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Serum Hepatocyte Growth Factor Activator (HGFA) in Benign Prostatic Hyperplasia and Prostate Cancer
European Urology
European Urology 48 (2005) 686–690
Translational Research: From Lab to Clinic
Serum Hepatocyte Growth Factor Activator (HGFA) in Benign Prostatic Hyperplasia and Prostate Cancer Osamu Nagakawaa,*, Toshiya Yamagishib, Yasuyoshi Fujiuchia, Akira Junichoa, Takuya Akashia, Kazuhiro Nagaikeb, Hideki Fusea a
Department of Urology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan Mitsubishi Chemical Group Science and Technology Research Center, 1000 Kamoshida Aoba-ku, Yokohama 227-8502, Japan
b
Accepted 31 May 2005 Available online 29 June 2005
Abstract Objectives: Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor (HGF). We attempted to clarify whether serum levels of HGFA could be used as a marker for prostate cancer. Material and methods: Serum levels of total HGF and HGFA were measured by enzyme-linked immunosorbent assay in 99 healthy controls, 27 patients with benign prostatic hyperplasia (BPH) and 119 patients with prostate cancer. Results: The mean S.D. serum levels of HGFA in untreated prostate cancer and BPH cases were 0.42 0.24 and 0.50 0.26 ng/ml, respectively (no significant difference). Serum HGFA was significantly elevated in hormonerefractory prostate cancer (stage D3) compared to other stages, while HGF did not significantly differ with regard to clinical stage. Conclusions: Serum HGFA tends was elevated in patients with advanced stage prostate cancer. Further studies in large groups of patients are needed to clarify the clinical value of HGFA. # 2005 Elsevier B.V. All rights reserved. Keywords: HGF; HGFA; Prostate cancer
1. Introduction Hepatocyte growth factor (HGF) is considered to play an important role in functional regulation in the microenvironment of the tissues in physiological states. HGF shows pleiotropic activities such as mitogenic [1], motogenic [2] and angiogenic [3] activities for various types of cells, and acts through a specific membrane receptor encoded by c-met proto-oncogene [4]. It also induces the migration and invasion of various types of cancer cells, including those from colon, stomach, lung, gall bladder and hepatocelluar carcinoma. Both HGF and c-MET were expressed in prostatic tissues and hormone independent cancer cell * Corresponding author. Tel. +81 76 434 7372; Fax: +81 76 434 5039. E-mail address: [email protected] (O. Nagakawa).
lines [5,6]. HGF induced cellular proliferation and scattering in DU-145 prostate cancer cells [5]. HGF also increased the invasive capacity of DU-145 prostate cancer cells [7]. However, the mechanism of the effect of HGF on tumor invasion of prostate cancer cells remains unclear. We demonstrated that HGF promoted the invasiveness of DU-145 and PC-3 prostate cancer cells through the enhancement of the expression of urokinase-type plasminogen activator (u-PA), its receptor (uPAR), matrix metalloproteinase (MMP)-1 and 9 and membrane-type 1-MMP (MT1-MMP) [8,9]. HGF activator is secreted as an inactive singlechain precursor form (pro-HGF). HGF activator (HGFA) is a factor XII-like serine protease that catalyzes proteolytic conversion of pro-HGF to the active two-chain form in response to tissue injury [10,11]. Although HGFA is mainly produced by
0302-2838/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.eururo.2005.05.020
O. Nagakawa et al. / European Urology 48 (2005) 686–690
hepatocytes, recent studies revealed extrahepatic production in the gastrointestinal tract and central nervous system [12]. The breast cancer [13] and renal cell carcinoma [14] specimens also expressed a significantly higher level of HGFA compared with normal tissues. Elevated serum HGF levels have been reported in patients with metastatic prostate cancer [15], but very little is known about HGFA in prostatic carcinoma. In the present study, we attempted to clarify whether serum levels of HGFA could be a useful marker in patients with prostate cancer.
687
the reaction was stopped by the addition of 100 ml/well of 1N H2SO4, and the plates were read at 490 nm in a E-max Micro-plate Reader (Molecular Devices, Palo Alto, CA). Serum total HGF and HGFA levels were considered elevated when they exceeded 2.65 ng/ml and 0.76 ng/ml, respectively, which were the mean + 2 standard deviation of values in healthy subjects. All statistical analyses were carried out with the Statview 5.0 program (Abacus Concepts, Berkely, CA, USA). Differences in the serum levels of HGF or HGFA between BPH and prostate cancer patients were calculated by the Mann-Whitney U-test. Correlative analyses between clinical stages and tumor grades were performed using a one-way analysis of variance (ANOVA) and multiple comparison tests. A Spearman rank test was used to analyze the correlation between HGF and HGFA. A difference with p < 0.05 was considered significant.
2. Materials and methods A retrospective analysis of 95 patients with untreated prostate cancer (age 54–94 years, median 73) and 27 patients with benign prostatic hyperplasia (BPH) (age 54–82 years, median 70) between 2000 and 2004 in Toyama Medical and Pharmaceutical University Hospital was carried out. Diagnosis was confirmed pathologically by transrectal ultrasonography (TRUS) guided systematic biopsy or transurethral resection. The stage was determined according to the Jewett staging system, and the histological grade was determined by WHO classification. Of the 95 patients with prostate cancer, 43 had clinical stage B, 30 stage C, 22 stage D2. Sera were also collected from 24 patients with prostate cancer in progression after endocrine therapy (stage D3) (age 54–92 years, median 76) and 99 healthy males (age 54–84 years, median 68) who had no suspicious findings of prostate cancer in tests including digital rectal examination, TRUS and measurement of serum PSA levels. Informed consent was obtained from all patients for measuring serum HGF and HGFA. Blood samples were taken from the patients, and sera were immediately frozen and stored at 80 8C until analysis. The assay for total HGF (pro-HGF + mature HGF) and HGFA was carried out with an enzyme-linked immunosorbent assay (ELISA). Polystyrene 96-well plates (Costar, Cambridge, MA) were coated with 100 ml/well of 0.05 M carbonic acid-bicarbonate buffer, pH 9.6, containing a primary antibody such as rabbit polyclonal antibody to human total HGF or HGFA, at a concentration of 10 mg/ml for 12 h at 4 8C. After removing the buffer from the plates, the plates were blocked with 300 ml/well of 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 12 h at 4 8C. After removing the PBS from the plates, 50 ml/well of 20 mM phosphoric acid sodium buffer solution containing 0.15 M NaCl and 0.1% BSA and 50 ml/well of serum samples were added to the plates and incubated for 1 h at room temperature. The plates were washed 5 times with 20 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl and 0.05% Tween-20, and then incubated with 100 ml/ well of PBS a containing biotin labeled secondary antibody at a concentration of 1 mg/ml, such as anti-total HGF goat polyclonal antibody or anti-HGFA rabbit polyclonal antibody, respectively, and 1% BSA for 1 h at room temperature. The plates were washed with 20 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl and 0.05% Tween-20, and then incubated with 100 ml/well of PBS containing a 1:3,000 dilution of horseradish peroxidase (HRP) (Amasham Biosciences, Tokyo, Japan) labeled streptavidin and 1% BSA for 1 h at room temperature. The wells washed well and developed with a 0.4 mg/ml O-phenylenediamine dehydrochloride substrate (Shigma-Aldrich Japan, Tokyo, Japan) in 0.1 M of citric acid buffer, pH 5.0, with 0.015% H2O2 at room temperature. Then
3. Results 3.1. Serum HGF and HGFA levels In healthy controls, the mean S.D. serum levels of HGF and HGFA were 1.19 0.73 ng/ml and 0.28 0.24 ng/ml, respectively (Table 1). There were no significant differences in serum HGF or HGFA levels between healthy controls and patients with BPH. The mean serum levels of HGF in patients with BPH and untreated prostate cancer were 1.07 0.41 and 1.56 0.72 ng/ml, respectively, and those of HGFA were 0.42 0.24 and 0.50 0.26 ng/ml, respectively. There were no significant differences in serum HGFA levels between patients with BPH and untreated prostate cancer (p = 0.1296). In contrast, serum levels of HGF in patients with untreated prostate cancer were significantly higher than those in patients with BPH (p = 0.0002). The mean and the distribution of serum total HGF and HGFA according to clinical stage are shown in Figs. 1 and 2. While HGF did not differ among prostate cancer subgroups, serum HGFA was increased in patients with stage D3 compared with stage B, C or D2 (p = 0.0001, p = 0.0009 and p = 0.0224, respectively). In addition, the differences in serum total HGFA levels between patients with BPH and stage D3 prostate cancer were also significant (p < 0.0001). There were no significant differences in serum HGF or HGFA levels among untreated prostate cancer subgroups according to tumor grade. 3.2. Elevation of serum HGF and HGFA Serum HGF was elevated in 0 patients with BPH, 6 patients (14.0%) with stage B, 3 patients (10.0%) with stage C, 1 patient (4.8%) with stage D2 and 4 patients (16.7%) with stage D3. Serum HGFA was elevated in 3 patients (11.1%) with BPH, 2 patients (4.7%) with stage B, 4 patients (11.8%) with stage C, 8 patients
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O. Nagakawa et al. / European Urology 48 (2005) 686–690
Table 1 Patient characteristics n
Age Mean S.D.
HGF (ng/ml) Mean S.D. (range)
HGFA (ng/ml) Mean S.D. (range)
Healthy controls BPH
99 27
67.4 7.1 69.4 8.5
1.19 0.73 (0.21–3.33) 1.07 0.41 (0.48–2.29)
0.28 0.24 (0.08–1.90) 0.42 0.24 (0.14–1.12)
Prostate cancer Untreated
95
73.5 8.3
1.56 0.72 (0.59–4.37)
0.50 0.26 (0.17–1.71)
465.7 2082.9 (3.1–18900)
43 30 22
1.63 1.20 (0.71–3.61) 1.53 0.76 (0.59–4.37) 1.44 0.59 (0.81–3.12)
0.43 0.26 (0.17–1.71) 0.46 0.19 (0.17–0.91) 0.68 0.27 (0.20–1.28)
11.8 14.0 (3.1–93.1) 291.9 635.1 (8.1–2940) 1663.1 4113.4 (6.9–18900)
23 39 33 24
1.67 0.60 1.64 0.87 1.41 0.60 1.83 1.08
0.51 0.33 0.44 0.20 0.57 0.26 1.58 2.08
Clinical stage B C D2 Tumor grade Well Moderately Poorly Hormone-refractory (stage D3)
76.0 7.5
(36.4%) with stage D2 and 14 patients (58.3%) with stage D3. 3.3. Correlation between HGF and HGFA A positive correlation was found between HGF and HGFA in patients with untreated prostate cancer (r = 0.467, p < 0.0001) or those with stage D3 (r = 0.443, p = 0.0338).
4. Discussion HGF and c-met proto-oncogene are overexpressed in prostatic carcinoma [5,16] and HGF stimulates the growth and invasion and cell motility in prostate cancer cells. Moreover, serum HGF level was elevated in men with prostate cancer compared with those without
Fig. 1. Serum total HGF levels according to clinical stage in patients with prostate cancer and frequencies of their elevation. Each bar represents the mean S.D. (*) individual serum HGF values.
(0.76–2.97) (0.71–4.37) (0.59–3.12) (0.71–4.87)
(0.17–1.71) (0.20–0.95) (0.17–1.28) (0.29–8.27)
PSA(ng/ml) Mean S.D. (range) 0.7 0.4 (0.2–1.7) 4.3 3.1 (0.4–13.3)
9.4 5.6 (3.1–24.2) 350.4 1083.5 (3.3–6303) 918.9 3312.5 (3.6–18900) 531.2 1241.2 (5.1–6026)
prostate cancer and in men with metastatic disease compared with those with localized disease [15]. In the present study, serum levels of HGF in patients with untreated prostate cancer were also significantly higher than those in patients with BPH. In contrast to a previous report [15], we did not find a significant association between serum HGF levels and clinical stage. We do not know the exact reason for these contradictory results. In the present study, we measured the serum levels of total HGF (pro-HGF + mature HGF). If we could measure only mature HGF levels, a positive correlation might be found between mature HGF and clinical stage.
Fig. 2. Serum HGFA levels according to clinical stage in patients with prostate cancer and frequencies of their elevation. Each bar represents the mean S.D. *p < 0.05, **p < 0.01, ***p < 0.001; (*); individual serum HGFA values.
O. Nagakawa et al. / European Urology 48 (2005) 686–690
HGF is secreted as an inactive single-chain precursor called pro-HGF. Pro-HGF is converted to an active two-chain form by HGFA, matriptase [17] and plasminogen activators [18]. Among these converters of HGF, HGFA is the most potent enzyme in this processing [12]. HGFA is produced mainly by hepatocytes as an inactive form (pro-HGFA) and is activated by thrombin [19], but can also be produced by various types of cancer cells such as breast cancer [13], renal cell carcinoma [14], colorectal carcinoma [20] and hepatocelluar carcinoma [21]. In addition, tumor tissues from node-positive patients expressed a higher level of HGFA than from those without nodal involvement in breast cancer [13]. However the extrahepatic expression of HGFA in prostate cancers has not yet been determined. In the present study, we demonstrated for the first time, that serum HGFA levels were elevated in patients with advanced-stage prostate cancer, especially hormone- resistant prostate cancer (stage D3). An increase of serum HGFA levels in prostate cancer would allow for a more potent influence from HGF, because a higher level of pro-HGF would be processed. Significant correlations were observed between total HGF and HGFA in patients with untreated prostate cancer, and those with stage D3. In the present study, serum HGFA was elevated in patients with advanced
689
stage prostate cancer. Therefore, we need to examine the source of elevated serum HGFA and whether the HGFA level could be used an indicator of the progression of prostate cancer after endocrine treatment. HGFA activity is controlled in a pericellular microenvironment by two new serine protease inhibitors. These inhibitors are called HGFA inhibitor type 1 (HAI-1) [22] and type 2 (HAI-2) [23]. HAI-1 mRNA was expressed in normal tissues such as the kidney, pancreas, prostate, small intestine, lungs and placenta and many cancer cells. HAI-1 protein was also expressed in PC-3 and DU-145 prostate cancer cells [13]. The balance between HGFA and HAI is a critical factor in pericellular HGF activation in tumors. Therefore, the relationship between HGFA and HAI in prostate cancer will be needed to be determined.
5. Conclusions The mean serum levels of HGFA in patients with hormone resistant prostate cancer were significantly elevated compared with those in patients with untreated prostate cancer. Additional studies in large groups of patients are needed to define the clinical value of HGFA.
References [1] Sakata H, Takayama H, Sharp R, Rubin JS, Merlino G, LaRochelle WJ. Hepatocyte growth factor/scatter factor overexpression induces growth, abnormal development, and tumor formation in transgenic mouse livers. Cell Growth Differ 1996;7:1513–23. [2] Tajima H, Matsumoto K, Nakamura T. Regulation of cell growth and motility by hepatocyte growth factor and receptor expression in various cell species. Exp Cell Res 1992;202:423–31. [3] Bussolino F, Di Renzo MF, Ziche M, Bocchietto E, Olivero M, Naldini L, et al. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 1992;119:629–41. [4] Bottaro DP, Rubin JS, Faketto DL, Chan AM, Kmiecik TE, Vande Woude GF, et al. Identification of the hepatocyte growth factor as the c-MET proto-oncogene product. Science 1991;251:802–4. [5] Humphrey AP, Zhu X, Zanegar R, Swanson PE, Ratliff TL, Vollmer RT, et al. Hepatocyte growth factor and its receptor (c-MET) in prostatic carcinoma. Am J Pathol 1995;147:386–96. [6] Kurimoto S, Moriyama N, Horie S, Sakai M, Kameyama S, Akimoto Y, et al. Co-expression of hepatocyte growth factor and its receptor in human prostate cancer. Histochem J 1998;30:27–32. [7] Nishimura K, Kitamura M, Takada S, Nonomura N, Tsujimura A, Matsumiya K, et al. Regulation of invasive potential of human prostate cancer cell lines by hepatocyte growth factor. Int J Urol 1998;5:276–81. [8] Nagakawa O, Murakami K, Yamaura T, Fujiuchi Y, Murata J, Fuse H, et al. Expression of membrane-type 1 matrix metalloproteinase(MT1MMP) on prostate cancer cell lines. Cancer Lett 2000;155:173–9. [9] Fujiuchi Y, Nagakawa O, Murakami K, Fuse H, Saiki I. Effect of hepatocyte growth factor on invasion of prostate cancer cells. Oncol Rep 2003;10:1001–6.
[10] Miyazawa K, Shimomura T, Kitamura A, Kondo J, Morimoto Y, Kitamura N. Molecular cloning and sequence analysis of the cDNA for a human serine protease responsible for activation of hepatocyte growth factor. Structural similarity for the protease precursor to blood coagulation factor XII. J Biol Chem 1993;268:10024–8. [11] Miyazawa K, Shimomura T, Kitamura A. Activation of hepatocyte growth factor in the injured tissues is mediated by hepatocyte growth factor activator. J Biol Chem 1996;271:3615–8. [12] Kataoka H, Miyata S, Uchinokura S, Itoh H. Roles of hepatocyte growth factor (HGF) activator and HGF activator inhibitor in the pericellular activation of HGF/scatter factor. Cancer Metastasis Rev 2003;22:223–36. [13] Parr C, Watkins G, Mansel RE, Jiang WG. The hepatocyte growth factor regulatory factors in human breast cancer. Clin Cancer Res 2004;10:202–11. [14] Yamauchi M, Kataoka H, Itoh H, Seguchi T, Hsaui Y, Osada Y. Hepatocyte growth factor activator inhibitor type 1 and 2 are expressed by tubular epithelium in kidney and down regulated in renal cell carcinoma. J Urol 2004;171:890–6. [15] Naughton M, Picus J, Zhu X, Catalona WJ, Vollmer RT, Humphrey PA. Scatter factor-hepatocyte growth factor elevation in the serum of patients with prostate cancer. J Urol 2001;165:1325–8. [16] Zhu X, Humphrey PA. Overexpression and regulation of expression of scatter factor/hepatocyte growth factor in prostatic carcinoma. Urology 2000;56:1071–4. [17] Lee S, Dickson RB, Lin CY. Activation of hepatocyte growth factor and urokinase/plasminogen activator by matriptase, an epithelial membrane serine protease. J Biol Chem 2000;275: 36720–5.
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[18] Naldini L, Tamagnone L, Vigna E, Sachs M, Hartmann G, Birchmeier W, et al. Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor. EMBO J 1992;11:4825–33. [19] Shimomura T, Kondo J, Ochiai M, Naka D, Miyazawa K, Morimoto Y, et al. Activation of the zymogen of hepatocyte growth factor activator by thrombin. J Biol Chem 1993;268: 22927–32. [20] Kataoka H, Hamasuna R, Itoh H, Kitamura N, Koono M. Activation of hepatocyte growth factor/scatter factor in colorectal carcinoma. Cancer Res 2000;60:6148–59.
[21] Nagata K, Hirono S, Ido A, Kataoka H, Moriuchi A, Shimomura T, et al. Expression of hepatocyte growth factor activator and hepatocyte growth factor activator inhibitor type 1 in human hepatocellular carcinoma. Biochem Biophys Res Commun 2001;289:205–11. [22] Shimomura T, Denda K, Kitamura A, Kwaguchi T, Kito M, Kondo J, et al. Hepatocyte growth factor activator inhibitor, a novel Kunitz-type serine protease inhibitor. J Biol Chem 1997;272:6370–6. [23] Kawaguchi T, Qin L, Shimomura T, Kondo K, Matsumoto K, Denda K, et al. Purification and cloning of hepatocyte growth factor activator inhibito rtype 2, a Kunitz-type serine protease inhibitor. J Biol Chem 1997;272:27558–64.
European Urology 48 (2005) 686–690
Translational Research: From Lab to Clinic
Serum Hepatocyte Growth Factor Activator (HGFA) in Benign Prostatic Hyperplasia and Prostate Cancer Osamu Nagakawaa,*, Toshiya Yamagishib, Yasuyoshi Fujiuchia, Akira Junichoa, Takuya Akashia, Kazuhiro Nagaikeb, Hideki Fusea a
Department of Urology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan Mitsubishi Chemical Group Science and Technology Research Center, 1000 Kamoshida Aoba-ku, Yokohama 227-8502, Japan
b
Accepted 31 May 2005 Available online 29 June 2005
Abstract Objectives: Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor (HGF). We attempted to clarify whether serum levels of HGFA could be used as a marker for prostate cancer. Material and methods: Serum levels of total HGF and HGFA were measured by enzyme-linked immunosorbent assay in 99 healthy controls, 27 patients with benign prostatic hyperplasia (BPH) and 119 patients with prostate cancer. Results: The mean S.D. serum levels of HGFA in untreated prostate cancer and BPH cases were 0.42 0.24 and 0.50 0.26 ng/ml, respectively (no significant difference). Serum HGFA was significantly elevated in hormonerefractory prostate cancer (stage D3) compared to other stages, while HGF did not significantly differ with regard to clinical stage. Conclusions: Serum HGFA tends was elevated in patients with advanced stage prostate cancer. Further studies in large groups of patients are needed to clarify the clinical value of HGFA. # 2005 Elsevier B.V. All rights reserved. Keywords: HGF; HGFA; Prostate cancer
1. Introduction Hepatocyte growth factor (HGF) is considered to play an important role in functional regulation in the microenvironment of the tissues in physiological states. HGF shows pleiotropic activities such as mitogenic [1], motogenic [2] and angiogenic [3] activities for various types of cells, and acts through a specific membrane receptor encoded by c-met proto-oncogene [4]. It also induces the migration and invasion of various types of cancer cells, including those from colon, stomach, lung, gall bladder and hepatocelluar carcinoma. Both HGF and c-MET were expressed in prostatic tissues and hormone independent cancer cell * Corresponding author. Tel. +81 76 434 7372; Fax: +81 76 434 5039. E-mail address: [email protected] (O. Nagakawa).
lines [5,6]. HGF induced cellular proliferation and scattering in DU-145 prostate cancer cells [5]. HGF also increased the invasive capacity of DU-145 prostate cancer cells [7]. However, the mechanism of the effect of HGF on tumor invasion of prostate cancer cells remains unclear. We demonstrated that HGF promoted the invasiveness of DU-145 and PC-3 prostate cancer cells through the enhancement of the expression of urokinase-type plasminogen activator (u-PA), its receptor (uPAR), matrix metalloproteinase (MMP)-1 and 9 and membrane-type 1-MMP (MT1-MMP) [8,9]. HGF activator is secreted as an inactive singlechain precursor form (pro-HGF). HGF activator (HGFA) is a factor XII-like serine protease that catalyzes proteolytic conversion of pro-HGF to the active two-chain form in response to tissue injury [10,11]. Although HGFA is mainly produced by
0302-2838/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.eururo.2005.05.020
O. Nagakawa et al. / European Urology 48 (2005) 686–690
hepatocytes, recent studies revealed extrahepatic production in the gastrointestinal tract and central nervous system [12]. The breast cancer [13] and renal cell carcinoma [14] specimens also expressed a significantly higher level of HGFA compared with normal tissues. Elevated serum HGF levels have been reported in patients with metastatic prostate cancer [15], but very little is known about HGFA in prostatic carcinoma. In the present study, we attempted to clarify whether serum levels of HGFA could be a useful marker in patients with prostate cancer.
687
the reaction was stopped by the addition of 100 ml/well of 1N H2SO4, and the plates were read at 490 nm in a E-max Micro-plate Reader (Molecular Devices, Palo Alto, CA). Serum total HGF and HGFA levels were considered elevated when they exceeded 2.65 ng/ml and 0.76 ng/ml, respectively, which were the mean + 2 standard deviation of values in healthy subjects. All statistical analyses were carried out with the Statview 5.0 program (Abacus Concepts, Berkely, CA, USA). Differences in the serum levels of HGF or HGFA between BPH and prostate cancer patients were calculated by the Mann-Whitney U-test. Correlative analyses between clinical stages and tumor grades were performed using a one-way analysis of variance (ANOVA) and multiple comparison tests. A Spearman rank test was used to analyze the correlation between HGF and HGFA. A difference with p < 0.05 was considered significant.
2. Materials and methods A retrospective analysis of 95 patients with untreated prostate cancer (age 54–94 years, median 73) and 27 patients with benign prostatic hyperplasia (BPH) (age 54–82 years, median 70) between 2000 and 2004 in Toyama Medical and Pharmaceutical University Hospital was carried out. Diagnosis was confirmed pathologically by transrectal ultrasonography (TRUS) guided systematic biopsy or transurethral resection. The stage was determined according to the Jewett staging system, and the histological grade was determined by WHO classification. Of the 95 patients with prostate cancer, 43 had clinical stage B, 30 stage C, 22 stage D2. Sera were also collected from 24 patients with prostate cancer in progression after endocrine therapy (stage D3) (age 54–92 years, median 76) and 99 healthy males (age 54–84 years, median 68) who had no suspicious findings of prostate cancer in tests including digital rectal examination, TRUS and measurement of serum PSA levels. Informed consent was obtained from all patients for measuring serum HGF and HGFA. Blood samples were taken from the patients, and sera were immediately frozen and stored at 80 8C until analysis. The assay for total HGF (pro-HGF + mature HGF) and HGFA was carried out with an enzyme-linked immunosorbent assay (ELISA). Polystyrene 96-well plates (Costar, Cambridge, MA) were coated with 100 ml/well of 0.05 M carbonic acid-bicarbonate buffer, pH 9.6, containing a primary antibody such as rabbit polyclonal antibody to human total HGF or HGFA, at a concentration of 10 mg/ml for 12 h at 4 8C. After removing the buffer from the plates, the plates were blocked with 300 ml/well of 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 12 h at 4 8C. After removing the PBS from the plates, 50 ml/well of 20 mM phosphoric acid sodium buffer solution containing 0.15 M NaCl and 0.1% BSA and 50 ml/well of serum samples were added to the plates and incubated for 1 h at room temperature. The plates were washed 5 times with 20 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl and 0.05% Tween-20, and then incubated with 100 ml/ well of PBS a containing biotin labeled secondary antibody at a concentration of 1 mg/ml, such as anti-total HGF goat polyclonal antibody or anti-HGFA rabbit polyclonal antibody, respectively, and 1% BSA for 1 h at room temperature. The plates were washed with 20 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl and 0.05% Tween-20, and then incubated with 100 ml/well of PBS containing a 1:3,000 dilution of horseradish peroxidase (HRP) (Amasham Biosciences, Tokyo, Japan) labeled streptavidin and 1% BSA for 1 h at room temperature. The wells washed well and developed with a 0.4 mg/ml O-phenylenediamine dehydrochloride substrate (Shigma-Aldrich Japan, Tokyo, Japan) in 0.1 M of citric acid buffer, pH 5.0, with 0.015% H2O2 at room temperature. Then
3. Results 3.1. Serum HGF and HGFA levels In healthy controls, the mean S.D. serum levels of HGF and HGFA were 1.19 0.73 ng/ml and 0.28 0.24 ng/ml, respectively (Table 1). There were no significant differences in serum HGF or HGFA levels between healthy controls and patients with BPH. The mean serum levels of HGF in patients with BPH and untreated prostate cancer were 1.07 0.41 and 1.56 0.72 ng/ml, respectively, and those of HGFA were 0.42 0.24 and 0.50 0.26 ng/ml, respectively. There were no significant differences in serum HGFA levels between patients with BPH and untreated prostate cancer (p = 0.1296). In contrast, serum levels of HGF in patients with untreated prostate cancer were significantly higher than those in patients with BPH (p = 0.0002). The mean and the distribution of serum total HGF and HGFA according to clinical stage are shown in Figs. 1 and 2. While HGF did not differ among prostate cancer subgroups, serum HGFA was increased in patients with stage D3 compared with stage B, C or D2 (p = 0.0001, p = 0.0009 and p = 0.0224, respectively). In addition, the differences in serum total HGFA levels between patients with BPH and stage D3 prostate cancer were also significant (p < 0.0001). There were no significant differences in serum HGF or HGFA levels among untreated prostate cancer subgroups according to tumor grade. 3.2. Elevation of serum HGF and HGFA Serum HGF was elevated in 0 patients with BPH, 6 patients (14.0%) with stage B, 3 patients (10.0%) with stage C, 1 patient (4.8%) with stage D2 and 4 patients (16.7%) with stage D3. Serum HGFA was elevated in 3 patients (11.1%) with BPH, 2 patients (4.7%) with stage B, 4 patients (11.8%) with stage C, 8 patients
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O. Nagakawa et al. / European Urology 48 (2005) 686–690
Table 1 Patient characteristics n
Age Mean S.D.
HGF (ng/ml) Mean S.D. (range)
HGFA (ng/ml) Mean S.D. (range)
Healthy controls BPH
99 27
67.4 7.1 69.4 8.5
1.19 0.73 (0.21–3.33) 1.07 0.41 (0.48–2.29)
0.28 0.24 (0.08–1.90) 0.42 0.24 (0.14–1.12)
Prostate cancer Untreated
95
73.5 8.3
1.56 0.72 (0.59–4.37)
0.50 0.26 (0.17–1.71)
465.7 2082.9 (3.1–18900)
43 30 22
1.63 1.20 (0.71–3.61) 1.53 0.76 (0.59–4.37) 1.44 0.59 (0.81–3.12)
0.43 0.26 (0.17–1.71) 0.46 0.19 (0.17–0.91) 0.68 0.27 (0.20–1.28)
11.8 14.0 (3.1–93.1) 291.9 635.1 (8.1–2940) 1663.1 4113.4 (6.9–18900)
23 39 33 24
1.67 0.60 1.64 0.87 1.41 0.60 1.83 1.08
0.51 0.33 0.44 0.20 0.57 0.26 1.58 2.08
Clinical stage B C D2 Tumor grade Well Moderately Poorly Hormone-refractory (stage D3)
76.0 7.5
(36.4%) with stage D2 and 14 patients (58.3%) with stage D3. 3.3. Correlation between HGF and HGFA A positive correlation was found between HGF and HGFA in patients with untreated prostate cancer (r = 0.467, p < 0.0001) or those with stage D3 (r = 0.443, p = 0.0338).
4. Discussion HGF and c-met proto-oncogene are overexpressed in prostatic carcinoma [5,16] and HGF stimulates the growth and invasion and cell motility in prostate cancer cells. Moreover, serum HGF level was elevated in men with prostate cancer compared with those without
Fig. 1. Serum total HGF levels according to clinical stage in patients with prostate cancer and frequencies of their elevation. Each bar represents the mean S.D. (*) individual serum HGF values.
(0.76–2.97) (0.71–4.37) (0.59–3.12) (0.71–4.87)
(0.17–1.71) (0.20–0.95) (0.17–1.28) (0.29–8.27)
PSA(ng/ml) Mean S.D. (range) 0.7 0.4 (0.2–1.7) 4.3 3.1 (0.4–13.3)
9.4 5.6 (3.1–24.2) 350.4 1083.5 (3.3–6303) 918.9 3312.5 (3.6–18900) 531.2 1241.2 (5.1–6026)
prostate cancer and in men with metastatic disease compared with those with localized disease [15]. In the present study, serum levels of HGF in patients with untreated prostate cancer were also significantly higher than those in patients with BPH. In contrast to a previous report [15], we did not find a significant association between serum HGF levels and clinical stage. We do not know the exact reason for these contradictory results. In the present study, we measured the serum levels of total HGF (pro-HGF + mature HGF). If we could measure only mature HGF levels, a positive correlation might be found between mature HGF and clinical stage.
Fig. 2. Serum HGFA levels according to clinical stage in patients with prostate cancer and frequencies of their elevation. Each bar represents the mean S.D. *p < 0.05, **p < 0.01, ***p < 0.001; (*); individual serum HGFA values.
O. Nagakawa et al. / European Urology 48 (2005) 686–690
HGF is secreted as an inactive single-chain precursor called pro-HGF. Pro-HGF is converted to an active two-chain form by HGFA, matriptase [17] and plasminogen activators [18]. Among these converters of HGF, HGFA is the most potent enzyme in this processing [12]. HGFA is produced mainly by hepatocytes as an inactive form (pro-HGFA) and is activated by thrombin [19], but can also be produced by various types of cancer cells such as breast cancer [13], renal cell carcinoma [14], colorectal carcinoma [20] and hepatocelluar carcinoma [21]. In addition, tumor tissues from node-positive patients expressed a higher level of HGFA than from those without nodal involvement in breast cancer [13]. However the extrahepatic expression of HGFA in prostate cancers has not yet been determined. In the present study, we demonstrated for the first time, that serum HGFA levels were elevated in patients with advanced-stage prostate cancer, especially hormone- resistant prostate cancer (stage D3). An increase of serum HGFA levels in prostate cancer would allow for a more potent influence from HGF, because a higher level of pro-HGF would be processed. Significant correlations were observed between total HGF and HGFA in patients with untreated prostate cancer, and those with stage D3. In the present study, serum HGFA was elevated in patients with advanced
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stage prostate cancer. Therefore, we need to examine the source of elevated serum HGFA and whether the HGFA level could be used an indicator of the progression of prostate cancer after endocrine treatment. HGFA activity is controlled in a pericellular microenvironment by two new serine protease inhibitors. These inhibitors are called HGFA inhibitor type 1 (HAI-1) [22] and type 2 (HAI-2) [23]. HAI-1 mRNA was expressed in normal tissues such as the kidney, pancreas, prostate, small intestine, lungs and placenta and many cancer cells. HAI-1 protein was also expressed in PC-3 and DU-145 prostate cancer cells [13]. The balance between HGFA and HAI is a critical factor in pericellular HGF activation in tumors. Therefore, the relationship between HGFA and HAI in prostate cancer will be needed to be determined.
5. Conclusions The mean serum levels of HGFA in patients with hormone resistant prostate cancer were significantly elevated compared with those in patients with untreated prostate cancer. Additional studies in large groups of patients are needed to define the clinical value of HGFA.
References [1] Sakata H, Takayama H, Sharp R, Rubin JS, Merlino G, LaRochelle WJ. Hepatocyte growth factor/scatter factor overexpression induces growth, abnormal development, and tumor formation in transgenic mouse livers. Cell Growth Differ 1996;7:1513–23. [2] Tajima H, Matsumoto K, Nakamura T. Regulation of cell growth and motility by hepatocyte growth factor and receptor expression in various cell species. Exp Cell Res 1992;202:423–31. [3] Bussolino F, Di Renzo MF, Ziche M, Bocchietto E, Olivero M, Naldini L, et al. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 1992;119:629–41. [4] Bottaro DP, Rubin JS, Faketto DL, Chan AM, Kmiecik TE, Vande Woude GF, et al. Identification of the hepatocyte growth factor as the c-MET proto-oncogene product. Science 1991;251:802–4. [5] Humphrey AP, Zhu X, Zanegar R, Swanson PE, Ratliff TL, Vollmer RT, et al. Hepatocyte growth factor and its receptor (c-MET) in prostatic carcinoma. Am J Pathol 1995;147:386–96. [6] Kurimoto S, Moriyama N, Horie S, Sakai M, Kameyama S, Akimoto Y, et al. Co-expression of hepatocyte growth factor and its receptor in human prostate cancer. Histochem J 1998;30:27–32. [7] Nishimura K, Kitamura M, Takada S, Nonomura N, Tsujimura A, Matsumiya K, et al. Regulation of invasive potential of human prostate cancer cell lines by hepatocyte growth factor. Int J Urol 1998;5:276–81. [8] Nagakawa O, Murakami K, Yamaura T, Fujiuchi Y, Murata J, Fuse H, et al. Expression of membrane-type 1 matrix metalloproteinase(MT1MMP) on prostate cancer cell lines. Cancer Lett 2000;155:173–9. [9] Fujiuchi Y, Nagakawa O, Murakami K, Fuse H, Saiki I. Effect of hepatocyte growth factor on invasion of prostate cancer cells. Oncol Rep 2003;10:1001–6.
[10] Miyazawa K, Shimomura T, Kitamura A, Kondo J, Morimoto Y, Kitamura N. Molecular cloning and sequence analysis of the cDNA for a human serine protease responsible for activation of hepatocyte growth factor. Structural similarity for the protease precursor to blood coagulation factor XII. J Biol Chem 1993;268:10024–8. [11] Miyazawa K, Shimomura T, Kitamura A. Activation of hepatocyte growth factor in the injured tissues is mediated by hepatocyte growth factor activator. J Biol Chem 1996;271:3615–8. [12] Kataoka H, Miyata S, Uchinokura S, Itoh H. Roles of hepatocyte growth factor (HGF) activator and HGF activator inhibitor in the pericellular activation of HGF/scatter factor. Cancer Metastasis Rev 2003;22:223–36. [13] Parr C, Watkins G, Mansel RE, Jiang WG. The hepatocyte growth factor regulatory factors in human breast cancer. Clin Cancer Res 2004;10:202–11. [14] Yamauchi M, Kataoka H, Itoh H, Seguchi T, Hsaui Y, Osada Y. Hepatocyte growth factor activator inhibitor type 1 and 2 are expressed by tubular epithelium in kidney and down regulated in renal cell carcinoma. J Urol 2004;171:890–6. [15] Naughton M, Picus J, Zhu X, Catalona WJ, Vollmer RT, Humphrey PA. Scatter factor-hepatocyte growth factor elevation in the serum of patients with prostate cancer. J Urol 2001;165:1325–8. [16] Zhu X, Humphrey PA. Overexpression and regulation of expression of scatter factor/hepatocyte growth factor in prostatic carcinoma. Urology 2000;56:1071–4. [17] Lee S, Dickson RB, Lin CY. Activation of hepatocyte growth factor and urokinase/plasminogen activator by matriptase, an epithelial membrane serine protease. J Biol Chem 2000;275: 36720–5.
690
O. Nagakawa et al. / European Urology 48 (2005) 686–690
[18] Naldini L, Tamagnone L, Vigna E, Sachs M, Hartmann G, Birchmeier W, et al. Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor. EMBO J 1992;11:4825–33. [19] Shimomura T, Kondo J, Ochiai M, Naka D, Miyazawa K, Morimoto Y, et al. Activation of the zymogen of hepatocyte growth factor activator by thrombin. J Biol Chem 1993;268: 22927–32. [20] Kataoka H, Hamasuna R, Itoh H, Kitamura N, Koono M. Activation of hepatocyte growth factor/scatter factor in colorectal carcinoma. Cancer Res 2000;60:6148–59.
[21] Nagata K, Hirono S, Ido A, Kataoka H, Moriuchi A, Shimomura T, et al. Expression of hepatocyte growth factor activator and hepatocyte growth factor activator inhibitor type 1 in human hepatocellular carcinoma. Biochem Biophys Res Commun 2001;289:205–11. [22] Shimomura T, Denda K, Kitamura A, Kwaguchi T, Kito M, Kondo J, et al. Hepatocyte growth factor activator inhibitor, a novel Kunitz-type serine protease inhibitor. J Biol Chem 1997;272:6370–6. [23] Kawaguchi T, Qin L, Shimomura T, Kondo K, Matsumoto K, Denda K, et al. Purification and cloning of hepatocyte growth factor activator inhibito rtype 2, a Kunitz-type serine protease inhibitor. J Biol Chem 1997;272:27558–64.