Original Paper
Vascular endothelial growth factor is implicated in early invasion in cervical cancer

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Abstract

The association between the expression of vascular endothelial growth factor (VEGF) and clinicopathological factors has scarcely been examined in cervical cancer. This study examines the level of VEGF messenger RNA (mRNA) expression in invasive cervical cancer and its association with clinicopathological features including microvessel density. The level of VEGF mRNA was assessed by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) using β-actin as an internal control in 66 patients with stages Ia–IVb invasive cervical cancer. In 42 patients who underwent surgery, the microvessel count was also assessed by immunostaining for factor VIII-related antigen in the most neovascularised area of the specimen. The highest level of VEGF mRNA expression was observed in early invasive cervical cancers. Except for stage IVb, the stage of the disease inversely correlated with the level of VEGF mRNA (P<0.05). There was no significant difference in the level of VEGF mRNA with respect to histological cell types. 38 patients with stages Ib–IIb cervical cancer underwent radical hysterectomy and pelvic lymphadenectomy. There was no significant difference in the level of VEGF mRNA with respect to lymph node metastasis, depth of stromal invasion, tumour size, parametrial involvement or vaginal involvement among these patients. A significant relationship was found between the microvessel density and the level of VEGF mRNA (P<0.01). These findings provide evidence that the expression of VEGF is involved in the promotion of angiogenesis in cervical cancer and plays an important role in early invasion.

Introduction

Angiogenesis, the development of new blood vessels, is essential in tissue development, reproduction and wound healing[1]. Solid tumours require angiogenesis for progression and metastasis. In fact, tumour growth beyond 1–2 mm is strictly dependent on angiogenesis[2]. Angiogenesis also contributes to the metastatic process, carrying cancer cells into the circulation[3].

Tumour tissues secrete angiogenic factors that activate neovascularisation around tumours[1]. Vascular endothelial growth factor (VEGF) was originally detected in the conditioned medium of bovine pituitary folliculostellate cells. Vascular permeability factor (VPF) was identified in tumour ascites and was found to be identical to VEGF4, 5. VEGF is detectable in a number of tumour cell lines and tumour tissues and is thought to be a selective growth factor for endothelial cells[6]. Four molecular isoforms of VEGF are generated by alternative splicing, rendering proteins containing 206-, 189-, 165- and 121-amino acid residues[7]. The two shorter isoforms, VEGF165 and VEGF121, are secreted proteins which may act as diffusible agents, whereas the longer isoforms remain cell associated8, 9.

Recently, it was reported that microvessel density correlates with immunoreactivity of VEGF in stages Ib—IIb cervical cancer[10]. There has been no study investigating the level of VEGF mRNA in invasive cervical cancer. The present study examines both the expression of VEGF mRNA in stage Ia–IVb invasive cervical cancer tissues by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) and its association with clinicopathological features including microvessel density.

Section snippets

Tissue samples

The patient population consisted of 66 patients with a diagnosis of invasive cervical cancer at the Department of Obstetrics and Gynecology of Okayama University Medical School, Okayama, Japan, who underwent treatment from 1995 to 1996. Biopsy specimens obtained by colposcopy were available at the time of admission and each specimen was bisected. One portion was snap frozen and stored at −80°C until RNA was extracted. The other portion was fixed in 10% formaldehyde solution for

VEGF mRNA expression and clinicopathological features

Although VEGF 206 transcripts were not amplified, VEGF 189, 165 and 121 were routinely detected in this series of cervical cancer. The VEGF/β-actin (V/A) ratios ranged from 0.6 to 9.7, with an average of 3.9±2.6. Fig. 1 shows a representative final RT-PCR product for VEGF and β-actin and the corresponding values of the V/A ratios. The highest expression of VEGF mRNA was observed in early invasive cervical cancer. The stage of disease correlated significantly with a decrease in the V/A ratio,

Discussion

Several angiogenic factors have been described, such as acidic and basic fibroblast growth factor (bFGF), transforming growth factor-α (TGF-α), transforming growth factor-β, VEGF, placenta growth factor (PlGF), interleukin-8, tumour necrosis factor-α and platelet-derived endothelial cell growth factor/thymidine phosphorylase14, 15, 16, 17, 18. In invasive cervical cancer, the association between the expression of the most important angiogenic factor, VEGF, and clinicopathological factors

Acknowledgements

This work was partially supported in part by Grants-in-Aid for Scientific Research (09771277 and 09671684) from the Ministry of Education, Sport, Science and Culture, Japan. We thank Drs A. Dusso and T. Pavlopoulos for their assistance in the preparation of the manuscript.

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