Abstract
OBJECTIVE Epidermal growth factor receptor (EGFR) is dysregulated in many human malignancies and is a potential target for therapeutic intervention, but there is a major disagreement among researchers about both the frequency and possible clinical importance of EGFR overexpression in ovarian cancer. We investigated the expression and significance of the EGFR mRNA and vascular endothelial growth factor (VEGF) mRNA in ovarian carcinoma.
METHODS Reverse transcription polymerase chain reaction (RT-PCR) was employed to determine the expression of EGFR mRNA and VEGF mRNA in 79 ovarian specimen (including 15 normal, 13 benign and 51 malignant, from 79 patients). The relationship between EGFR and VEGF expression was analyzed.
RESULTS The positive rates of the expression of EGFR mRNA and VEGF mRNA were significantly higher in the patients with ovarian carcinoma than those in both the patients with benign ovarian tumors and in the normal controls. There was correlation between EGFR mRNA expression and clinical stages. The positive rate of the expression of EGFR mRNA in Stage III-IV was higher than that in Stage I-II of ovarian carcinoma (P < 0.05). The expression of VEGF mRNA was correlated with the clinical stages and lymph node metastasis. The expression levels of VEGF mRNA in Stage III-IV and in the group with lymph node metastasis were significantly higher than those in Stage I-II and in the group without lymph node metastasis, respectively (P < 0.05). The expression of EGFR mRNA was positively correlated with the expression of VEGF mRNA (r = 0.438, P < 0.05).
CONCLUSION The expressions of EGFR mRNA and VEGF mRNA are positively correlated to the occurrence of ovarian carcinoma and its metastasis. The detection of EGFR and VEGF may be helpful for the targeted chemotherapy.
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Introduction
Ovarian cancer is the most common cause of death among the gynecologic malignancies in most countries, and therapies over the last 30 years have not improved the cure rates[1]. To develop a novel effective therapy for ovarian cancer, further understanding of the processes and molecules leading to the initiation and progression of ovarian cancer is required. HER1/epidermal growth factor receptor (EGFR) is dysregulated in many human malignancies and is a potential target for therapeutic intervention[2-5]. The HER1/EGFR signaling pathway plays a pivotal role in controlling tumor growth progression, apoptosis, and angiogenesis. The growth and metastasis of neoplasms, including ovarian carcinoma, depend on the formation of adequate vasculature. Vascular endothelial growth factor (VEGF) is one of the many proangiogenic factors. The expression of VEGF is substantially increased in solid cancers, leading to greater microvascular density and a poor prognosis[6]. In this study, a reverse transcription polymerase chain reaction (RT-PCR) was employed to detect the expressions of EGFR mRNA and VEGF mRNA in primary ovarian cancers. We analyzed the relationship between EGFR and VEGF genes, the relationship between EGFR mRNA and VEGF mRNA expressions, and the different clinicopathological features.
Materials and Methods
Specimens
All 79 patients who underwent the surgery were collected at the Department of Gynecology, the Affiliated Hospital of Qingdao University Medical College (Qingdao, China) from April 2004 to October 2007 in this study. All the ovarian cancer specimens were obtained from 51 patients ranged from 23-75 years old (median age of 50.5 years), comprising 18 cases with Stage I-II ovarian cancer classified by the International Federation of Gynecology and Obstetrics and 33 cases with Stage III-IV. Initial histologic grades in the 51 cases were as the followings: well differentiated in 3 cases, moderately differentiated in 8, and poorly differentiated in 40. Thirty-nine out of 51 patients had tumors of serous histology. None of the 51 patients had received chemotherapy before surgery. Thirteen specimens of the benign epithelial ovarian tumors and 15 pieces of normal ovarian tissues as the normal controls were collected at the same period of time. The normal ovarian tissues were removed from the patients with benign tumors (such as patients had uterine fibroids whose ovarian tissue were removed and stained by H&E in order to be identified). After dissection, all of the fresh tumor tissue specimens were immediately stored at -80°C until used.
RNA preparation and RT-PCR procedure
Total RNA was prepared from fresh specimens using RNAiso reagent (TaKaRa Biotechnology Co. Lid) according to the manufacturer’s protocol. RNA integrity was confirmed by using agarose gel electrophoresis and ethidium bromide staining. The total RNA concentration was determined by the spectrophotometric analysis at A260/280. The first-strand cDNA synthesis was done with 1.0 ug total RNA in which SuperScript II reverse transcriptase (TaKaRa) was used following the manufacturer’s protocol. The synthesized cDNA was used as a template for PCR amplification. A semi-quantitative PCR amplification was carried out through the procedures of denaturing at 94°C for 1min, annealing at 55°C for 45 s, extension at 70°C for 1 min, and a final extension at 72°C for 1 min, using a thermal cycler. The primers were designed to amplify EGFR mRNA and VEGF mRNA based on the sequences of human EGFR and VEGF, which was published in the literature. In addition, amplification of human glyceraldehydes phosphate dehydrogenase (GAPDH) was performed using specific primers to rule out the possibility of RNA degradation, and was used to control variation in the mRNA amount in PCR. The primers used in this quantitative analysis of EGFR, VEGF and GAPDH were: for EGFR, 5’-AAC ACA GTG GAG CGA ATT CCT TT-3’ and 5’-GGA AGT CCA TCG ACA TGT TGC T-3’ that generated a specific 262-bp EGFR PCR product; for VEGF, 5’-CAG CTA CTG CCA TCC AAT CGA-3’, and 5’-CTG GCC TTG GTG AGG TTT GAT-3’ that generated a specific 199-bp VEGF PCR product; for GAPDH, 5’-TCA TGG GTG TGA ACC ATG AGA A-3’ and 5’-GGC ATG GAC TGT GGT CAT GAG-3’ that generated a specific 146-bp product. The PCR amplifications were performed in 20 μl PCR mixture containing 10 × PCR buffer, 0.2 mM each dNTP, 50 pmol specific primers, each cDNA template, and 0.2 unit Taq polymerase. PCR products (10 μl) were analyzed by agarose gel electrophoresis (2%) and the bands were visualized with ethidium bromide and photographed with a camera staining, and the product sizes were estimated by comparison to DNA molecular weight markers.
Statistical analysis
Statistical analysis was done with SPSS version 14.0. Categorical variables were analyzed with χ2 test and t test. Correlation between the mRNA expressions of molecules was analyzed using Pearson’s correlation analysis. Statistical significance was based on two-tailed test for the statistical analyses, and a P value < 0.05 was considered as statistically significant.
Results
The expressions of EGFR mRNA and VEGF mRNA in normal ovaries and ovarian cancer
The predicted PCR products of EGFR, VEGF and GAPDH were obtained as 262-bp, 199-bp and 146-bp respectively. Using RT-PCR, significant differences in the mRNA expression index of EGFR and VEGF between the normal ovaries and ovarian cancer were found. The EGFR mRNA and VEGF mRNA expression levels in the ovarian cancer were significantly higher than those in the benign tumors and in the normal ovaries (Fig. 1). The positive rates of EGFR mRNA and VEGF mRNA expression in ovarian cancer were 70.6% and 100% respectively, which were significantly higher than those in the normal ovaries (20.0% and 20.0% respectively) and in the benign ones (30.8% and 38.5% respectively) (P < 0.05, Table 1).
The expression of EGFR mRNA and VEGF mRNA in ovarian carcinoma. M, DNA Maeker; 1-5, The expression of GAPDH mRNA in ovarian carcinoma; 6-7, The expression of EGFR mRNA or VEGF mRNA in ovarian carcinoma; 8-9, The expression of EGFR mRNA or VEGF mRNA in benign ovarian tumor; 10, The expression of EGFR mRNA or VEGF mRNA in normal ovary; 11, Negative control.
The expression of EGFR and VEGF gene in normal ovaries, benign ovarian tumors and ovarian cancer
The expressions of EGFR and VEGF and clinicopathologic features of ovarian cancer
The EGFR mRNA expression was related to clinical stages, but not to patients’ age, histological types, differentiation and lymph node metastasis. Furthermore, the positive expression rate of EGFR gene in the patients with Stage III-IV was 84.8% that was higher than that in Stage I-II of the ovarian cancer (44.4%). We also found that the level of EGFR gene expression was increased in the patients with advanced ovarian cancer (P < 0.05, Table 2). The expression of VEGF mRNA was correlated with the clinical stages and lymph node metastasis, but there were no significant differences between VEGF mRNA expression and patients’age, histological types, and the level of differentiation. The expression level of VEGF mRNA in Stage III-IV and in the group with lymph node metastasis (0.94 ± 0.22, 1.00 ± 0.15) were significantly higher than those in Stage I-II and in the group without lymph node metastasis (0.60 ± 0.21, 0.74 ± 0.27) respectively (P < 0.05, Table 3).
Relationship between the EGFR mRNA expression and clinicopathologic features in ovarian cancer
Relationship between the VEGF mRNA expression and clinicopathologic features in ovarian carcinoma
The relationship between the expression indices of EGFR and VEGF in ovarian cancer
In 51 cases with ovarian cancer, the expressions of EGFR mRNA and VEGF mRNA were positively correlated. The difference between the expressions of EGFR mRNA and VEGF mRNA was statistically significant tested by Pearson’s correlation analysis (r = 0.438, P < 0.05).
Discussion
The EGFR, a transmembrane tyrosine kinase, is one of four members of the HER receptor family. This receptor is overexpressed in a number of solid tumors originated from the ectoderm, including ovarian carcinoma. Elevated levels of the EGFR have been detected in ovarian tumors, in ascites, and in the urine of ovarian cancer patients[2,7]. EGFR overexpression has been correlated with disease progression, poor prognosis and reduced sensitivity to chemotherapy[8]. In the present study, we have shown that the positive rate of EGFR in ovarian cancer was 70.6% that shows a high expression state of EGFR. Furthermore, the EGFR gene expression was significantly higher in the patients with ovarian cancer in Stage III-IV than that in Stage I-II (P < 0.05), which suggests that EGFR maybe related to an increased probability of the recurrence and progression of the tumor.
Tumor angiogenesis is the process leading to the formation of blood vessels within a tumor and plays a key role in cancer cell survival of the local tumor and in the development of distant metastases[9]. VEGF is a potent and specific mitogen for endothelial cells, which activates the angiogenic switch in vivo and enhances vascular permeability[10,11]. VEGF plays many roles in cancer biology including migration, tumor vessel formation, and metastasis[12]. In this study, it was found that the level of VEGF mRNA expression in ovarian cancer is related to the clinical stages and lymph node metastasis, and indicates that the VEGF gene is important in the invasion of the ovarian cancer. VEGF is likely to promote the metastasis of tumors.
Tissues regulate vascular architecture by signaling to endothelial cells through potent angiogenic agents such as VEGF. Previously, it has been shown that the expression of proangiogenic molecules, such as VEGF, in tumor cells can be stimulated by EGF signaling[13]. Indeed, antiangiogenic effects, such as decreased tumor cell production of proangiogenic molecules and inhibition of tumor-associated angiogenesis, have been described in several ErbB family inhibitors[14]. We have shown that in ovarian cancer patients, EGFR mRNA expression level has positive association with VEGF mRNA level, which suggests that EGFR may modulate the expression of VEGF.
In conclusion, the expressions of EGFR and VEGF are positively correlated to the occurrence and metastasis of the ovarian carcinoma. The detection of EGFR and VEGF may be helpful for targeted chemotherapy.
- Received November 25, 2008.
- Accepted March 2, 2009.
- Copyright © 2009 by Tianjin Medical University Cancer Institute & Hospital and Springer
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