The significance of placenta growth factor in angiogenesis and clinical outcome of human gastric cancer

Cancer Letters 213 (2004) 73–82 www.elsevier.com/locate/canlet

The significance of placenta growth factor in angiogenesis and clinical outcome of human gastric cancer Chiung-Nien Chena,d, Fon-Jou Hsiehb,d, Yunn-Ming Chengc, Wen-Fan Chengb,d, Yi-Ning Sub, King-Jen Changa,d, Po-Huang Leea,d,* a

b

Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan, ROC Department of Gynecology and Obstetrics, National Taiwan University Hospital, Taipei, Taiwan, ROC c Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan, ROC d Angiogenesis Research Center, National Taiwan University, Taipei, Taiwan, ROC Received 20 January 2004; received in revised form 12 April 2004; accepted 3 May 2004

Abstract Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family of proangiogenic factors and its overexpression has been linked to pathological angiogenesis. We studied the relationship between the expression of PlGF and VEGF in human gastric cancer tissues and microvessel density (MVD), as well as clinical outcome in 79 patients with gastric cancer by using an enzyme immunoassay for PlGF and VEGF expression levels in gastric cancers and surrounding noncancerous mucosa. PlGF protein levels were significantly higher in tumor than in the corresponding non-tumorous mucosa (median value 48.5 vs 9.8 pg/mg, P!0.001). In contrast, VEGF protein levels were not (66.7 vs 80.7 pg/mg, PZ0.522). VEGF expression level was not significantly correlated with MVD, patient survival, and clinicopathological factors except Lauren classification in this study. PlGF may be an important angiogenic factor in human gastric cancer, and PlGF expression level was significantly correlated with serosal invasion, positive lymph node metastases, tumor stages, and patient survival. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Vascular endothelial growth factor; Microvessel density; Survival; Clinicopathological factor

1. Introduction Angiogenesis is a very complex phenomenon and essential for the growth of solid tumors measuring * Corresponding author. Address: Department of Surgery, National Taiwan University Hospital, National Taiwan University College of Medicine, No. 7, Chung-Shan S Road, Taipei, Taiwan, ROC. Tel.: C886-2-2312-3446x5104; fax: C886-2-2356-8810. E-mail address: [email protected] (P.-H. Lee).

more than a few millimeters [1]. It permits rapid tumor growth and potential presence of tumor metastasis [2,3]. Tumor angiogenesis is a significant predictor of prognosis and hematogenous metastasis of patients with gastric cancer [4,5]. Angiogenesis is not a passive process and is driven by many angiogenic factors produced by tumor. Of the known angiogenic factors, vascular endothelial growth factor (VEGF) have been shown to play an important role not only in angiogenesis and prognosis

0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.05.020

74

C.-N. Chen et al. / Cancer Letters 213 (2004) 73–82

of different human tumors [6–18], but also in physiological [19,20] and nonmalignant pathological conditions [21]. Placenta growth factor (PlGF) is a member of the VEGF family of proangiogenic factors [22]. VEGF binds with high affinity to two tyrosine kinase receptors: VEGF receptor-1 (Flt-1) and VEGFR-2 (Flk-1) [23–25]. PlGF binds with high affinity to Flt-1 but not to Flk-1 [26]. In contrast to VEGF, PlGF is not regulated by hypoxia and its physiological and pathological roles are largely unknown [27]. PlGF is upregulated in placenta [28] and during the active angiogenic phase of wound healing [29]. Recent basic studies in mouse model [30] has shown PlGF cooperated with VEGF could enhance angiogenesis in pathological conditions such as tumor and ischemic tissue. Its upregulation has been found in human meningiomas, hemangioblastomas, melanoma, cervical squamous cell carcinoma and associated with angiogenesis in renal cell carcinoma [31–36]. On the contrary, PlGF was down regulated in thyroid carcinoma, germ cell tumor, and cervical adenocarcinoma [36–38]. To our knowledge, there is no report on the significance of PlGF expression in the clinical outcome of human gastric cancer. In order to elucidate the clinical significance of PlGF expression, this study was conducted to quantify expression of PlGF and VEGF in human gastric cancer tissue and correlate them with microvessel density (MVD), clinicopathological factors and patient survival.

2. Materials and methods 2.1. Patients A total of 79 patients with gastric cancer, who had undergone radical gastrectomy at our institution from July 1995 to March 1999, were included in this study. They were all proved to have adenocarcinomas by panendoscopic biopsies. They were staged according to TNM system. Criteria for consideration as curative resection were the complete removal of a primary gastric tumor, D2 dissection of regional lymph nodes, and no macroscopic tumor being left behind. They had no detectable metastasis in liver, peritoneum and distant organ at the time of surgery. No other previous or concomitant primary cancer was present. No patient had received chemotherapy and radiotherapy before

surgery. Clinicopathologic factors including age, sex, gross types of tumors (Borrmann classification), histological types of tumors (Lauren classification), depth of tumor invasion, and status of lymph node metastasis documented with histologic findings were reviewed and stored in patients’ database. The patients were followed up from 3 to 46 months after surgery. The follow-up intervals were calculated as survival intervals after surgery. 2.2. Microvessel, PlGF, VEGF staining and evaluation The paraffinized tumor blocks of 79 patients whose gastric cancers were stained for endothelial cell CD34 antigen using the labelled streptavidin–biotin after antigen retrieval (Fig. 2). Briefly, deparaffinized sections were heated in a pressure cooker. After endogenous peroxidase was blocked with 3% hydrogen peroxide in the section, each section was incubated with nonimmuned horse serum. The sections were incubated in anti-CD34 monoclonal antibody (Santa Cruz Biotecnology, Inc., Santa Cruz, CA) at a dilution of 1:20, or the control nonimmune serum at 4 8C overnight. The sections were incubated with link antibodies followed by peroxidase conjugated streptavidin complex (LSAB kit, DAKO Corporation, Carpinteria, CA). The peroxidase activity was visualized with diaminobenzidine tetrahydroxychloride solution (DAB, DAKO corporation, Carpinteria, CA) as the substrate. The sections were lightly counterstained with hematoxylin. After screening the areas with intense neovascularized spots at low power field (100!), microvessels in the area with the highest number of discrete microvessels were counted in a 400! field. Three separate intense neovascularized areas were assessed, and the mean was calculated as MVD of each tumor evaluated. PlGF and VEGF were stained using the same method as microvessel staining with anti-PlGF and anti-VEGF monoclonal antibody (Santa Cruz Biotecnology, Inc., Santa Cruz, CA) at a dilution of 1:40 and 1:80, respectively. 2.3. Extraction of the tumor cytosols Protein lysate from each specimen was prepared using 10 mg tissue cut into tiny pieces, suspended in cell lysis buffer (0.15 M NaCl; 0.1 M Tris, pH 8.0;

C.-N. Chen et al. / Cancer Letters 213 (2004) 73–82

1 mM EDTA, pH 8.0; 1 mM PMSF) and mechanically homogenized with a polytron PT 3000 (30,000 rpm for 1 min). 2.4. Quantification of PlGF and VEGF levels in cancer and non-tumor tissue of the same resected stomach Concentrations of VEGF in human gastric cancer and non-tumor gastric tissue cytosolic extracts were quantified using a ‘Quantikine’ human VEGF immunoassay (R&D Systems, Inc., Minneapolis, MN). Cytosols were stored at K80 8C before measurement of VEGF levels. Diluted cytosols were incubated in triplicates overnight at 4 8C on microtiter plates coated with a murine monoclonal antibody against human VEGF. Unbound proteins were washed off, and an enzyme-linked polyclonal antibody specific for VEGF was added to ‘sandwich’ the VEGF immobilized during the first incubation. A substrate solution for horseradish peroxidase was added, and color developed in proportion to the amount of antibody-bound VEGF. The absordance of the color was read at 450 nm. A standard curve, consisting of known amounts of VEGF, was carried through the above procedure, and the concentrations of VEGF in the unknown samples were determined from this standard curve. Concentrations of VEGF were expressed as picograms per milligram cytosolic protein. Concentrations of PlGF in human gastric cancer and non-tumor gastric tissue cytosolic extracts were quantified using a ‘Quantikine’ human PlGF immunoassay (R&D Systems, Inc., Minneapolis, MN). The procedure was the same as that performed for VEGF. PlGF and VEGF expression levels of gastric cancers were represented as tumor protein levels, which are the concentration values of PlGF and VEGF in the tumor tissues directly measured by enzyme immunoassay (EIA), and the expression ratio defined as a ratio of protein level in the tumor to that in the corresponding non-tumor tissue. Therefore, both tumor protein levels and expression ratios of PlGF and VEGF were used to perform statistical calculations with MVD and clinical outcomes, respectively, and results were compared. 2.5. Statistics Protein levels of PlGF and VEGF between nontumorous and tumorous tissues were determined by

75

Wilcoxon rank sum test. The relationship between PlGF, VEGF, and the various clinicopathological factors was examined by Wilcoxon rank sum test. A multivariate analysis for correlation of expression ratio of PlGF and VEGF with clinicopathological factors and MVD was performed by linear regression method. Correlation of tumor protein level and expression ratio of PlGF and VEGF with tumor stages was determined by Kruskal Willis test. Univariate survival analysis was calculated with Kaplan–Meier method, and the differences were analyzed by the Log-rank test. A multivariate survival analysis was performed using Cox proportional hazards model to investigate the independent prognostic factors. Statistical significance was defined as P!0.05.

3. Results 3.1. VEGF and PlGF protein levels in gastric adenocarcinoma Protein levels of VEGF and PlGF in gastric cancer tissue ranged from 1.2 to 672.7 pg/mg, and 1.6 to 239 pg/mg, and the median values were 66.7 and 48.5 pg/mg, respectively. Protein levels of VEGF and PlGF in corresponding noncancerous mucosa ranged from 4.2 to 549.5 pg/mg, and 0.7 to 58.5 pg/mg, and the median values were 80.7 and 9.8 pg/mg, respectively. PlGF protein levels of tumors were significantly higher than those of the corresponding non-tumorous mucosa (P!0.001). In contrast, VEGF protein levels of the tumors were similar to those of the corresponding non-tumorous mucosa (PZ0.522; Fig. 1).

3.2. Localization of VEGF and PlGF in the gastric adenocarcinoma tissues Immunohistochemical studies of gastric cancer tissues revealed that both PlGF and VEGF were localized in the cytoplasm of cancer cells. In addition to cancer cells, VEGF immunoreactivity was also present in some fibroblasts, smooth muscle cells,

76

C.-N. Chen et al. / Cancer Letters 213 (2004) 73–82

Fig. 1. The protein level of PlGF, but not VEGF, is significantly higher in gastric cancer tissues than in non-tumor corresponding gastric mucosa. The left scale is for PlGF concentration, and the right scale is for VEGF.

inflammatory cells and vascular endothelial cells (Fig. 2). 3.3. Correlation of PlGF and VEGF with MVD and clinicopathologic factors 3.3.1. Tumor protein level as a parameter VEGF protein level of intestinal type gastric cancer was significantly higher than that of diffuse type gastric cancer (PZ0.017). There was no other significant association between VEGF protein levels, MVD and clinicopathological factors as tested in this study. However, PlGF protein level in the patients with T3 and T4 lesions, positive lymph node metastases, and greater MVD (O32) was significantly higher than in those with T1 and T2 lesions (PZ 0.026), negative lymph node metastases (PZ0.022), less MVD (!32; PZ0.029). 3.3.2. Expression ratio as a parameter Expression ratios of PlGF were not correlated significantly with those of VEGF (Pearson coefficient rZK0.048, PZ0.686). VEGF and PlGF expression ratios ranged from 0.01 to 82.43 and 0.10 to 58.62, respectively. VEGF and PlGF expression ratios that were greater than one were observed in 48.1% (38/79) and 88.6% (70/79) of patients, respectively. The median values for VEGF and PlGF expression ratios

of these patients were 1.160 and 5.640, respectively. Therefore, we classified them into two subgroups: high VEGF and PlGF groups, for which the ratios were greater than 1.160 and 5.640, respectively, and low VEGF and PlGF groups, for which the ratios were lower than 1.160 and 5.640, respectively. These two cutoff points were used to perform further statistical calculation correlated with clinicopathologic factors and survival. VEGF expression ratio of intestinal type gastric cancer was significantly higher than that of diffuse type gastric cancer (PZ0.033). There was no other significantly association between VEGF expression ratios, MVD and clinicopathological factors as tested in this study. However, PlGF expression ratio in the patients with T3 and T4 lesions, positive lymph node metastases, and greater MVD (O32) was significantly higher than in those with T1 and T2 lesions (PZ0.026), negative lymph node metastases (PZ0.002), less MVD (!32). A multiple linear regression analysis showed only PlGF expression ratio was significantly correlated with MVD (PZ0.0228; Table 1). Results of clinical correlation study using PlGF and VEGF protein levels in gastric cancer were consistent with those using PlGF and VEGF expression ratios. 3.4. Correlation of PlGF and VEGF with tumor stages 3.4.1. Tumor protein level as a parameter PlGF protein levels of the tumors were significantly associated with tumor stages (PZ0.024), but VEGF protein levels were not (PZ0.228; Fig. 3A). 3.4.2. Expression ratio as a parameter PlGF expression ratios were significantly associated with tumor stages (PZ0.011), but VEGF expression ratios were not (PZ0.586; Fig. 3B). 3.5. Correlation between PlGF or VEGF and survival 3.5.1. Tumor protein level as a parameter Using a PlGF protein level, O120 pg/mg (the upper 30% of PlGF protein level), as a cutoff point showed significant survival difference (PZ0.0470). VEGF protein level did not show any survival difference.

C.-N. Chen et al. / Cancer Letters 213 (2004) 73–82

77

Fig. 2. Immunohistochemical staining for PlGF and VEGF. The normal gastric epithelial cells are negative for PlGF (A). Strong diffuse cytoplasmic staining of PlGF is seen in cancer cells (B). In non-tumorous gastric mucosa, activated lymphocytes in germinal center are positive for VEGF (C). Strong diffuse cytoplasmic staining of VEGF in tumor cells is seen (D). Positive staining of VEGF is seen in endothelial cells (E). Positive staining of VEGF is seen in fibroblasts (F). Smooth muscular cells in gastric wall are positive for VEGF (G). (Original magnification A: 100!, B:200!, C:400!, D:200!, E:400!, F:400!, G:200!).

78

C.-N. Chen et al. / Cancer Letters 213 (2004) 73–82

Table 1 Association of microvessel density with clinicopathological factors, PlGF and VEGF expression ratios determined by multivariate analysis Variables

B

SE

Exp(b)

Sig

Borrmann type (type I and II or type III and IV) Lauren classification (intestinal or diffuse type) Depth of invasion (T1 T2 or T3 T4) Nodal status (negative or positive) PlGF expression ratio (high or low) VEGF expression ratio (high or low)

0.1327 0.2424 1.0043 1.3730 1.2957 1.0621

0.3469 0.3773 0.8964 0.9414 0.5690 0.6926

101419 1.2744 2.7299 3.9472 3.6535 2.8925

0.7020 0.5204 0.2626 0.1447 0.0228 0.1252

B, b regression coefficient; SE, standard error; Exp(b), exponent b; Sig, significance.

3.5.2. Expression ratio as a parameter The survival rates were calculated using the Kaplan–Meier method. The survival rate of the group with high PlGF expression ratio was significantly lower than that with low one (30.5% vs 51.4%, PZ 0.0475), however, VEGF expression ratio did not show significant difference of survival rate (37.5% vs 45.7%, PZ0.6053; Table 2). The effects of variables presumably associated with patient survival were studied by multivariate analysis using Cox proportional hazards model. As a result, MVD (PZ0.0226) and depth of tumor invasion (PZ0.0434) were independent prognostic factors in this study (Table 3).

4. Discussion The results of a large clinical trial presented in the annual meeting of American Society of Clinical Oncology in 2003 showed that an antiangiogenesis drug, the anti-VEGF antibody Avastin, prolonged the lives of patients with advanced colon cancer but did not in the patients with advanced breast cancer [39]. This suggests that tumors from different patients and tissue origin may use different repertoires of VEGF and VEGF-related molecules or other angiogenic factors to stimulate angiogenesis. That is why it is crucial to elucidate interaction of signaling pathway of these molecules and to evaluate whether there is a relation between the expression of VEGF-related molecules, the density of microvessels in histological section, and the aggressiveness of the tumor, as reported for VEGF before [40]. The expression of PlGF is restricted to the placenta and not observed in the majority of normal adult tissues [26]. The role of PlGF as an angiogenic factor has been debated for a long time.

Fig. 3. (A) The tumor protein level of PlGF is significantly correlated with increasing tumor stage but that of VEGF is not. The right scale is for PlGF concentration, and the left scale is for VEGF concentration. (B) The expression ratio of PlGF is significantly correlated with increasing tumor stage but that of VEGF is not.

C.-N. Chen et al. / Cancer Letters 213 (2004) 73–82 Table 2 Clinicopathological factors affecting survival rate by univariate analysis Variable Borrmann type I and II III and IV Lauren classification Intestinal type Diffuse type Depth of invasion T1 and T2 T3 and T4 Lymph node metastasis Negative Positive VEGF expression ratio Low High PlGF expression ratio Low High MVD !32 R32

No. of patient

3-year survival rate (%)

P value 0.3819

14 65

52.5 41.9 0.0061

33 46

66.1 26.7 0.0000

21 58

83.7 23.0 0.0001

21 58

82.2 25.0 0.6053

41 38

45.7 37.5 0.0475

40 39

51.4 30.5

44 35

67.5 17.1

0.0001

However, increasing evidence has shown that by upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the ‘angiogenic switch’ in many pathologic disorders [30], and can also

79

potentiate the activity of suboptimal concentrations of VEGF [41]. Recent published observations from PlGF knockout mice studies indicate that PlGF receptor-mediated signaling may have a significant role in adult pathological angiogenesis, and loss of PlGF impairs angiogenesis in ischemic diseases and in cancer, without affecting physiological angiogenesis [30]. Overexpression of PlGF in a transgenic mouse model demonstrated that it resulted in substantial increase in the number, branching and size of blood vessels [42]. Up to now, several mechanisms were proposed to explain enhanced pathological angiogenesis by upregulation of PlGF. PlGF displaces VEGF from Flt-1 and makes more VEGF available to bind and activate Flk-1 [41]. PlGF transmits signals through Flt-1, independently of cross talk with Flk-1 [43]. PlGF activates Flt-1, leading to intermolecular transphosphorylation of Flk-1 and enhances Flk-1-phosphotyrosine levels. PlGF forms a heterodimer with VEGF (VEGF/ PlGF), which can activate and transmit angiogenic signals through the Flk-1/Flt-1 heterodimer receptor complex. PlGF displaces VEGF homodimers, which stimulate Flk-1/Flt-1 heterodimerization, from Flt-1 [44]. Among the known angiogenic factors, VEGF has emerged as a central regulator of the angiogenic process in physiological and pathological conditions as in various human malignancies [23,45]. In this study, VEGF expression level of gastric cancer tissue was comparable with that of corresponding non-tumor mucosa, and this observation was the same as that reported by Kido et al., who also assess VEGF expression of gastric cancer using EIA [46]. PlGF was over-expressed in nearly 90% of gastric cancers compared with that of corresponding non-tumor

Table 3 Association of various factors with overall survival determined by the Cox proportional hazards model Variables

B

SE

Exp(b)

Sig

Borrmann type (type I and II or type III and IV) Lauren classification (intestinal or diffuse type) Depth of invasion (T1 T2 or T3 T4) Nodal status (negative or positive) PlGF expression ratio (high or low) VEGF expression ratio (high or low) Microvessel density (!32 or R32)

0.2370 0.2765 1.3567 0.7708 K0.3921 K0.4929 0.9359

0.2488 0.2748 0.7024 0.7098 0.3801 0.4459 0.4106

1.2674 1.3185 3.8833 2.1616 0.6756 0.6108 2.5495

0.3408 0.3144 0.0434 0.2775 0.3023 0.2690 0.0226

B, b regression coefficient; SE, standard error; Exp(b), exponent b; Sig, significance.

80

C.-N. Chen et al. / Cancer Letters 213 (2004) 73–82

mucosa. To better represent individual difference, the ratio of tumor to non-tumor expression was adapted to normalize individual variation, and both tumor protein level and expression ratio showed similar correlation with clinical outcome. Although the expression of VEGF was associated with patient’s survival and tumor vascularity in some reports [15–17], our present study and other reports [18,30,46] did not show any significant association. This discordance may reflect the difference in patient cohort, histological types, tumor stages and assay technique used. In this study, VEGF of intestinal type gastric cancer was significantly higher than that of diffuse type, and patients with intestinal type tumor survived significantly longer than those with diffuse type tumor. Thus, this may be one of the reasons why VEGF expression did not correlate with overall survival of patients, and this result is consistent with that of Tanigawa et al. [18]. Expression levels of VEGF in gastric cancer reported by Kido et al. [46], breast cancer by Relf et al. [47], and renal cell carcinoma by Slaton et al. [48] were measured with EIA rather than immunohistochemical stain, as in the most of similar study, and did not show any association between VEGF protein level and clinical outcome. EIA can give an assessment with less sampling bias, and can measure various growth factors that present within tumor mass. It not only provides data of non-tumor tissues as control but also an average of expression levels of growth factors produced by various cell types such as stromal cells and macrophages in a larger mass of tumor, not a focal expression as evaluated with subjective immunohistochemical grading. Based on the VEGF/VEGF-related molecule profiles reported by Salven et al. [40], tumors can be divided into different groups depending on which factors predominate. VEGF predominated in the early stages of tumor development, while other angiogenic factors such as PlGF, fibroblast growth factors and transforming growth factor beta played more additional roles than VEGF did in advanced stages. Tumors that do not produce significant amounts of VEGF may use VEGF-related molecules to stimulate angiogenesis [39,47]. In this study, most of the tumors (81%) were in advanced stages and VEGF-related molecule, PlGF, was significantly upregulated in gastric cancer tissue and was significantly correlated with MVD-evaluated angiogenesis and tumor stages,

however, VEGF kept stationary expression level in various angiogenic grading and different tumor stages. This seemed to be in line with the report that PlGF enhanced VEGF to activate tumor angiogenesis in mouse models [30,42,44]. Thus, these may be the reasons PlGF expression, not VEGF, was correlated with MVD and overall patient survival in this clinical setting. In conclusions, PlGF expression was associated with depth of invasion, lymph node metastasis, TNM stages and patient survival and strongly correlated with MVDevaluated angiogenesis in human gastric cancer. We suggested that PlGF may play an important role in the angiogenesis of human gastric cancer.

Acknowledgements Supported by grant from National Science Council and Department of Industrial Technology, Ministry of Economic Affairs, Taipei, Taiwan.

References [1] J. Folkman, D. Long, F. Becker, Growth and metastasis of tumor in organ culture, Cancer 16 (1963) 453–457. [2] J. Folkman, What is the evidence that tumors are angiogenesis dependent?, J. Natl Cancer Inst. 82 (1990) 4–6. [3] R. Bicknell, A.L. Harris, Novel growth regulatory factors and tumor angiogenesis, Eur. J. Cancer 27 (1991) 781–784. [4] K. Maeda, Y.S. Chung, S. Takatsuka, Y. Ogawa, T. Sawada, Y. Yamashita, et al., Tumor angiogenesis as a predictor of recurrence in gastric carcinoma, J. Clin. Oncol. 13 (1995) 477–481. [5] N. Tanigawa, H. Amaya, M. Matsumura, T. Shimomatsuya, T. Horiuchi, R. Muraoka, et al., Extent of tumor vascularization correlates with prognosis and hematogenous metastasis in gastric carcinomas, Cancer Res. 56 (1996) 2671–2676. [6] D.W. Leung, G. Cachianes, W.J. Kuang, D.V. Goeddel, N. Ferrara, Vascular endothelial growth factor is a secreted angiogenic mitogen, Science 246 (1989) 306–1309. [7] N. Ferrara, K. Houck, L. Jakeman, D.W. Leung, Molecular and biological properties of the vascular endothelial growth factor family of proteins, Endocr. Rev. 13 (1992) 18–32. [8] R.A. Berkman, M.J. Merrill, W.C. Reinhold, W.T. Monnaci, A. Saxena, W.C. Clark, et al., Expression of the vascular permeability factor/vascular endothelial growth factor gene in central nervous system neoplasms, J. Clin. Invest. 91 (1992) 153–159. [9] K.H. Plate, G. Breier, H.A. Weich, W. Risau, et al., Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo, Nature 359 (1992) 845–848.

C.-N. Chen et al. / Cancer Letters 213 (2004) 73–82 [10] B. Berse, L.F. Brown, L. Van de Water, H.F. Dvorak, D.R. Senger, et al., Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors, Mol. Biol. Cell 3 (1992) 211–220. [11] T.A. Olson, D. Mohanraj, L.F. Carson, S. Ramakrishnan, Vascular permeability factor gene expression in normal and neoplastic human overies, Cancer Res. 54 (1994) 276–280. [12] H.T. Zhang, P. Craft, P.A. Scott, M. Ziche, H.A. Weich, A.L. Harris, et al., Enhancement of tumor growth and vascular density by transfection of vascular endothelial cell growth factor into MCF-7 human breast carcinoma cells, J. Natl Cancer Inst. 87 (1995) 213–219. [13] Y. Takahashi, Y. Kitadai, C.D. Bucana, K.R. Cleary, L.M. Ellis, Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer, Cancer Res. 55 (1995) 3964–3968. [14] L.F. Brown, B. Berse, R.W. Jackman, K. Tognazzi, E.J. Manseau, D.R. Senger, et al., Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract, Cancer Res. 53 (1993) 4727–4735. [15] Y. Takahashi, K.R. Cleary, M. Mai, Y. Kitadai, C.D. Bucana, L.M. Ellis, Significance of vessel count and vascular endothelial growth factor and its receptor (KDR) in intestinal-type gastric cancer, Clin. Cancer Res. 2 (1996) 1679– 1684. [16] H. Saito, S. Tsujitani, A. Kondo, M. Ikeguchi, M. Maeta, N. Kaibara, Expression of vascular endothelial growth factor correlates with hematogenous recurrence in gastric carcinoma, Surgery 125 (1999) 195–201. [17] K. Maeda, Y.S. Chung, Y. Ogawa, S. Takatsuka, S.M. Kang, M. Ogawa, et al., Prognostic value of vascular endothelial growth factor expression in gastric carcinoma, Cancer 77 (1996) 858–863. [18] N. Tanigawa, H. Amaya, M. Matsumura, T. Shimomatsuya, et al., Correlation between expression of vascular endothelial growth factor and tumor vascularity, and patient outcome in human gastric carcinoma, J. Clin. Oncol. 15 (1997) 826–832. [19] G. Breier, U. Albrecht, S. Sterrer, W. Risau, Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation, Development 114 (1992) 521–532. [20] D.S. Charnock, A.M. Sharkey, J. Rajput, D. Burch, J.P. Schofield, S.A. Fountain, et al., Identification and localization of alternatively spliced mRNAs for vascular endothelial growth factor in human uterus and estrogen regulation in endometrial cell lines, Biol. Reprod. 48 (1993) 1120–1128. [21] H. Grone, M. Simon, E. Grone, Expression of vascular endothelial growth factor in renal vascular disease and renal allograft, J. Pathol. 177 (1995) 259–267. [22] D. Maglione, V. Guerriero, G. Viglietto, P. Delli-Bovi, M.G. Persico, Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor, Proc. Natl Acad. Sci. USA 88 (1991) 9267–9271.

81

[23] N. Ferrara, T. David-Smyth, The biology of vascular endothelial growth factor, Endocr. Rev. 18 (1997) 4–25. [24] N. Vaisman, D. Gospodarowicz, G. Neufeld, Characterization of the receptors for vascular endothelial growth factor, J. Biol. Chem. 265 (1990) 19461–19469. [25] T. Mustonen, K. Alitalo, Endothelial receptor tyrosine kinases involved in angiogenesis, J. Cell Biol. 129 (1995) 895–898. [26] Y. Cao, R.J. Weidong, Q. Pang, A. Rosin, Y. Cao, Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing, Biochem. Biophys. Res. Commun. 235 (1997) 493–498. [27] V. Joukov, A. Kaipainen, M. Jeltsch, K. Pajusola, B. Olofsson, V. Kumar, et al., Vascular endothelial growth factors VEGF-B and VEGF-C, J. Cell Physiol. 173 (1997) 211–215. [28] A. Khaliq, C. Dunk, J. Jiang, M. Shams, X.F. Li, C. Acevedo, et al., Hypoxia down-regulates placenta growth factor (PlGF) while fetal growth restriction upregulates PlGF expression: molecular evidence for placental hyperoxia in IURG, Lab. Invest. 79 (1999) 151–170. [29] C.M. Failla, T. Odorisio, F. Cianfarani, C. Schietroma, P. Puddu, G. Zambruno, Placenta growth factor is induced in human keratinocytes during wound healing, J. Invest. Dermatol. 115 (2000) 388–395. [30] P. Carmeliet, L. Moons, A. Luttun, V. Vincenti, V. Compernolle, M. De Mol, et al., Synergism between vascular endothelial growth factor and placenta growth factor contributes to angiogenesis and plasma extravasation in pathological conditions, Nat. Med. 7 (2001) 575–583. [31] S. Donnini, M.R. Machein, K.H. Plate, H.A. Weich, Expression and localization of placenta growth factor and PlGF receptors in human meningiomas, J. Pathol. 189 (1999) 66–71. [32] E. Hatva, T. Bohling, J. Jaaskelainen, M.G. Persico, M. Haltia, K. Alitalo, Vascular growth factors and receptors in capillary hemangioblastomas and hemangiopericytomas, Am. J. Pathol. 148 (1996) 763–775. [33] P.M. Lacal, C.M. Failla, E. Pagani, T. Odorisio, C. Schietroma, S. Falcinelli, et al., Human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor, J. Invest. Dermatol. 115 (2000) 1000–1007. [34] M. Nomura, S. Yamagishi, S. Harada, T. Yamashima, J. Yamashita, H. Yamamoto, Placenta growth factor (PlGF) mRNA expression in brain tumors, J. Neurooncol. 40 (1998) 123–130. [35] A. Takahashi, H. Sasaki, S.J. Kim, K. Tobisu, T. Kakizoe, T. Tsukamoto, et al., Markedly increased amounts of messenger RNAs for vascular endothelial growth factor and placenta growth factor in renal cell carcinoma associated with angiogenesis, Cancer Res. 54 (1994) 4233–4237. [36] J. Kodama, N. Seki, K. Tokumo, Y. Nakanishi, M. Yoshinouchi, T. Kudo, Placenta growth factor is abundantly expressed in human cervical squamous cell carcinoma, Eur. J. Gynecol. Oncol. 18 (1997) 508–510. [37] G. Viglietto, D. Maglione, M. Rambaldi, J. Cerutti, A. Romano, F. Trapasso, et al., Upregulation of vascular endothelial growth factor (VEGF) and downregulation of

82

[38]

[39] [40]

[41]

[42]

[43]

C.-N. Chen et al. / Cancer Letters 213 (2004) 73–82 palcenta growth factor (PlGF) associated with malignancy in human thyroid tumors and cell lines, Oncogene 11 (1995) 1569–1579. G. Viglietto, A. Romano, D. Maglione, M. Rambaldi, I. Paoletti, C.T. Lago, et al., Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor, Oncogene 13 (1996) 577–587. J. Marx, A boost for tumor starvation, Science 301 (2003) 452–454. P. Salven, A. Lymboussaki, P. Heikkila, Jaaskela-Saari, K. Aase, G. von Euler, et al., Vascular endothelial growth factors VEGF-B and VEGF-C are expressed in human tumors, Am. J. Pathol. 153 (1998) 103–108. J.E. Park, H. Chen, J. Winer, K. Houck, N. Ferrara, Placenta growth factor: potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR, J. Biol. Chem. 269 (1994) 25646–25654. T. Odorisio, C. Schietroma, M.L. Zaccaria, F. Cianfarani, C. Tiveron, L. Tatangelo, et al., Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability, J. Cell Sci. 115 (2002) 2559–2567. A. Luttun, M. Tjwa, L. Moons, Y. Wu, A. AngelilloScherrer, F. Liao, et al., Revascularization of ischemic

[44]

[45]

[46]

[47]

[48]

tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt-1, Nat. Med. 8 (2002) 831–840. M. Autiero, J. Waltenberger, D. Communi, A. Kranz, L. Moons, D. Lambrechts, et al., Role of PlGF in the intraand intermolecular cross talk between the VEGF receptors Flt1 and Flk1, Nat. Med. 9 (2003) 936–943. M. Stimpfl, D. Tong, B. Fasching, E. Schuster, A. Obermair, S. Leodolter, et al., Vascular endothelial growth factor splice variants and their prognostic value in breast and ovarian cancer, Clin. Cancer Res. 8 (2002) 2253–2259. S. Kido, Y. Kitadai, N. Hattori, K. Haruma, T. Kido, M. Ohta, et al., Interleukin 8 and vascular endothelial growth factor— prognostic factors in human gastric carcinomas?, Eur. J. Cancer 37 (2001) 1482–1487. M. Relf, S. LeJeune, P.A.E. Scott, S. Fox, K. Smith, R. Leek, et al., Expression of the angiogenic factors vascular endothelial growth factor, acidic and basic fibroblast growth factor, tumor growth factor b-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation toangiogenesis, Cancer Res. 57 (1997) 963–969. J.W. Slaton, K. Inoue, P. Perrotte, A.K. El-Naggar, D.A. Swanson, I.J. Fidler, et al., Expression levels of genes that regulate metastasis and angiogenesis correlate with advanced pathological stage of renal cell carcinoma, Am. J. Pathol. 158 (2001) 735–743.