Elsevier

European Journal of Cancer

Volume 39, Issue 13, September 2003, Pages 1948-1956
European Journal of Cancer

Expression levels of vascular endothelial growth factor, matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 and 2 in the plasma of patients with ovarian carcinoma

https://doi.org/10.1016/S0959-8049(03)00427-1Get rights and content

Abstract

We measured the levels of the vascular endothelial growth factor (VEGF), matrix metalloproteinases type 2 and type 9 (MMP-2 and MMP-9) and tissue inhibitors of matrix metalloproteinase 1 and 2 (TIMP-1 and TIMP-2) in the plasma of patients with ovarian carcinoma (n=40), in other gynaecological pathologies (n=30) and in the plasma of healthy volunteers (n=26). MMP-2 and MMP-9 (pro and active forms) gelatinolytic activity was measured by zymography. Enzyme-linked immunosorbent assays (ELISA) were used to assay soluble VEGF and TIMPs. Preoperative plasma VEGF levels were significantly higher in patients with ovarian cancer than in healthy volunteers (P<0.0001) or patients with a benign gynaecological pathology (P<0.0001). The expression of pro-MMP-9 was higher in the plasma of ovarian cancer patients than in the plasma of women with non-malignant disease (P=0.01) or healthy women (P<0.0002). Pro-MMP-2 was detected in the plasma of ovarian cancer patients, but levels did not differ from those in non-malignant disease or healthy donor samples. Plasma TIMP-1 and TIMP-2 levels were significantly higher in patients with ovarian carcinomas than in healthy volunteers (P<0.0001 and P=0.006, respectively) or in the patients with a non-malignant pathology (P<0.0001 and P=0.002, respectively). Sub-group analysis showed that VEGF and pro-MMP-9 were higher in the plasma of patients with serous carcinomas than other histological types. Furthermore, plasma VEGF and pro-MMP-9 levels were higher in the plasma of cancer patients with thrombocytosis. Throughout the study, and in the univariate analysis, no correlation was found between the VEGF, MMP and TIMP levels. Only TIMP-1 was associated with a poor survival and mortality risk.

Introduction

Ovarian cancer is the most common gynaecological malignancy [1], with a prevalence of 40 cases per 100 000 women aged 50 years and over. Epithelial ovarian cancer has been described as a silent killer because when it is first diagnosed the disease has already spread outside the ovary and pelvis. Only 15% of ovarian cancers are diagnosed at stage I, when cure rates approach 90%. Cure rates at advanced stages are 30–35%. As a result, 50% of these patients die within 5 years 1, 2.

The most common route of dissemination of epithelial ovarian cancer into the peritoneal cavity is by exfoliation of malignant cells through the surface of the ovary capsule. The circulation of peritoneal fluid facilitates the dissemination of these cells onto the intraperitoneal surfaces [2]. The early and extensive metastatic dissemination of ovarian cancer suggests that angiogenesis is an important event in the progression of this disease 3, 4. The production of growth factors and cytokines and the activation of proteolytic enzymes are responsible for ovarian cancer-associated angiogenesis and tumour dissemination [5]. Their identification, therefore, might have important implications for prognosis and therapy 6, 7.

Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors in solid tumours 8, 9, 10. The expression of VEGF and its receptors (VEGFR) in ovarian carcinoma has been associated with growth and invasion [6]. High levels of VEGF have been found in serum or plasma and in ascitic fluid of ovarian cancer patients [9] and VEGF levels have been proposed as an additional prognostic factor 11, 12. Indeed VEGF, recognised as a vascular permeability factor, plays an important role in ascites formation. A correlation between ascites volume and VEGF levels has been reported in experimental models 13, 14. Inhibitors of VEGF activity reduce the formation of malignant ascites in human ovarian carcinoma xenograft models [15].

The invasion and metastatic capacity of ovarian cancer cells is related to their ability to degrade the extracellular matrix and components of the basement membrane [16]. The matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases able to degrade components of the extracellular matrix and basement membrane [17]. MMPs are secreted as inactive proenzymes and transformed into active forms after cleavage of a propeptide domain of the molecule [18]. Gelatinase-A (MMP-2) and gelatinase-B (MMP-9) are believed to be vital in the invasion of malignant tumours and angiogenesis. Expression of MMP-2 and MMP-9 is elevated in several human tumours and is related to tumour aggressiveness and overall survival 19, 20. The use of secreted metalloproteases as a serum marker of malignant cancer has also been evaluated [21]. MMP-2 and MMP-9 are expressed in ovarian cancer tissue, ascites and cultured cells 22, 23. Lengyel and colleagues showed that pro-MMP-9, but not active MMP-9 or MMP-2, serves as an independent prognostic factor in FIGO stage III ovarian cancer. Davidson and colleagues suggested that MMP-2 and MMP-9 were predictors of poor survival in advanced ovarian carcinoma [24]. Experimental studies have shown that MMP-2 and MMP-9 are important in favouring the invasion and metastasis of ovarian cancer cells, and animals bearing ovarian carcinoma xenografts treated with MMP inhibitors had less tumour burden and ascites formation, and a longer survival [25].

MMPs work in concert with tissue inhibitors of the matrix metalloproteinase (TIMPs), a family of endogenous proteins that consists of four homologous members, TIMPs 1–4 [26]. By inhibiting active MMPs, TIMPs inhibit cell invasion in vitro and tumorigenesis and metastasis in vivo[27]. TIMPs can also associate with proMMPs. TIMP-1 selectively binds proMMP-9. TIMP-2 associates with proMMP-2 in a crucial step in the cell-mediated activation of MMP-2, through the formation of a TIMP-2/MMP-2/MT1-MMP complex (membrane type-1 MMP) [28]. TIMPs have been studied in the plasma and tumour tissue of patients with various cancers [29]. There appears to be a positive correlation between high TIMP levels and a poor prognosis in human malignancies 19, 30.

Our study was designed to assay VEGF, MMP-2 and MMP-9 and TIMP-1 and TIMP-2 levels in plasma and ascitic fluid of patients with ovarian cancer. An initial analysis of the correlation with clinical parameters and outcome is described. MMP-2 and MMP-9, their inhibitors and VEGF, were chosen because they play an important role in tumour metastasis and angiogenesis.

Section snippets

Patients and methods

Peripheral venous blood samples were collected from 96 women: 26 healthy women, 30 with non-malignant gynecological disease, and 40 with ovarian carcinoma diagnosed at the Department of Gynecological Oncology, S. Gerardo Hospital, Monza, Italy, between January 1994 and June 2001. The local ethics committee approved peripheral venipuncture. Informed consent for the taking of venous blood was obtained from all of the patients. Healthy volunteers gave their permission verbally. The main clinical

VEGF

Plasma levels of VEGF were compared in patients with ovarian cancer, patients with non-malignant disease and healthy volunteers (Fig. 1). The median VEGF concentration was 109.1 pg/ml (range 0–2845.2 pg/ml) for ovarian carcinoma patients and 18.8 pg/ml (0–121.3 pg/ml) for non-malignant gynaecological patients. VEGF was also detectable in healthy individuals, but at lower levels (median 0 pg/ml; range 0–48.4 pg/ml). VEGF levels were significantly higher (P<0.0001) in the ovarian carcinoma

Discussion

Ovarian cancer almost inevitably presents with an extension of disease outside of the pelvis, with ascites and omental tumour implants. Pleural effusion or swelling of the inguinal or axillary lymph nodes may be a later event. Malignant ascitic fluid accumulates because of the hyperpermeability of microvessels lining the peritoneal cavity, the angiogenesis associated with marked peritoneal neovascularisation, and a weakened lymphatic recovery system 13, 14.

One of the emerging clinical

Acknowledgements

This work was supported, in part, by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) and Fondazione Italiana per la Ricerca sul Cancro (FIRC) to R.G. The generous contribution of the Nerina and Mario Mattioli foundation is gratefully acknowledged. D.B. is a recipient of a Consiglio Nazionale delle Ricerche (CNR) fellowship (bando no. 201.17.3). We thank Paola Allavena for supplying the ascitic fluid samples and Judy Baggott for editing the manuscript.

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