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Prothrombin promotes the invasiveness of melanoma cells by a different mechanism from thrombin
Fibrinolysis & Proteolysis (1999) 13 (6), 272–278 © Harcourt Publishers Ltd 1999 Article No. fipr. 2000.0040
Prothrombin promotes the invasiveness of melanoma cells by a different mechanism from thrombin H. Zhou,1 E. C. Gabazza,1,2 T. Sano,1 Y. Adachi,2 K. Suzuki1 1
The Department of Molecular Pathobiology, Mie University School of Medicine, Tsu-city, Mie 514, Japan Third Department of Internal Medicine, Mie University School of Medicine, Tsu-city, Mie 514, Japan
2
Summary The migration of tumor cells through the extravascular matrix is a crucial step in the process of tumor invasion and metastasis. It has been recognized that activation of blood coagulation may contribute to the growth and the invasive behavior of tumor cells. We have previously found that prothrombin and its derivative, fragment 1, bind to and stimulate the motility of melanoma cells by a different mechanism from thrombin. The present study showed that prothrombin and fragment 1 also significantly increase the invasive ability of melanoma cells in an in vitro matrigel system by a different mechanism from thrombin. The optimal concentration of each factor for stimulating invasiveness of tumor M2 cells was between 0.5~1 µM. The M2 cells with highly metastatic potential showed strong invasive ability compared to the CL10 cells with low metastatic potential in the presence of the same concentrations of each factor. Hirudin inhibited the stimulatory activity of thrombin but not that of prothrombin or fragment 1 on the invasive ability of M2 cells. The activity of plasmin and urokinase-type plasminogen activator (uPA) was significantly increased in conditioned media from M2 cells stimulated with thrombin, prothrombin or fragment 1; hirudin inhibited the stimulative activity of thrombin but not that of prothrombin or fragment 1. Western blot analysis showed that thrombin increases the generation of the active forms of gelatinase A on the surface of M2 cells; hirudin inhibited this stimulative activity of thrombin. Prothrombin also increases the active forms of gelatinase A, but hirudin did not inhibit this effect of prothrombin. The results of this study suggest that prothrombin enhances the invasiveness of melanoma cells in vitro by a mechanism different from thrombin. © 1999 Harcourt Publishers Ltd
INTRODUCTION Tumor metastasis is composed of a number of interrelated events involving multiple host-tumor interactions. These events include detachment of cancer cells from the primary tumor, infiltration of the local host tissue, entry into the vascular or lymphatic circulation, arrest in the capillary beds of distant organs, extravasation into the target organ interstitium and parenchyma, and growth as a secondary colony.1,2 A critical point for the completion of the multiple steps of the metastatic process is the invasion or infiltration of tumor cells into areas of normal tissue. The mechanism of local tumor invasion is poorly understood, but it appears to depend Received: 22 September 1999 Accepted after revision: 28 December 1999 Correspondence to: Koji Suzuki, PhD, Department of Molecular Pathobiology, Mie University School of Medicine, Edobashi 2–174, Tsu-city, Mie 514–8507, Japan. Tel: +81 59 231 5036; fax: +81 59 231 5209
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on the ability of tumor cells to attach and proteolyze components of the extracellular matrix and migrate into regions of the matrix modified by proteolysis.3 In this connection, abnormal cell adhesion to structural components of the basement membrane and stroma3–5 as well as increased secretion of hydrolytic enzymes6 have been documented in metastatic cells as compared to their nonmetastatic counterparts. Active tumor cell motility is another crucial step in the establishment of metastasis. Cell motility, coupled with proteolysis1,3 is required for the penetration of metastatic cells through subendothelial and interstitial matrices during the transition from in situ to invasive carcinoma and during intravasation and extravasation. Hemostatic abnormalities also have been implicated in the pathogenesis of tumor spread.7 Activation of coagulation factor intermediates assemble adjacent to viable tumor cells leading to generation of enzymatically active thrombin at the host-tumor interface.8 Thrombin,
Prothrombin promotes invasiveness of melanoma cells 273
besides its role in blood coagulation, has been also described to possess properties that may facilitate tumor dissemination, including its mitogenic activity on tumor cells, its abilities to enhance the proliferative response of tumor cells to growth factors and to induce the aggregation of platelets.9–11 Thrombin has been also reported to enhance the adhesion of tumor cells to endothelial cells and subendothelial matrix components and to stimulate the production and secretion of proteolytic enzymes by tumor cells.12–14 In a previous report we demonstrated that the precursor of thrombin, prothrombin, binds to the surface of melanoma cells through its fragment 1 region and stimulates their directional and random motility.15 In the present study, we investigated whether prothrombin is also able to stimulate the invasive ability of melanoma cells on the artificial basement membrane matrigel.
Preparation of prothrombin and its derivatives Prothrombin was purified from human plasma by methods previously described.20 Thrombin and fragment 1 of prothrombin were prepared as described.20 Protein and fragment preparations were homogeneous (more than 95% purity), as determined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). Residual thrombin activity was not detected in the purified prothrombin derivatives, but when present it was inactivated by treating with 1 mM PMSF in Tris-buffered saline (TBS) consisting of 50 mM Tris-HCl, 100 mM NaCl, pH 7.4 for 30 min, followed by dialysis in the same buffer. Each protein and fragment was dialysed against phosphate-buffered saline (PBS) consisting of 100 mM trisodium phosphate and 120 mM NaCl, pH 7.4, before using in the assays. Culture of melanoma cell lines
MATERIALS AND METHODS Materials Human serum albumin (HSA) was obtained from Bayer (Leverkusen, Germany) and Sephacryl S-200, DEAESephacel, Sephadex G-25M and sulfated Sephadex G-50 columns were from Pharmacia LKB Biotechnology (Uppsala, Sweden). Phenylmethyl-sulfonyl fluoride (PMSF) and bovine pancreas chymotypsin were from Wako Pure Chemicals (Osaka, Japan). The synthetic substrate of thrombin, D-Phe-pipecolyl-Arg-p-nitroanilide (S-2238) and that for uPA, Glu-Gly-Arg-pNA-HC1 (S-2444) were purchased from Chromogenix (Mölndal, Sweden) and that of plasmin, Boc-Val-Leu-Lys-MCA, from Peptide Institute (Osaka, Japan). Dulbecco’s modified Eagle’s minimum essential medium (DMEM), hirudin, bovine serum albumin (BSA), collagenase and the Russell’s viper venom (RVV) were from Sigma (St Louis, MO, USA). Plasminogen was purchased from Gelco Diagnostics (Shreveport, LA, USA). Penicillin and streptomycin were from Nakalai Tesque (Kyoto, Japan). Fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids, L-glutamine and vitamin solution were purchased from Gibco BRL (Gran Island, NY, USA). Gelatin was from Bio-Rad Laboratory (Richmond, CA, USA). Polyclonal anti-matrix metalloproteinase (MMP) –2 antibody was from Chemicon International Inc. (Temecula, CA, USA). Matrigel was purchased from Becton Dickinson Labware (Bedford, MA, USA). Factor X and factor V were purified from human plasma using methods previously described16,17 and they were activated by incubating with purified RVV-factor X activator and RVV-factor V activator, respectively.18,19 All other chemicals and reagents used were of the best quality commercially available. © Harcourt Publishers Ltd 1999
The K-1735 murine melanoma cell lines (M2 and CL10) were kind gifts from Dr Isaiah J. Fidler (University of Texas M.D. Anderson Cancer Center). The K-1735 cell lines were cultured in DMEM supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, L-glutamine and 2-fold vitamin solution. The cells were cultured in an atmosphere of 5% CO2-95% air at 37°C. All assays were carried out using cells at passages 3 to 10. Cells were harvested by a brief exposure to 0.25% trypsin/0.02% EDTA, and subcultured weekly. Invasion assay The invasion activity of murine melanoma cells was assayed following the method of Albini et al.21 with some modifications. Polycarbonate filters (Neuroprobe, Cabin Johnson, MD, USA) with 8-µm pore size were coated with matrigel following the manufacturer’s instructions. Matrigel was diluted to the desired concentration with cold DMEM (free of FBS), then applied to the filter, dried under a hood, and reconstituted with serum-free DMEM. Matrigel-coated filter was assembled in a 96-well Boyden chamber. Cells (0.5~1.0 × 106/ml) suspended in serumfree DMEM containing varying concentrations of prothrombin, fragment 1 or thrombin were added to the upper compartments of the chamber. As control, cells with serum-free DMEM were also added to the upper compartments. For making comparisons, cells suspended in DMEM containing a gradient concentrations of HSA were also added to the upper wells of the chamber. The lower wells of the chamber were filled with DMEM containing 0.1% HSA. The chamber was assembled and incubated at 37°C for 6 h in an atmosphere of 5%CO2-95% air. After incubation, the filters were removed, fixed in methanol and stained with Diff Quick (American Fibrinolysis & Proteolysis (1999) 13(6), 272–278
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Scientific Products, MacGraw Park, IL, USA). Cells that grew on the upper surface of the filter were mechanically removed, and those that migrated to the lower side of the filter were counted by light microscope under high power field (× 400). For each triplicate, the number of cells in five high power fields was determined and the counts were averaged. Invasion assay was also carried out in the presence of prothrombin, fragment 1 or thrombin that were pre-treated with 250 antithrombin units (ATU) of hirudin, a specific inhibitor of thrombin, at 37°C for 30 min.
Zymograghy Murine melanoma M2 cells cultured in 10 cm dish at 80% of confluency were washed three times with serumfree DMEM and then treated with 0.5 µM prothrombin, fragment 1 or thrombin for 48 h in the presence or absence of 250 ATU hirudin. The culture supernatants were then investigated for plasminogen activator activity using gel zymograghy as previously described.22 Briefly, aliquots of conditioned medium were mixed with gel loading buffer and they were electrophoresed on a 10% acrylamide gel containing 5% SDS, 1 mg/ml of gelatin in the presence of 10 µg/ml of plasminogen as substrate. After electrophoresis, the gel was incubated in 2.5% Triton X-100 at room temperature for one hour to remove SDS. Then, the gel was incubated in a buffer containing 100 mM Tris-HCl, pH 7.9, 30 mM CaCl2, and 1 µM ZnCl2 or 100 mM glycine/NaOH, pH 8.3, at 37°C for 16 h. The gel was then fixed in methanol: acetic acid: water (5:1:5) solution and stained with Coomassie blue, destained in 7% acetic acid solution and photographed. The lytic bands were quantified by densitometry using the public domain NIH image program (Wayne Rasband, NIH, Research Service Branch, NIMH, USA).
Western blotting SDS-PAGE of conditioned medium of murine melanoma M2 cells treated with prothrombin or thrombin was performed followed by blotting onto nitrocellulose membrane (Bio-Rad Laboratory). The membrane was incubated with blocking solution containing 100 mM PBS, pH 7.5, 5% BSA and 0.5% Tween 20 and then exposed to a 1/2000 dilution of rabbit polyclonal antiMMP-2 antibody for one hour while shaking at room temperature. After washing the membrane with PBS containing 0.1% BSA and 0.5% Tween 20, the membrane was incubated with a 1/2000 dilution of anti-rabbit IgG antibody conjugated to horse-raddish peroxidase. The membrane was washed and quantified using an enhanced chemiluminescence detection system (ECL, Amersham, Buckinghamshire, UK) as described previously.23 The Fibrinolysis & Proteolysis (1999) 13(6), 272–278
bands were quantified by densitometry by the NIH image program. Activity of plasmin and uPA in culture-conditioned medium Murine melanoma M2 cells grown in 10 cm dishes at 80 ~ 90% of confluency were washed twice with serum-free DMEM and then treated with 0.5 µM prothrombin, fragment 1 or thrombin for 48 h in the presence or absence of 250 ATU hirudin. Plasmin activity in cultureconditioned medium was analyzed using the fluorogenic substrate, Boc-Val-Leu-Lys-MCA, as previously described.24 In brief, aliquots of culture-conditioned medium were added to PBS containing 100 nM plasminogen and incubated a 37°C for 2 h; then, the release of aminomethyl coumarine (AMC) from the substrate was followed by measuring the increase in fluorescence intensity with a spectrophotometer (Shimadzu, RF-510). The uPA activity in culture-conditioned medium was measured using the chromogenic substrate S-2444. Aliquots of culture-conditioned medium added to TBS were incubated with 0.25 mM S-2444 at room temperature for 5 min; then the release of p-nitroaniline was determined at absorbance of 405 nm using an ELISA plate reader (EAR 340, Salzburg, Austria). Statistical analysis The results of invasion assays were expressed as invasion index; this was determined by dividing the number of cells migrating in response to prothrombin or its derivatives by the number of cells migrating in response to culture medium alone. Data were expressed as the mean index plus minus the standard error of the mean (SEM). The difference between the invasion index induced by ligands and control buffer (medium with increasing concentrations of HSA) was calculated by analysis of variance (Dunnett’s test) using the StatView 4.1 package software (ABACUS CONCEPTS, Berkeley, CA, USA) for the Macintosh. A P < 0.05 was considered as statistically significant.
RESULTS Prothrombin, fragment 1 and thrombin elicited a dosedependent invasion of murine melanoma M2 cells, a cell line with high metastatic potential (Fig. 1). At concentrations above 0.5 nM, prothrombin, fragment 1 or thrombin significantly increased the invasive ability of these cells as compared to controls (P < 0.05). The maximal effect was induced at concentrations between 0.5 ~ 1 µM of each attractant (P < 0.01 vs control). To evaluate whether the motile response of M2 cells to thrombin © Harcourt Publishers Ltd 1999
Prothrombin promotes invasiveness of melanoma cells 275
Fig. 1 Matrigel invasion of melanoma M2 cells stimulated with prothrombin (●), fragment 1 (◆), thrombin (▲), and control buffer (■ ■ ) and with those in the presence of hirudin (● ●, ◆, ▲). Invasion assay was carried out as described in Materials and Methods. Data are expressed as the mean of invasion index ± SEM. Each point represents the mean of five high power magnification (× 400) fields from each filter from three separate experiments. *; P < 0.05 as compared to control buffer. **; P < 0.01 as compared to thrombin only. Statistical analysis was carried out by Dunnett’s test.
Fig. 2 Matrigel invasion of melanoma cells with high (M2) (closed column) and low (CL10) (open column) metastatic potential in response to 1 µM prothrombin (column 2), fragment 1 (column 3), thrombin (column 4) and control buffer (column 1). Data are expressed as the mean of invasion index ± SEM. Bars; indicate mean values; *; P < 0.05 as compared to M2 cells. Statistical analysis was carried out by Dunnett’s test.
depends on the catalytic site of this protease, and that to prothrombin or fragment 1 on the presence of residual thrombin, both prothrombin and its derivatives were incubated with hirudin for 30 min before the invasion assay. As shown in Figure 1, treatment with hirudin inhibited more than 90% of thrombin-induced invasive activity of M2 cells (P < 0.01) but not that induced by prothrombin or fragment 1. We also evaluated the invasive ability of CL10 cells, a melanoma cell line with low metastatic potential, in © Harcourt Publishers Ltd 1999
Fig. 3 Activity of plasmin and uPA in culture-conditioned media from M2 cells. M2 cells were cultured for 24 h with 0.5 µM prothrombin or fragment 1 or thrombin in the presence or absence of 250 ATU hirudin and then aliquots of conditioned medium were analyzed. Plasmin activity (closed column) was measured using the fluorogenic substrate Boc-Val-Leu-Lys-MCA by spectrophotometry. uPA activity (open column) was measured using the chromogenic substrate S-2444. Column 1, serum-free medium from M2 cells; column 2, medium from prothrombin treated cells; column 3, medium from prothrombin and hirudin treated cells; column 4, medium from fragment 1 treated cells; column 5, medium from fragment 1 and hirudin treated cells; column 6, medium from thrombin-treated cells; column 7, medium from thrombin and hirudin treated cells. *; P < 0.05 as compared to control. Statistical analysis was carried out by Dunnett’s test.
response to prothrombin and its derivatives. The CL10 cells showed weak invasive ability compared to high metastatic M2 cells in the presence of the same concentrations of prothrombin or its derivatives (P < 0.05) (Fig. 2). The activity of plasmin and uPA in culture-conditioned medium from M2 cells treated with each ligand was also evaluated. As shown in Figure 3, the activity of plasmin and uPA was significantly enhanced in conditioned medium of M2 cells treated with prothrombin, fragment 1 or thrombin as compared to control medium. Hirudin inhibited the production of uPA induced by thrombin but not that induced by prothrombin or fragment 1. The activity of plasminogen activator in culture-conditioned medium of M2 cells treated with prothrombin, fragment 1 or thrombin was also evaluated by gel zymography. Conditioned medium of cells treated with prothrombin, fragment 1 or thrombin significantly increased the activity of plasminogen activator (high molecular weight uPA [56 kDa] and lower molecular weight uPA [35 kDa] bands) as shown by the gel zymography and densitometric analysis (Figure 4A B). Stimulation of M2 cells with thrombin resulted in increased generation of the active form (62 kDa) from the inactive gelatinase A (72 kDa) as shown by Western blotting (Fig. 5). The apparent decrease in 68 kDa intermediate form in thrombin-treated cells may be secondary to increased generation of the active forms. Incubation of melanoma cells with thrombin in the presence of hirudin decreased the generation of the 62 kDa active forms of Fibrinolysis & Proteolysis (1999) 13(6), 272–278
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gelatinase A. Prothrombin increased the production of gelatinase A and the generation of its active forms (62-kDa). These effects of prothrombin were not inhibited by hirudin (Fig. 5).
A. kDa 56 55 1
2
3
4
5
6
7
Fig. 4 Gel zymography showing the activity of plasminogen activator of culture-conditioned media from M2 cells. M2 cells were cultured for 24 h with 0.5 µM prothrombin or fragment 1 or thrombin in the presence or absence of 250 ATU hirudin and then each conditioned medium was run on SDS-PAGE. (A) Lytic bands: lane 1, serum-free medium from M2 cells; lane 2, medium from thrombin treated cells; lane 3, medium from thrombin and hirudin treated cells; lane 4, medium from prothrombin treated cells; lane 5, medium from prothrombin and hirudin treated cells; lane 6, medium from fragment 1 treated cells; lane 7, medium from fragment 1 and hirudin treated cells (B) Densitometric analysis of lytic bands. The intensity of each band was calculated using the NIH software. *; P < 0.05 as compared to control. Statistical analysis was carried out by Dunnett’s test.
kDa
72 68 64 62 1
2
3
4
5
Fig. 5 Western blotting of culture-conditioned media from M2 cells. M2 cells were cultured for 24 h with 0.5 µM prothrombin or thrombin in the presence or absence of 250 ATU hirudin and then each conditioned medium was run on SDS-PAGE. Lane 1, serumfree medium from M2 cells; lane 2, medium from thrombin treated cells; lane 3, medium from thrombin and hirudin treated cells; lane 4, medium from prothrombin treated cells; lane 5, medium from prothrombin and hirudin treated cells.
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DISCUSSION The role of components of the coagulation cascade in the pathogenesis of tumor spread has been well documented.7,25 Thrombin may contribute to tumor stroma formation by converting fibrinogen to fibrin or may support tumor cell proliferation because of its growth factor activity.8,26,27 Thrombin may also enhance the invasive phenotype of cancer cells by increasing their motility and their adhesion to endothelial cells, platelets and extracellular matrix proteins.12,14 The results of this study showed that, in addition to thrombin, its precursor prothrombin also promotes the invasion of melanoma cells on the artificial basement membrane matrigel. The effect of each factor was significant above concentrations of 0.5 nM and it was maximal at 1 µM. These findings have pathophysiological implications because prothrombin which circulates normally at plasma concentrations between 1 and 2 µM, may stimulate the intravascular migration of tumor cells invading the perivascular regions of host tissues, or vice versa, locally extravasated prothrombin may stimulate the extravasation of malignant cells arrested on capillary walls during their intravascular dissemination. Several lines of evidence suggest that local tumor invasion depends on the ability of tumor cells to attach and proteolyze components of the extracellular matrix and to migrate into regions of the matrix modified by proteolysis.28,29 Various proteolytic enzymes secreted by tumor cells have been implicated in the process of basement membrane and stromal matrix degradation during invasion and metastasis;30 those principally involved are serine proteases (plasminogen activator, plasmin, thrombin, trypsin, cathepsin G), cystein proteinase (cathepsin B) and a group of metalloproteinases.31 This latter group comprises the membrane-bound type MMP-1 (MT-MMP1), interstitial collagenase (MMP-1), stromelysin (MMP-3) and the type IV collagenases which include the gelatinase A (MMP-2) and gelatinase B (MMP-9).32–35 In the present study, it was shown that culture of melanoma cells in the presence of thrombin enhances the conversion of pro-gelatinase A (72 kDa) to its active forms (62 kDa) as shown by the Western blot analysis. The mechanism of gelatinase conversion to its active form by thrombin is unclear. The membrane-bound type (MT) MMP-1 has been previously reported to activate gelatinase A.35 Thus, it is conceivable that thrombin promotes gelatinase A activation by increasing the expression of MT-MMP-1 by melanoma cells. Thrombin stimulatory activity on © Harcourt Publishers Ltd 1999
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MT-MMP-1 expression likely proceeds via a proteolytically activation mechanism. Thrombin probably cleaves the external loop of its receptor on melanoma cells leading to activation of G-protein coupled signal transduction pathways and to increased secretion of MTMMP-1.36,37 The fact that hirudin inhibited the effects of thrombin suggests that the cellular effect of this protease is mediated by proteolytic activity on its receptor. In an attempt to explain the mechanism by which the zymogen prothrombin promotes tumor cell invasiveness, we evaluated the activity of plasmin and uPA in cultureconditioned media of M2 cells. The results showed that besides thrombin, prothrombin and its fragment 1 also significantly enhanced the activity of both plasmin and uPA in culture-conditioned media of M2 cells. In addition, the gel zymography showed that treatment with prothrombin increases the secretion of gelatinase A from M2 melanoma cells and the formation of the active forms of this metalloproteinase. Previous studies have shown that plasmin and plasminogen activator enhance the activation of gelatinase A and gelatinase B.38,39 Thus, it is conceivable that the increased generation of the active forms of gelatinase A as shown by the results of the Western blotting, was due to the increased secretion of plasminogen activator induced by prothrombin or fragment 1 in M2 cells. Interestingly, the cellular effects of thrombin were inhibited by hirudin but not those induced by prothrombin or fragment 1, suggesting that prothrombin and its fragment 1 exert their effect on melanoma cells by different mechanism from thrombin. Specific properties have been described to characterize tumor cell clones with increased ability to generate metastasis: major genetic instability, increased sialylation of cell surface, greater number of membrane receptors for extracellular matrix components, major resistant to host immune responses, enhanced secretion of adhesion molecules, proteolytic enzymes, cytokines and abnormal expression of the so called metastatic genes.40 In the present study, we also evaluated whether melanoma cells (M2 and CL10 cells) with established differences in metastasis potential demonstrate comparable differences in cell invasive ability in response to prothrombin and thrombin. The results showed that highly metastatic melanoma cell lines (M2) responded to each prothrombin and thrombin more significantly that the low metastatic cell lines (CL10). Difference in the number of surface receptors for prothrombin and thrombin may be a possible explanation for the different invasive response of M2 and CL10 cells in the presence of each ligand. In brief, the results of this study showed for the first time (1) that the zymogen prothrombin enhances the invasiveness of melanoma cells in vitro; (2) that both thrombin and prothrombin increase the plasmin and uPA activities and the activation of gelatinase A in © Harcourt Publishers Ltd 1999
culture-conditioned media from murine melanoma cells; and (3) that invasive responses induced by both prothrombin and thrombin differ according to the metastatic potential of melanoma cells. REFERENCES 1. Richard PH. Metastasis. In: Tannock IF, Hill RP, ed. The Basic Science of Oncology, ed 2. Canada: McGraw-Hill, Inc. Health Professions Division Press, 1992: 178–195. 2. Schottenfeld D. Epidemiology of lung cancer. In: Pass HI, Mitchell JB, Johnson DH, Turrisi AT, eds. Lung Cancer: Principles and Practice, Philadelphia: Lippincott-Raven Publishers Press, 1996: 305–321. 3. Liotta LA. Tumor invasion and metastases – role of the extracellular matrix: rhoads memorial award lecture. Cancer Res 1986; 46: 1–7. 4. Nakajima M, Irimura T, DiFerrante D, DiFerrante N, Nicolson G. Heparan sulfate degradation: relation to tumor invasion and metastatic properties of mouse B-16 melanoma sublines. Science 1983; 220: 611–613. 5. Robertson NP, Starkey JR, Meadows GG. Tumor cell invasion of three-dimensional matrices of defined composition: evidence for a specific role for heparan sulfate in rodent cell lines. Cancer Res 1989; 49: 1816–1823. 6. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shafie S. Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 1980; 284: 65–66. 7. Gasic GJ, Tuszynski GP, Gorelik E. Interaction of the hemostatic and immune system in the metastatic spread of tumor cells. Int Rev Exp Pathol 1986; 29: 173–212. 8. Dvorak HF, Harvey VS, Estrella P, Brown LF, Mcdonagh J, Dvorak AM. Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Lab Invest 1987; 57: 673–686. 9. Bruhn HD, Zorborn KH. Influences of clotting factors (thrombin, factor XIII) and of fibronectin on the growth of tumor cells and leukemic cells in vitro. Blut 1983; 46: 85–88. 10. Medrano EE, Cafferata EGA, Cancher F. Role of thrombin in the proliferative response of T-47D mamary tumor cells. Exp Cell Res 1987; 172: 354–364. 11. Nierodzik ML, Kajumo F, Karpatkin S. Effect of thrombin treatment of cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo. Cancer Res 1992; 52: 3267–3272. 12. Nierodzik ML, Plotkin A, Kajumo F, Karpatki S. Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo. J Clin Invest 1991; 87: 229–236. 13. Wojtukiewicz MZ, Tang DG, Nelson KK, Walz DA, Diglio CA, Honn KV. Thrombin enhances tumor cell adhesive and metastatic properties via increased aIIb expression on the cell surface. Thromb Res 1992; 68: 233–245. 14. Wojtukiewicz MZ, Tang DG, Ciarelli JJ et al. Thrombin increases the metastatic potential of tumor cells. Int J Cancer 1993; 54: 793–806. 15. Zhou H, Gabazza EC, Takeya H et al. Prothrombin and its derivatives stimulate motility of melanoma cells. Thromb Haemost 1998; 80: 407–12. 16. Miletich JP, Broze GJ, Majerus PW. The synthesis of sulfated dextran beads for isolation of human plasma coagulation factors II, IX, and X. Anal Biochem 1980; 105: 304–310. 17. Suzuki K, Stenflo J, Dahlbäck B, Teodorsson B. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914–1920.
Fibrinolysis & Proteolysis (1999) 13(6), 272–278
278 Zhou et al.
18. Suzuki K, Dahlbäck B, Stenflo J. Thrombin-catalyzed activation of human coagulation factor V. J Biol Chem 1982; 257: 6556–6564. 19. DiScipio RG, Hermodson MA, Davie EW. Activation of human factor X (Stuart factor) by a protease from Russells viper venom. Biochemistry 1977; 16: 5253–5260. 20. Deguchi H, Takeya H, Gabazza EC, Nishioka J, Suzuki K. Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex. Biochem J 1997; 321: 729–735. 21. Albini A, Iwamoto Y, Kleinman HK et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 1987; 47: 3239–3245. 22. Heussen C, Dowdle EB. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem 1980; 102: 196–202. 23. Smine A, Plantner JJ. Membrane type-1 matrix metalloproteinase in human ocular tissues. Curr Eye Res 1997; 16: 925–929. 24. Suzuki K, Nishioka J, Hashimoto S. Assay method of plasma plasminogen by means of a synthetic fluorogenic peptide substrate (3104-V). Rinsho Byori 1980; 28: 804–807. 25. Rickles FR, Edwards R. Activation of blood coagulation in cancer: Trousseau’s syndrome revisited. Blood 1983; 62: 14–31. 26. Olander JV, Bremer ME, Marasa JC, Feder J. Fibrin enhanced endothelial cell organization. J Cell Physiol 1985; 125: 1–9. 27. Walz DA, Fenton JW. The role of thrombin in tumor cell metastasis. Invas Metast 1994–95; 14: 303–308. 28. Melchiori A, Albini A, Ray JM, Stetler-Stevenson WG. Inhibition of tumor cell invasion by a highly conserved peptide sequence from the matrix metalloproteinase enzyme prosegment. Cancer Res 1992; 52: 2353–2356. 29. Ruddon RW. Cancer Biology ed 3: Biology of tumor metastasis. Oxford University. New York Press, 1995; 410–412. 30. Mignatti P, Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. Physiol Reviews 1993; 73: 161–195.
Fibrinolysis & Proteolysis (1999) 13(6), 272–278
31. Evans CW. The Metastatic Cell: behaviour and biochemistry. Chapman and Hall. Great Britain Press, 1991. 32. Brown PD, Levy AT, Margulies IMK, Liotta LA, StetlerStevenson WG. Independent expression and cellular processing of Mr 72,000 type IV collagenase and interstitial collagenase in human tumorigenic cell lines. Cancer Res 1990; 50: 6184–6191. 33. Okada Y, Morodomi T, Enghild J et al. Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Eur J Biochem 1990; 194: 721–730. 34. Okada Y, Gonoji Y, Naka K et al. Matrix metalloproteinase 9 (92-kDa) gelatinase/type IV collagenase) from HT1080 human fibrosarcoma cells. J Biol Chem 1992; 267: 21712–21719. 35. Sato T, Kondo T, Seiki M, Ito A, Cell type-specific involvement of furin in membrane type 1 matrix metalloproteinase-mediated progelatinase A activation. Ann N Y Acad Sci. 1999; 878: 713–5. 36. Vu T-K H, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057–1068. 37. Fischer EG, Ruf W, Mueller BM. Tissue factor-initiated thrombin generation activates the signaling thrombin receptor on malignant melanoma cells. Cancer Res 1995; 55: 1629–1632. 38. Mazzieri R, Masiero L, Zanetta L et al. Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants. EMBO J 1997; 16: 2319–2332. 39. Baramova EN, Bajou K, Remacle A et al. Involvement of PA/ plasmin system in the processing of pro-MMP-9 and in the second step of pro-MMP-2 activation. FEBS Lett 1997; 405: 157–162. 40. Yabkowitz R, Mansfield PJ, Dixit VM, Suchard SJ. Motility of human carcinoma cells in response to thrombospondin: relationship to metastatic potential and thrombospondin structural domains. Cancer Res 1993; 53: 378–387.
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Prothrombin promotes the invasiveness of melanoma cells by a different mechanism from thrombin H. Zhou,1 E. C. Gabazza,1,2 T. Sano,1 Y. Adachi,2 K. Suzuki1 1
The Department of Molecular Pathobiology, Mie University School of Medicine, Tsu-city, Mie 514, Japan Third Department of Internal Medicine, Mie University School of Medicine, Tsu-city, Mie 514, Japan
2
Summary The migration of tumor cells through the extravascular matrix is a crucial step in the process of tumor invasion and metastasis. It has been recognized that activation of blood coagulation may contribute to the growth and the invasive behavior of tumor cells. We have previously found that prothrombin and its derivative, fragment 1, bind to and stimulate the motility of melanoma cells by a different mechanism from thrombin. The present study showed that prothrombin and fragment 1 also significantly increase the invasive ability of melanoma cells in an in vitro matrigel system by a different mechanism from thrombin. The optimal concentration of each factor for stimulating invasiveness of tumor M2 cells was between 0.5~1 µM. The M2 cells with highly metastatic potential showed strong invasive ability compared to the CL10 cells with low metastatic potential in the presence of the same concentrations of each factor. Hirudin inhibited the stimulatory activity of thrombin but not that of prothrombin or fragment 1 on the invasive ability of M2 cells. The activity of plasmin and urokinase-type plasminogen activator (uPA) was significantly increased in conditioned media from M2 cells stimulated with thrombin, prothrombin or fragment 1; hirudin inhibited the stimulative activity of thrombin but not that of prothrombin or fragment 1. Western blot analysis showed that thrombin increases the generation of the active forms of gelatinase A on the surface of M2 cells; hirudin inhibited this stimulative activity of thrombin. Prothrombin also increases the active forms of gelatinase A, but hirudin did not inhibit this effect of prothrombin. The results of this study suggest that prothrombin enhances the invasiveness of melanoma cells in vitro by a mechanism different from thrombin. © 1999 Harcourt Publishers Ltd
INTRODUCTION Tumor metastasis is composed of a number of interrelated events involving multiple host-tumor interactions. These events include detachment of cancer cells from the primary tumor, infiltration of the local host tissue, entry into the vascular or lymphatic circulation, arrest in the capillary beds of distant organs, extravasation into the target organ interstitium and parenchyma, and growth as a secondary colony.1,2 A critical point for the completion of the multiple steps of the metastatic process is the invasion or infiltration of tumor cells into areas of normal tissue. The mechanism of local tumor invasion is poorly understood, but it appears to depend Received: 22 September 1999 Accepted after revision: 28 December 1999 Correspondence to: Koji Suzuki, PhD, Department of Molecular Pathobiology, Mie University School of Medicine, Edobashi 2–174, Tsu-city, Mie 514–8507, Japan. Tel: +81 59 231 5036; fax: +81 59 231 5209
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on the ability of tumor cells to attach and proteolyze components of the extracellular matrix and migrate into regions of the matrix modified by proteolysis.3 In this connection, abnormal cell adhesion to structural components of the basement membrane and stroma3–5 as well as increased secretion of hydrolytic enzymes6 have been documented in metastatic cells as compared to their nonmetastatic counterparts. Active tumor cell motility is another crucial step in the establishment of metastasis. Cell motility, coupled with proteolysis1,3 is required for the penetration of metastatic cells through subendothelial and interstitial matrices during the transition from in situ to invasive carcinoma and during intravasation and extravasation. Hemostatic abnormalities also have been implicated in the pathogenesis of tumor spread.7 Activation of coagulation factor intermediates assemble adjacent to viable tumor cells leading to generation of enzymatically active thrombin at the host-tumor interface.8 Thrombin,
Prothrombin promotes invasiveness of melanoma cells 273
besides its role in blood coagulation, has been also described to possess properties that may facilitate tumor dissemination, including its mitogenic activity on tumor cells, its abilities to enhance the proliferative response of tumor cells to growth factors and to induce the aggregation of platelets.9–11 Thrombin has been also reported to enhance the adhesion of tumor cells to endothelial cells and subendothelial matrix components and to stimulate the production and secretion of proteolytic enzymes by tumor cells.12–14 In a previous report we demonstrated that the precursor of thrombin, prothrombin, binds to the surface of melanoma cells through its fragment 1 region and stimulates their directional and random motility.15 In the present study, we investigated whether prothrombin is also able to stimulate the invasive ability of melanoma cells on the artificial basement membrane matrigel.
Preparation of prothrombin and its derivatives Prothrombin was purified from human plasma by methods previously described.20 Thrombin and fragment 1 of prothrombin were prepared as described.20 Protein and fragment preparations were homogeneous (more than 95% purity), as determined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). Residual thrombin activity was not detected in the purified prothrombin derivatives, but when present it was inactivated by treating with 1 mM PMSF in Tris-buffered saline (TBS) consisting of 50 mM Tris-HCl, 100 mM NaCl, pH 7.4 for 30 min, followed by dialysis in the same buffer. Each protein and fragment was dialysed against phosphate-buffered saline (PBS) consisting of 100 mM trisodium phosphate and 120 mM NaCl, pH 7.4, before using in the assays. Culture of melanoma cell lines
MATERIALS AND METHODS Materials Human serum albumin (HSA) was obtained from Bayer (Leverkusen, Germany) and Sephacryl S-200, DEAESephacel, Sephadex G-25M and sulfated Sephadex G-50 columns were from Pharmacia LKB Biotechnology (Uppsala, Sweden). Phenylmethyl-sulfonyl fluoride (PMSF) and bovine pancreas chymotypsin were from Wako Pure Chemicals (Osaka, Japan). The synthetic substrate of thrombin, D-Phe-pipecolyl-Arg-p-nitroanilide (S-2238) and that for uPA, Glu-Gly-Arg-pNA-HC1 (S-2444) were purchased from Chromogenix (Mölndal, Sweden) and that of plasmin, Boc-Val-Leu-Lys-MCA, from Peptide Institute (Osaka, Japan). Dulbecco’s modified Eagle’s minimum essential medium (DMEM), hirudin, bovine serum albumin (BSA), collagenase and the Russell’s viper venom (RVV) were from Sigma (St Louis, MO, USA). Plasminogen was purchased from Gelco Diagnostics (Shreveport, LA, USA). Penicillin and streptomycin were from Nakalai Tesque (Kyoto, Japan). Fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids, L-glutamine and vitamin solution were purchased from Gibco BRL (Gran Island, NY, USA). Gelatin was from Bio-Rad Laboratory (Richmond, CA, USA). Polyclonal anti-matrix metalloproteinase (MMP) –2 antibody was from Chemicon International Inc. (Temecula, CA, USA). Matrigel was purchased from Becton Dickinson Labware (Bedford, MA, USA). Factor X and factor V were purified from human plasma using methods previously described16,17 and they were activated by incubating with purified RVV-factor X activator and RVV-factor V activator, respectively.18,19 All other chemicals and reagents used were of the best quality commercially available. © Harcourt Publishers Ltd 1999
The K-1735 murine melanoma cell lines (M2 and CL10) were kind gifts from Dr Isaiah J. Fidler (University of Texas M.D. Anderson Cancer Center). The K-1735 cell lines were cultured in DMEM supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, L-glutamine and 2-fold vitamin solution. The cells were cultured in an atmosphere of 5% CO2-95% air at 37°C. All assays were carried out using cells at passages 3 to 10. Cells were harvested by a brief exposure to 0.25% trypsin/0.02% EDTA, and subcultured weekly. Invasion assay The invasion activity of murine melanoma cells was assayed following the method of Albini et al.21 with some modifications. Polycarbonate filters (Neuroprobe, Cabin Johnson, MD, USA) with 8-µm pore size were coated with matrigel following the manufacturer’s instructions. Matrigel was diluted to the desired concentration with cold DMEM (free of FBS), then applied to the filter, dried under a hood, and reconstituted with serum-free DMEM. Matrigel-coated filter was assembled in a 96-well Boyden chamber. Cells (0.5~1.0 × 106/ml) suspended in serumfree DMEM containing varying concentrations of prothrombin, fragment 1 or thrombin were added to the upper compartments of the chamber. As control, cells with serum-free DMEM were also added to the upper compartments. For making comparisons, cells suspended in DMEM containing a gradient concentrations of HSA were also added to the upper wells of the chamber. The lower wells of the chamber were filled with DMEM containing 0.1% HSA. The chamber was assembled and incubated at 37°C for 6 h in an atmosphere of 5%CO2-95% air. After incubation, the filters were removed, fixed in methanol and stained with Diff Quick (American Fibrinolysis & Proteolysis (1999) 13(6), 272–278
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Scientific Products, MacGraw Park, IL, USA). Cells that grew on the upper surface of the filter were mechanically removed, and those that migrated to the lower side of the filter were counted by light microscope under high power field (× 400). For each triplicate, the number of cells in five high power fields was determined and the counts were averaged. Invasion assay was also carried out in the presence of prothrombin, fragment 1 or thrombin that were pre-treated with 250 antithrombin units (ATU) of hirudin, a specific inhibitor of thrombin, at 37°C for 30 min.
Zymograghy Murine melanoma M2 cells cultured in 10 cm dish at 80% of confluency were washed three times with serumfree DMEM and then treated with 0.5 µM prothrombin, fragment 1 or thrombin for 48 h in the presence or absence of 250 ATU hirudin. The culture supernatants were then investigated for plasminogen activator activity using gel zymograghy as previously described.22 Briefly, aliquots of conditioned medium were mixed with gel loading buffer and they were electrophoresed on a 10% acrylamide gel containing 5% SDS, 1 mg/ml of gelatin in the presence of 10 µg/ml of plasminogen as substrate. After electrophoresis, the gel was incubated in 2.5% Triton X-100 at room temperature for one hour to remove SDS. Then, the gel was incubated in a buffer containing 100 mM Tris-HCl, pH 7.9, 30 mM CaCl2, and 1 µM ZnCl2 or 100 mM glycine/NaOH, pH 8.3, at 37°C for 16 h. The gel was then fixed in methanol: acetic acid: water (5:1:5) solution and stained with Coomassie blue, destained in 7% acetic acid solution and photographed. The lytic bands were quantified by densitometry using the public domain NIH image program (Wayne Rasband, NIH, Research Service Branch, NIMH, USA).
Western blotting SDS-PAGE of conditioned medium of murine melanoma M2 cells treated with prothrombin or thrombin was performed followed by blotting onto nitrocellulose membrane (Bio-Rad Laboratory). The membrane was incubated with blocking solution containing 100 mM PBS, pH 7.5, 5% BSA and 0.5% Tween 20 and then exposed to a 1/2000 dilution of rabbit polyclonal antiMMP-2 antibody for one hour while shaking at room temperature. After washing the membrane with PBS containing 0.1% BSA and 0.5% Tween 20, the membrane was incubated with a 1/2000 dilution of anti-rabbit IgG antibody conjugated to horse-raddish peroxidase. The membrane was washed and quantified using an enhanced chemiluminescence detection system (ECL, Amersham, Buckinghamshire, UK) as described previously.23 The Fibrinolysis & Proteolysis (1999) 13(6), 272–278
bands were quantified by densitometry by the NIH image program. Activity of plasmin and uPA in culture-conditioned medium Murine melanoma M2 cells grown in 10 cm dishes at 80 ~ 90% of confluency were washed twice with serum-free DMEM and then treated with 0.5 µM prothrombin, fragment 1 or thrombin for 48 h in the presence or absence of 250 ATU hirudin. Plasmin activity in cultureconditioned medium was analyzed using the fluorogenic substrate, Boc-Val-Leu-Lys-MCA, as previously described.24 In brief, aliquots of culture-conditioned medium were added to PBS containing 100 nM plasminogen and incubated a 37°C for 2 h; then, the release of aminomethyl coumarine (AMC) from the substrate was followed by measuring the increase in fluorescence intensity with a spectrophotometer (Shimadzu, RF-510). The uPA activity in culture-conditioned medium was measured using the chromogenic substrate S-2444. Aliquots of culture-conditioned medium added to TBS were incubated with 0.25 mM S-2444 at room temperature for 5 min; then the release of p-nitroaniline was determined at absorbance of 405 nm using an ELISA plate reader (EAR 340, Salzburg, Austria). Statistical analysis The results of invasion assays were expressed as invasion index; this was determined by dividing the number of cells migrating in response to prothrombin or its derivatives by the number of cells migrating in response to culture medium alone. Data were expressed as the mean index plus minus the standard error of the mean (SEM). The difference between the invasion index induced by ligands and control buffer (medium with increasing concentrations of HSA) was calculated by analysis of variance (Dunnett’s test) using the StatView 4.1 package software (ABACUS CONCEPTS, Berkeley, CA, USA) for the Macintosh. A P < 0.05 was considered as statistically significant.
RESULTS Prothrombin, fragment 1 and thrombin elicited a dosedependent invasion of murine melanoma M2 cells, a cell line with high metastatic potential (Fig. 1). At concentrations above 0.5 nM, prothrombin, fragment 1 or thrombin significantly increased the invasive ability of these cells as compared to controls (P < 0.05). The maximal effect was induced at concentrations between 0.5 ~ 1 µM of each attractant (P < 0.01 vs control). To evaluate whether the motile response of M2 cells to thrombin © Harcourt Publishers Ltd 1999
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Fig. 1 Matrigel invasion of melanoma M2 cells stimulated with prothrombin (●), fragment 1 (◆), thrombin (▲), and control buffer (■ ■ ) and with those in the presence of hirudin (● ●, ◆, ▲). Invasion assay was carried out as described in Materials and Methods. Data are expressed as the mean of invasion index ± SEM. Each point represents the mean of five high power magnification (× 400) fields from each filter from three separate experiments. *; P < 0.05 as compared to control buffer. **; P < 0.01 as compared to thrombin only. Statistical analysis was carried out by Dunnett’s test.
Fig. 2 Matrigel invasion of melanoma cells with high (M2) (closed column) and low (CL10) (open column) metastatic potential in response to 1 µM prothrombin (column 2), fragment 1 (column 3), thrombin (column 4) and control buffer (column 1). Data are expressed as the mean of invasion index ± SEM. Bars; indicate mean values; *; P < 0.05 as compared to M2 cells. Statistical analysis was carried out by Dunnett’s test.
depends on the catalytic site of this protease, and that to prothrombin or fragment 1 on the presence of residual thrombin, both prothrombin and its derivatives were incubated with hirudin for 30 min before the invasion assay. As shown in Figure 1, treatment with hirudin inhibited more than 90% of thrombin-induced invasive activity of M2 cells (P < 0.01) but not that induced by prothrombin or fragment 1. We also evaluated the invasive ability of CL10 cells, a melanoma cell line with low metastatic potential, in © Harcourt Publishers Ltd 1999
Fig. 3 Activity of plasmin and uPA in culture-conditioned media from M2 cells. M2 cells were cultured for 24 h with 0.5 µM prothrombin or fragment 1 or thrombin in the presence or absence of 250 ATU hirudin and then aliquots of conditioned medium were analyzed. Plasmin activity (closed column) was measured using the fluorogenic substrate Boc-Val-Leu-Lys-MCA by spectrophotometry. uPA activity (open column) was measured using the chromogenic substrate S-2444. Column 1, serum-free medium from M2 cells; column 2, medium from prothrombin treated cells; column 3, medium from prothrombin and hirudin treated cells; column 4, medium from fragment 1 treated cells; column 5, medium from fragment 1 and hirudin treated cells; column 6, medium from thrombin-treated cells; column 7, medium from thrombin and hirudin treated cells. *; P < 0.05 as compared to control. Statistical analysis was carried out by Dunnett’s test.
response to prothrombin and its derivatives. The CL10 cells showed weak invasive ability compared to high metastatic M2 cells in the presence of the same concentrations of prothrombin or its derivatives (P < 0.05) (Fig. 2). The activity of plasmin and uPA in culture-conditioned medium from M2 cells treated with each ligand was also evaluated. As shown in Figure 3, the activity of plasmin and uPA was significantly enhanced in conditioned medium of M2 cells treated with prothrombin, fragment 1 or thrombin as compared to control medium. Hirudin inhibited the production of uPA induced by thrombin but not that induced by prothrombin or fragment 1. The activity of plasminogen activator in culture-conditioned medium of M2 cells treated with prothrombin, fragment 1 or thrombin was also evaluated by gel zymography. Conditioned medium of cells treated with prothrombin, fragment 1 or thrombin significantly increased the activity of plasminogen activator (high molecular weight uPA [56 kDa] and lower molecular weight uPA [35 kDa] bands) as shown by the gel zymography and densitometric analysis (Figure 4A B). Stimulation of M2 cells with thrombin resulted in increased generation of the active form (62 kDa) from the inactive gelatinase A (72 kDa) as shown by Western blotting (Fig. 5). The apparent decrease in 68 kDa intermediate form in thrombin-treated cells may be secondary to increased generation of the active forms. Incubation of melanoma cells with thrombin in the presence of hirudin decreased the generation of the 62 kDa active forms of Fibrinolysis & Proteolysis (1999) 13(6), 272–278
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gelatinase A. Prothrombin increased the production of gelatinase A and the generation of its active forms (62-kDa). These effects of prothrombin were not inhibited by hirudin (Fig. 5).
A. kDa 56 55 1
2
3
4
5
6
7
Fig. 4 Gel zymography showing the activity of plasminogen activator of culture-conditioned media from M2 cells. M2 cells were cultured for 24 h with 0.5 µM prothrombin or fragment 1 or thrombin in the presence or absence of 250 ATU hirudin and then each conditioned medium was run on SDS-PAGE. (A) Lytic bands: lane 1, serum-free medium from M2 cells; lane 2, medium from thrombin treated cells; lane 3, medium from thrombin and hirudin treated cells; lane 4, medium from prothrombin treated cells; lane 5, medium from prothrombin and hirudin treated cells; lane 6, medium from fragment 1 treated cells; lane 7, medium from fragment 1 and hirudin treated cells (B) Densitometric analysis of lytic bands. The intensity of each band was calculated using the NIH software. *; P < 0.05 as compared to control. Statistical analysis was carried out by Dunnett’s test.
kDa
72 68 64 62 1
2
3
4
5
Fig. 5 Western blotting of culture-conditioned media from M2 cells. M2 cells were cultured for 24 h with 0.5 µM prothrombin or thrombin in the presence or absence of 250 ATU hirudin and then each conditioned medium was run on SDS-PAGE. Lane 1, serumfree medium from M2 cells; lane 2, medium from thrombin treated cells; lane 3, medium from thrombin and hirudin treated cells; lane 4, medium from prothrombin treated cells; lane 5, medium from prothrombin and hirudin treated cells.
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DISCUSSION The role of components of the coagulation cascade in the pathogenesis of tumor spread has been well documented.7,25 Thrombin may contribute to tumor stroma formation by converting fibrinogen to fibrin or may support tumor cell proliferation because of its growth factor activity.8,26,27 Thrombin may also enhance the invasive phenotype of cancer cells by increasing their motility and their adhesion to endothelial cells, platelets and extracellular matrix proteins.12,14 The results of this study showed that, in addition to thrombin, its precursor prothrombin also promotes the invasion of melanoma cells on the artificial basement membrane matrigel. The effect of each factor was significant above concentrations of 0.5 nM and it was maximal at 1 µM. These findings have pathophysiological implications because prothrombin which circulates normally at plasma concentrations between 1 and 2 µM, may stimulate the intravascular migration of tumor cells invading the perivascular regions of host tissues, or vice versa, locally extravasated prothrombin may stimulate the extravasation of malignant cells arrested on capillary walls during their intravascular dissemination. Several lines of evidence suggest that local tumor invasion depends on the ability of tumor cells to attach and proteolyze components of the extracellular matrix and to migrate into regions of the matrix modified by proteolysis.28,29 Various proteolytic enzymes secreted by tumor cells have been implicated in the process of basement membrane and stromal matrix degradation during invasion and metastasis;30 those principally involved are serine proteases (plasminogen activator, plasmin, thrombin, trypsin, cathepsin G), cystein proteinase (cathepsin B) and a group of metalloproteinases.31 This latter group comprises the membrane-bound type MMP-1 (MT-MMP1), interstitial collagenase (MMP-1), stromelysin (MMP-3) and the type IV collagenases which include the gelatinase A (MMP-2) and gelatinase B (MMP-9).32–35 In the present study, it was shown that culture of melanoma cells in the presence of thrombin enhances the conversion of pro-gelatinase A (72 kDa) to its active forms (62 kDa) as shown by the Western blot analysis. The mechanism of gelatinase conversion to its active form by thrombin is unclear. The membrane-bound type (MT) MMP-1 has been previously reported to activate gelatinase A.35 Thus, it is conceivable that thrombin promotes gelatinase A activation by increasing the expression of MT-MMP-1 by melanoma cells. Thrombin stimulatory activity on © Harcourt Publishers Ltd 1999
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MT-MMP-1 expression likely proceeds via a proteolytically activation mechanism. Thrombin probably cleaves the external loop of its receptor on melanoma cells leading to activation of G-protein coupled signal transduction pathways and to increased secretion of MTMMP-1.36,37 The fact that hirudin inhibited the effects of thrombin suggests that the cellular effect of this protease is mediated by proteolytic activity on its receptor. In an attempt to explain the mechanism by which the zymogen prothrombin promotes tumor cell invasiveness, we evaluated the activity of plasmin and uPA in cultureconditioned media of M2 cells. The results showed that besides thrombin, prothrombin and its fragment 1 also significantly enhanced the activity of both plasmin and uPA in culture-conditioned media of M2 cells. In addition, the gel zymography showed that treatment with prothrombin increases the secretion of gelatinase A from M2 melanoma cells and the formation of the active forms of this metalloproteinase. Previous studies have shown that plasmin and plasminogen activator enhance the activation of gelatinase A and gelatinase B.38,39 Thus, it is conceivable that the increased generation of the active forms of gelatinase A as shown by the results of the Western blotting, was due to the increased secretion of plasminogen activator induced by prothrombin or fragment 1 in M2 cells. Interestingly, the cellular effects of thrombin were inhibited by hirudin but not those induced by prothrombin or fragment 1, suggesting that prothrombin and its fragment 1 exert their effect on melanoma cells by different mechanism from thrombin. Specific properties have been described to characterize tumor cell clones with increased ability to generate metastasis: major genetic instability, increased sialylation of cell surface, greater number of membrane receptors for extracellular matrix components, major resistant to host immune responses, enhanced secretion of adhesion molecules, proteolytic enzymes, cytokines and abnormal expression of the so called metastatic genes.40 In the present study, we also evaluated whether melanoma cells (M2 and CL10 cells) with established differences in metastasis potential demonstrate comparable differences in cell invasive ability in response to prothrombin and thrombin. The results showed that highly metastatic melanoma cell lines (M2) responded to each prothrombin and thrombin more significantly that the low metastatic cell lines (CL10). Difference in the number of surface receptors for prothrombin and thrombin may be a possible explanation for the different invasive response of M2 and CL10 cells in the presence of each ligand. In brief, the results of this study showed for the first time (1) that the zymogen prothrombin enhances the invasiveness of melanoma cells in vitro; (2) that both thrombin and prothrombin increase the plasmin and uPA activities and the activation of gelatinase A in © Harcourt Publishers Ltd 1999
culture-conditioned media from murine melanoma cells; and (3) that invasive responses induced by both prothrombin and thrombin differ according to the metastatic potential of melanoma cells. REFERENCES 1. Richard PH. Metastasis. In: Tannock IF, Hill RP, ed. The Basic Science of Oncology, ed 2. Canada: McGraw-Hill, Inc. Health Professions Division Press, 1992: 178–195. 2. Schottenfeld D. Epidemiology of lung cancer. In: Pass HI, Mitchell JB, Johnson DH, Turrisi AT, eds. Lung Cancer: Principles and Practice, Philadelphia: Lippincott-Raven Publishers Press, 1996: 305–321. 3. Liotta LA. Tumor invasion and metastases – role of the extracellular matrix: rhoads memorial award lecture. Cancer Res 1986; 46: 1–7. 4. Nakajima M, Irimura T, DiFerrante D, DiFerrante N, Nicolson G. Heparan sulfate degradation: relation to tumor invasion and metastatic properties of mouse B-16 melanoma sublines. Science 1983; 220: 611–613. 5. Robertson NP, Starkey JR, Meadows GG. Tumor cell invasion of three-dimensional matrices of defined composition: evidence for a specific role for heparan sulfate in rodent cell lines. Cancer Res 1989; 49: 1816–1823. 6. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shafie S. Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 1980; 284: 65–66. 7. Gasic GJ, Tuszynski GP, Gorelik E. Interaction of the hemostatic and immune system in the metastatic spread of tumor cells. Int Rev Exp Pathol 1986; 29: 173–212. 8. Dvorak HF, Harvey VS, Estrella P, Brown LF, Mcdonagh J, Dvorak AM. Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Lab Invest 1987; 57: 673–686. 9. Bruhn HD, Zorborn KH. Influences of clotting factors (thrombin, factor XIII) and of fibronectin on the growth of tumor cells and leukemic cells in vitro. Blut 1983; 46: 85–88. 10. Medrano EE, Cafferata EGA, Cancher F. Role of thrombin in the proliferative response of T-47D mamary tumor cells. Exp Cell Res 1987; 172: 354–364. 11. Nierodzik ML, Kajumo F, Karpatkin S. Effect of thrombin treatment of cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo. Cancer Res 1992; 52: 3267–3272. 12. Nierodzik ML, Plotkin A, Kajumo F, Karpatki S. Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo. J Clin Invest 1991; 87: 229–236. 13. Wojtukiewicz MZ, Tang DG, Nelson KK, Walz DA, Diglio CA, Honn KV. Thrombin enhances tumor cell adhesive and metastatic properties via increased aIIb expression on the cell surface. Thromb Res 1992; 68: 233–245. 14. Wojtukiewicz MZ, Tang DG, Ciarelli JJ et al. Thrombin increases the metastatic potential of tumor cells. Int J Cancer 1993; 54: 793–806. 15. Zhou H, Gabazza EC, Takeya H et al. Prothrombin and its derivatives stimulate motility of melanoma cells. Thromb Haemost 1998; 80: 407–12. 16. Miletich JP, Broze GJ, Majerus PW. The synthesis of sulfated dextran beads for isolation of human plasma coagulation factors II, IX, and X. Anal Biochem 1980; 105: 304–310. 17. Suzuki K, Stenflo J, Dahlbäck B, Teodorsson B. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914–1920.
Fibrinolysis & Proteolysis (1999) 13(6), 272–278
278 Zhou et al.
18. Suzuki K, Dahlbäck B, Stenflo J. Thrombin-catalyzed activation of human coagulation factor V. J Biol Chem 1982; 257: 6556–6564. 19. DiScipio RG, Hermodson MA, Davie EW. Activation of human factor X (Stuart factor) by a protease from Russells viper venom. Biochemistry 1977; 16: 5253–5260. 20. Deguchi H, Takeya H, Gabazza EC, Nishioka J, Suzuki K. Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex. Biochem J 1997; 321: 729–735. 21. Albini A, Iwamoto Y, Kleinman HK et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 1987; 47: 3239–3245. 22. Heussen C, Dowdle EB. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem 1980; 102: 196–202. 23. Smine A, Plantner JJ. Membrane type-1 matrix metalloproteinase in human ocular tissues. Curr Eye Res 1997; 16: 925–929. 24. Suzuki K, Nishioka J, Hashimoto S. Assay method of plasma plasminogen by means of a synthetic fluorogenic peptide substrate (3104-V). Rinsho Byori 1980; 28: 804–807. 25. Rickles FR, Edwards R. Activation of blood coagulation in cancer: Trousseau’s syndrome revisited. Blood 1983; 62: 14–31. 26. Olander JV, Bremer ME, Marasa JC, Feder J. Fibrin enhanced endothelial cell organization. J Cell Physiol 1985; 125: 1–9. 27. Walz DA, Fenton JW. The role of thrombin in tumor cell metastasis. Invas Metast 1994–95; 14: 303–308. 28. Melchiori A, Albini A, Ray JM, Stetler-Stevenson WG. Inhibition of tumor cell invasion by a highly conserved peptide sequence from the matrix metalloproteinase enzyme prosegment. Cancer Res 1992; 52: 2353–2356. 29. Ruddon RW. Cancer Biology ed 3: Biology of tumor metastasis. Oxford University. New York Press, 1995; 410–412. 30. Mignatti P, Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. Physiol Reviews 1993; 73: 161–195.
Fibrinolysis & Proteolysis (1999) 13(6), 272–278
31. Evans CW. The Metastatic Cell: behaviour and biochemistry. Chapman and Hall. Great Britain Press, 1991. 32. Brown PD, Levy AT, Margulies IMK, Liotta LA, StetlerStevenson WG. Independent expression and cellular processing of Mr 72,000 type IV collagenase and interstitial collagenase in human tumorigenic cell lines. Cancer Res 1990; 50: 6184–6191. 33. Okada Y, Morodomi T, Enghild J et al. Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Eur J Biochem 1990; 194: 721–730. 34. Okada Y, Gonoji Y, Naka K et al. Matrix metalloproteinase 9 (92-kDa) gelatinase/type IV collagenase) from HT1080 human fibrosarcoma cells. J Biol Chem 1992; 267: 21712–21719. 35. Sato T, Kondo T, Seiki M, Ito A, Cell type-specific involvement of furin in membrane type 1 matrix metalloproteinase-mediated progelatinase A activation. Ann N Y Acad Sci. 1999; 878: 713–5. 36. Vu T-K H, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057–1068. 37. Fischer EG, Ruf W, Mueller BM. Tissue factor-initiated thrombin generation activates the signaling thrombin receptor on malignant melanoma cells. Cancer Res 1995; 55: 1629–1632. 38. Mazzieri R, Masiero L, Zanetta L et al. Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants. EMBO J 1997; 16: 2319–2332. 39. Baramova EN, Bajou K, Remacle A et al. Involvement of PA/ plasmin system in the processing of pro-MMP-9 and in the second step of pro-MMP-2 activation. FEBS Lett 1997; 405: 157–162. 40. Yabkowitz R, Mansfield PJ, Dixit VM, Suchard SJ. Motility of human carcinoma cells in response to thrombospondin: relationship to metastatic potential and thrombospondin structural domains. Cancer Res 1993; 53: 378–387.
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