal cells for E-, P- and L-selectins. Prostatic stromal cells (closed histogram) were reacted with anti-E, anti-P and anti-L-selectin antibody, followed by FITC-conjugated second antibody. Positive control cells (open histograms) shown are IL-1β-stimulated human lung microvascular endothelial (HMVEC-L) cells reacted with anti-E-selectin antibody and anti-P-selectin antibody and human peripheral blood lymphocytes reacted with anti-L-selectin antibody. (b) Effect of anti-selectin antibodies on adhesion of PC-3-FTIII cells to prostatic stromal cells. Adhesion assays were carried out in a similar manner as shown in Figure 6(b). 1, mock-transfected PC-3 cells; 2–5, PC-3-FTIII treated with none (2), anti-E-selectin (3), anti-P-selectin (4) and anti-L-selectin (5) antibody.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The present study demonstrates that expression of sialyl Lewis X on prostate cancer cells in human patients correlates positively with the progression of prostate cancer as determined by the Gleason scale. Our findings are consistent with results of previous reports indicating an association of sialyl Lewis X with tumor progression and its prognostic significance in patients with prostate cancer.24, 25

More importantly in the present study, we generated PC-3 cells expressing sialyl Lewis X through gene transfer of FucT-III and tested cell growth in vivo by inoculating them into the mouse prostate. While mock-transfected PC-3 cells without sialyl Lewis X on the cell surface produced small tumors, sialyl Lewis X expressor PC-3-FTIII cells grew to large tumors, exhibiting invasion. Sialyl Lewis X-dependent growth of PC-3-FTIII cells in vivo was supported by the following results: the size of tumors derived from PC-3-FTIII cells was reduced significantly by preincubation of PC-3-FTIII cells with anti-sialyl Lewis X antibody and in the presence of sialyl Lewis X oligosaccharides or selectin ligand mimic peptide. However, we found no difference between the PC-3-FTIII cells and mock-transfectaed PC-3 in growth potential, motility on fibronectin and invasion into Matrigel when these cells are cultured in vitro. This suggested that an in vivo environment enhanced preferentially the growth of PC-3-FTIII cells but not that of mock-transfected PC-3 upon inoculation of these cells into the mouse prostate.

Many studies described an importance of interactions between prostate cancer cells and stromal cells for prostate cancer progression.26, 27, 28, 40, 41, 42 These studies suggest that prostate cancer cells are stimulated by factors secreted by prostatic stromal cells.28, 40, 41, 42 In the present study, we demonstrated by in vitro adhesion assay that PC-3-FTIII cells adhere efficiently to stromal cells, while mock-transfected PC-3 cells do not adhere well to the stromal cells. This adhesion was abrogated by preincubation of PC-3-FTIII cells with anti-sialyl Lewis X antibody or by treatment with sialyl Lewis X oligosaccharide or selectin ligand mimic peptide. These results demonstrate that PC-3-FTIII cells exhibit an increased adhesion to stromal cells in a sialyl Lewis X-dependent manner. The increased adhesion of prostate cancer cells to stromal cells was not, however, inhibited by preincubation of stromal cells with antibodies specific to E-, P- or L-selectin, and the prostate stromal cells are negative for immunostaining of E-, P- and L-selectins. These results indicate that PC-3-FTIII cells adhere to prostate stromal cells through a sialyl Lewis X-dependent manner, but none of E-, P- or L-selectin is involved in this adhesion. Nonetheless, it is likely that PC-3-FTIII cells are stimulated for cell growth by adhering to the prostatic stromal cells.

Recently, it has been shown that mouse B16 and human MeWo melanoma cells can form lung tumor foci after transfection with FTIII and acquisition of sialyl Lewis X.32, 34 Similarly, acquiring sialyl Lewis X by transfection of fucosyltransferase VII results in increased tumor formation by a lung carcinoma cell line.43 It has been shown that coinoculation of A431 epidermoid cancer cells and F-2 endothelial cells resulted in larger tumors than inoculation of A431 cells alone.43 The interaction between A431 cells and F-2 cells is sialyl Lewis X-dependent, and anti-sialyl Lewis X antibody treatment reduced the tumor size. In another study, the size of tumors formed by human colon or lung adenocarcinoma cell lines in the immune-deficient mouse was significantly reduced by the pretreatment of the cancer cells with the acetylated GlcNAcβ1[RIGHTWARDS ARROW]3Gal disaccharide that reduces the expression of silayl Lewis X.44 The inhibition of sialyl Lewis X formation by colon cancer cells with antisense RNA treatment resulted in the diminishment of tumor formation.18 These results collectively indicate that sialyl Lewis X expressed on cancer cells facilitate tumor progression. On the other hand, there was no clear consensus whether selectins or proteins other than selectins are involved in recognition of sialyl Lewis X on cancer cells. It has been shown that the number of tumor foci significantly decreased in P- and L-selectin-deficient mice compared to wild-type mice.19 By contrast, sialyl Lewis X-dependent tumor colonization of the lung by B16-FTIII or MeWo-FTIII cells is equally efficient in mice deficient in E- and P-selectins as in wild-type mice.23 It has also been reported that ex vivo adhesion of human colon cancer cells to liver tissues is inhibited by anti-sialyl Lewis X antibody but not by anti-E-selectin antibody.45 These results indicate that sialyl Lewis X-dependent tumor formation can be facilitated by an as yet unidentified adhesion molecule. The present study extends this hypothesis, showing that prostate cancer cells adhere to stromal cells in a sialyl Lewis X-dependent manner by a mechanism independent from selectins. Further studies are necessary to determine if a common adhesion molecule that is distinct from selectins plays a role in tumor formation in the lung, liver and prostate.

Taken together, the results obtained in the present study indicate that upregulation of sialyl Lewis X may be an important step in prostate cancer progression. The aggressive potential exhibited by sialyl Lewis X expressing cells may be initiated by the increased adhesion of prostate cancer cells to prostate stromal cells. Such adhesion may stimulate growth of prostate cancer cells through growth factors secreted from stromal cells and other cells in vivo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank Dr. Kenji Nishimura for the gift of prostate stromal cells, Dr. Elise Lamar for critically reading the article and Ms. Thu Gruenberg for organizing it.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  • 1
    Reiter RE, Dekernion JB. Epimiology, etiology, and prevention of prostate cancer. In: WalshPC, RetikAB, VaughanED, WeinAJ, eds. Campbell's urology, 8th ed. Philadelphia: Saunders, 2000. 30324.
  • 2
    Stamey TA, Yang N, Hay AR, McNeal JE, Freiha FS, Redwine E. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N Engl J Med 1987; 317: 90916.
  • 3
    Benson MC, Whang IS, Pantuck A, Ring K, Kaplan SA, Olsson CA, Cooner WH. Prostate specific antigen density: a means of distinguishing benign prostatic hypertrophy and prostate cancer. J Urol 1992; 147: 8156.
  • 4
    Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 5770.
  • 5
    Abate-Shen C, Shen MM. Molecular genetics of prostate cancer. Genes Dev 2000; 14: 241034.
  • 6
    Fukuda M. Possible roles of tumor-associated carbohydrate antigens. Cancer Res 1996; 56: 223744.
  • 7
    Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci USA 2002; 99: 102313.
  • 8
    Renkonen J, Paavonen T, Renkonen R. Endothelial and epithelial expression of sialyl Lewis (x) and sialyl Lewis (a) in lesions of breast carcinoma. Int J Cancer 1997; 74: 296300.
  • 9
    Fukushima K, Hirota M, Terasaki PI, Wakasaka A, Togashi H, Chia D, Fukushi Y, Nudelman E, Hakomori S. Characterization of sialylated Lewis X as a new tumor-associated antigen. Cancer Res 1984; 44: 527985.
  • 10
    Nakamori S, Kameyama M, Imaoka S, Furukawa H, Ishikawa O, Sasaki Y, Kabuto T, Iwanaga T, Matsushita Y, Irimura T. Increased expression of sialyl Lewis X antigen correlates with poor survival in patients with cororectal carcinoma: clinicopathological and immunohistochemical study. Cancer Res 1993; 53: 36327.
  • 11
    Shimodaira K, Nakayama J, Nakamura N, Hasebe O, Katsuyama T, Fukuda M. Carcinoma-associated expression of core 2 beta 1-6-N-acetylglucosaminyltransferase gene in human colorectal cancer: role of O-glycans in tumor progression. Cancer Res 1997; 57: 52016.
  • 12
    Machida E, Nakayama J, Amano J, Fukuda M. Clinicopathological significance of core 2 beta 1, 6-N-acetylglucosaminyltransferase messenger RNA expressed in the pulmonary adenocarcinoma determined by in situ hybridization. Cancer Res 2001; 61: 222631.
  • 13
    Magnani JL, Brockhaus M, Smith DF, Ginsburg V, Blaszczyk M, Michell KF, Steplewski Z, Koprowski H. A monosialoganglioside is a monoclonal antibody-defined antigen of colon carcinoma. Science 1981; 212: 556.
  • 14
    Ono M, Sakamoto M, Ino Y, Moriya Y, Sugihara K, Muto T, Hirohashi S. Cancer cell morphology at the invasive front and expression of cell adhesion-related carbohydrate in the primary lesion of patients with colorectal carcinoma with liver metastasis. Cancer 1996; 78: 117986.
  • 15
    Rice GE, Bevilacqua MP. An inducible endothelial cell surface glycoprotein mediates melanoma adhesion. Science 1989; 246: 13036.
  • 16
    Takada A, Ohmori K, Yoneda T, Tsuyuoka K, Hasegawa A, Kiso M, Kannagi R. Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium. Cancer Res 1993; 53: 35461.
  • 17
    Sawada R, Tsuboi S, Fukuda M. Differential E-selectin-dependent adhesion efficiency in sublines of a human colon cancer exhibiting distinct metastatic potentials. J Biol Chem 1994; 269: 142531.
  • 18
    Weston BW, Hiller KM, Mayben JP, Manousos GA, Bendt KM, Liu R, Kusack JC. Expression of human alpha(1,3)fucosyltransferase antisense sequences inhibits selectin-mediated adhesion and liver metastasis of colon carcinoma cells. Cancer Res 1999; 59: 212735
  • 19
    Borsig L, Wong R, Hynes RO, Varki NM, Varki A. Synergistic effects of L- and P-selectin in facilitating tumor metastasis can involve non-mucin ligands and implicate leukocytes as enhancers of metastasis. Proc Natl Acad Sci USA 2002; 99: 21938.
  • 20
    Honeister JW, Lowe JB. Carbohydrate recognition in leukocyte-endothelial interactions. In: FukudaM, HindsgaulO, eds. Molecular cellular glycobiology. Oxford: Oxford University Press, 2000. 62115.
  • 21
    Khatib AM, Kontogiannea M, Fallavollita L, Jamison B, Meterissian S, Brodt P. Rapid induction of cytokine and E-selectin expression in the liver in response to metastatic tumor cells. Cancer Res 1999; 59: 135661.
  • 22
    Minami S, Furui J, Kanematsu T. Role of carcinoembryonic antigen in the progression of colon cancer cells that express carbohydrate antigen. Cancer Res 2001; 61: 27325.
  • 23
    Zhang J, Nakayama J, Ohyama C, Suzuki M, Suzuki A, Fukuda M, Fukuda MN. Sialyl Lewis X-dependent lung colonization of B16 melanoma cells through a selectin-like endothelial receptor distinct from E- or P-selectin. Cancer Res 2002; 62: 41948.
  • 24
    Jorgensen T, Berner A, Kaalhus O, Tveter KJ, Danielsen HE, Bryne M. Up-regulation of the oligosaccharide sialyl LewisX: a new prognostic parameter in metastatic prostate cancer. Cancer Res 1995; 55: 18179.
  • 25
    Idikio HA. Sialyl-Lewis-X, Gleason grade and stage in non-metastatic human prostate cancer. Glycoconj J 1997; 14: 8757.
  • 26
    Janvier R, Sourla A, Koutsilieris M, Doillon CJ. Stromal fibroblasts are required for PC-3 human prostate cancer cells to produce capillary-like formation of endothelial cells in a three-dimensional co-culture system. Anticancer Res 1997; 17: 15517.
  • 27
    Krill D, Shuman M, Thompson MT, Becich MJ, Strom SC. A simple method for the isolation and culture of epithelial and stromal cells from benign and neoplastic prostates. Urology 1997; 49: 9818.
  • 28
    Tuxhorn JA, McAlhany SJ, Dang TD, Ayala GE, Rowley DR. Stromal cells promote angiogenesis and growth of human prostate tumors in a differential reactive stroma (DRS) xenograft model. Cancer Res 2002; 62: 3298307.
  • 29
    Gleason DF, Mellinger GT. Prediction of prognosis for prostatic adenocarcinoma by combined histological grading and clinical staging. J Urol 1974; 111: 5864.
  • 30
    Kukowska-Latallo JF, Larsen RD, Nair RP, Lowe JB. A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group alpha(1,3/1,4)fucosyltransferase. Genes Dev 1990; 4: 1288303.
  • 31
    Ohyama C, Smith PL, Angata K, Fukuda MN, Lowe JB, Fukuda M. Molecular cloning and expression of GDP-D-mannose-4,6-dehydratase, a key enzyme for fucose metabolism defective in Lec13 cells. J Biol Chem 1998; 273: 145827.
  • 32
    Ohyama C, Tsuboi S, Fukuda M. Dual roles of sialyl Lewis X oligosaccharides in tumor metastasis and rejection by natural killer cells. EMBO J 1999; 18: 151625.
  • 33
    Weitz-Schmidt G, Stokmaier D, Scheel G, Nifant'ev NE, Tuzikov AB, Bovin N. An E-selectin binding assay based on a polyacrylamide-type glycoconjugate. Anal Biochem 1996; 238: 18490.
  • 34
    Ohyama C, Kanto S, Kato K, Nakano O, Arai Y, Kato T, Chen S, Fukuda MN, Fukuda M. Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides. Proc Natl Acad Sci USA 2002; 99: 1378994.
  • 35
    Fukuda MN, Ohyama C, Lowitz K, Matsuo O, Pasqualini R, Ruoslahti E, Fukuda M. A peptide mimic of E-selectin ligand inhibits sialyl Lewis X-dependent lung colonization of tumor cells. Cancer Res 2000; 60: 4506.
  • 36
    Gmyrek GA, Walburg M, Webb CP, Yu HM, You X, Vaughan ED, Vande Wounde GF, Knudsen BS. Normal and malignant prostate epithelial cells differ in their response to hepatocyte growth factor/scatter factor. Am J Pathol 2001; 159: 57990.
  • 37
    Nishimura K, Kitamura M, Miura H, Nonomura N, Takada S, Takahara S, et al. Prostate stromal cell-derived hepatocyte growth factor induces invasion of prostate cancer cell line DU145 through tumor-stromal interaction. Prostate 1999; 41: 14553.
  • 38
    Tsuboi S, Srivastava OP, Palcic MM, Hindsgaul O, Fukuda M. Acquisition of P-selectin binding activity by en bloc transfer of sulfo Le(x) trisaccharide to the cell surface: comparison to a sialyl Le(x) tetrasaccharide transferred on the cell surface. Arch Biochem Biophys 2000; 374: 1006.
  • 39
    Tsuboi S, Isogai Y, Hada N, King JK, Hindsgaul O, Fukuda M. 6′-sulfo sialyl Lex but not 6-sulfo sialyl Lex expressed on the cell surface supports L-selectin-mediated adhesion. J Biol Chem 1996; 271: 272136.
  • 40
    Gleave M, Hsieh JT, Gao CA, von Eschenbach AC, Chung LW. Acceleration of human prostate cancer growth in vivo by factors produced by prostate and bone fibroblasts. Cancer Res 1991; 51: 375361.
  • 41
    Song Z, Powell WC, Kasahara N, van Bokhoven A, Miller GJ, Roy-Burman P. The effect of fibroblast growth factor 8, isoform b, on the biology of prostate carcinoma cells and their interaction with stromal cells. Cancer Res 2000; 60: 67306.
  • 42
    Ropiquet F, Giri D, Kwabi-Addo B, Mansukhani A, Ittmann M. Increased expression of fibroblast growth factor 6 in human prostatic intraepithelial neoplasia and prostate cancer. Cancer Res 2000; 60: 424550.
  • 43
    Martin-Satue M, Marrugat R, Cancelas JA, Blanco J. Enhanced expression of alpha(1,3)-fucosyltransferase genes correlates with E-selectin-mediated adhesion and metastatic potential of human lung adenocarcinoma cells. Cancer Res 1998; 58: 154450.
  • 44
    Tei K, Kawakami-Kimura N, Taguchi O, Kumamoto K, Higashiyama S, Taniguchi N, Toda K, Kawata R, Hisa Y, Kannagi R. Roles of cell adhesion molecules in tumor angiogenesis induced by cotransplantation of cancer and endothelial cells to nude rats. Cancer Res 2002; 62: 628996.
  • 45
    Ota M, Takamura N, Irimura T. Involvement of cell surface glycans in adhesion of human colon carcinoma cells to liver tissue in a frozen section assay: role of endo-beta-galactosidase-sensitive structures. Cancer Res 2000; 60: 52618.