Zhong-Ping Gu, Yun-Jie Wang, Yong-An Zhou, Department of Thoracic Surgery, Jin-Ge Li, Department of Infectious Disease, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China
Correspondence to: Zhong Ping Gu, Department of Thoracic Surgery, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China. Zhongpg@pub.xaonline.com
Telephone: +86-29-3577737
Received 2001-08-09 Accepted 2001-10-23

Abstract
AIM: To investigate the effect of antisense RNA to vascular endothelial growth factor165 (VEGF₁₆₅) on human esophageal squamous cell carcinoma cell line EC109 and the feasibility of gene therapy for esophageal carcinoma.

METHODS: By using subclone technique, the full length of VEGF₁₆₅ amino acid cDNA, which was cut from pGEM-3Zf (+), was cloned inversely into the eukaryotic expression vector pCEP4 .The recombinant plasmid pCEP-AVEGF₁₆₅ was transfected into EC109 cell with lipofectamine. After a stable transfection, dot blot, enzyme-linked immunosorbent assay (ELISA), laser confocal imaging system analysis, transmission electron microscopy and flow cytometry were performed to determine the biological characteristics of EC109 cell line before and after transfection in vitro and whether there was a reversion in the tumorigenic properties of the EC109 cell in vivo.

RESULTS: The eukaryotic expression vector pCEP-AVEGF₁₆₅ was successfully constructed and transfected into EC109 cells. The expression of VEGF₁₆₅ was significantly decreased in the transfected cells while the biological characteristics of the cells were not influenced by the expression of antisense gene. The tumorigenic and angiogenic capabilities were greatly reduced in nude mice, as demonstrated by reduced tumor end volume (820±112.5)mm³ vs (7930±1035)mm³ and (7850±950)mm³,P＜0.01) and microvessel density(8.5±1.2)mm^-2 vs (44.3±9.4)mm^-2 and (46.4±12.6)mm^-2,P<0.01) in comparison between experimental groups empty vector transfected group and control group.

CONCLUSION: The angiogenesis and tumorigenicity of human esophageal squamous cell carcinoma were effectively inhibited by VEGF₁₆₅ antisense RNA. Antisense RNA to VEGF₁₆₅ can potentially be used as an adjuvant therapy for solid tumors.

Gu ZP, Wang YJ, Li JG, Zhou YA. VEGF₁₆₅ antisense RNA suppresses oncogenic properties of human esophageal squamous cell carcinoma. World J Gastroenterol 2002;8(1):44-48

INTRODUCTION
Angiogenesis, which is defined as the formation of new blood vessel from the pre-existing vascular bed, is essential for solid tumor growth, for the entrance of tumor cell into the circulation, and for the subsequent establishment and growth of metastasis. Many studies demonstrated that tumor angiogensis is associated with patient outcome and is an independent prognostic marker in almost all solid tumors, including esophageal carcinoma^[1-10]. Tumor angiogenesis is a complex process, involving growth factors and extracellular matrix enzymes. Among the many known triggers of tumor angiogenesis, vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell-specific mitogen and an angiogenesis inducer released by a variety of tumor cells and expressed in human tumors in situ. VEGF₁₆₅ is the most effective angiogenic factor in the VEGF family. Tumor cells engineered to express VEGF constitutively exhibit enhanced tumor growth and angiogenic phenotypes^[11-13]. Conversely, inhibition of the expression of VEGF₁₆₅ was considered as a therapeutic strategy for the treatment of solid tumors^[14-24].
      In this report, we constructed antisense RNA to VEGF₁₆₅ eukaryotic expression vector and applied gene transfer technology to modulate the expression in stably transfected human esophageal squamous cell carcinoma cells. We assessed the effects of down-regulation of VEGF expression on the biological characteristics in vitro , microvessel density and turmorigenic capability in nude mice.

MATERIALS AND METHODS
Cell line and vector
The EC109 human esophageal squamous cell carcinoma cell line was generously provided by Dr. Sun (Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM), high glucose media (Life Technoligies) and supplemented with 100mL·L^-1 fetal calf serum (HyClone Laboratories), penicillin, streptomycin, and nonessential amino acids (Life Technoligies). The vector pGEM-3Zf (+) (carrying the full length aminoacids cDNA of VEGF₁₆₅) was kindly provided by Dr. Abraham (Columbia University, USA) and vector pCEP4 was a gift from Dr. Li (Department of Infectious Disease, Tangdu Hospital, Fourth Military Medical University,China).

Plasmid construction
The expression vector for VEGF₁₆₅ antisense RNA was constructed by subcloning cDNA fragment that code for VEGF₁₆₅ into the eukaryotic expression vector pCEP4. pGEM-3Zf (+) was digested by Kpn Ⅰand Hind Ⅲ.The fragment was purified by gel. The VEGF₁₆₅ amino acids cDNA was cloned inversely in the Hind Ⅲ / Kpn Ⅰsite of pCEP4 to generate plasmid pCEP-AVEGF₁₆₅ (Figure 1).

Transfection and selection
The transfection and selection of the EC109 cells were carried out in a 6-well plate. When the cells reached 70% confluence, the transfection process began. Briefly, solution A was prepared by diluting 10μg of pCEP-AVEGF₁₆₅ into 200μL serum-free medium, and solution B was prepared by diluting 20μL Lipofectimine 2000 (Life Technoligies) into 200μLserum-free medium. The two solutions were combined for 20 min at room temperature, and then 0.6mL serum-free medium was added to the tube containing the complex, and subsequently added to the rinsed cells. The medium was replaced with fresh and complete medium 18 h after the start of transfection. Seventy-two hours after transfection, it was replaced again with the selective medium containing 200g·L^-1 hygromycin B (Boehrringer Mannheim). Once stable transfections were obtained, the cells were maintained in 100g·L^-1 of hygromycin B. The EC109 cells were transfected with either the empty pCEP4 vector or pCEP-AVEGF₁₆₅.

Figure 1(PDF) Diagram of the construction of the vector pCEP-AVEGF₁₆₅

Dot blot analysis
Total cellular RNA was extracted from the cultured cells using the Trizol isolation kit (Life Technoligies) according to the manufacturer's instruction. The recovered total RNA was redissolved in diethyl pyrocarbonate-treated water and 20μg was immobilized onto a gene screen plus membrane (DuPont) by gentle suction with a blotting manifold (Bethesad Research Laboratories). The membrane was then probed with a 5'-end-radiolabeled synthetic oligodeoxyribonucleotide complementary.

Flow cytometry analysis
Approximately 5×10⁶ centrifugal sedimentation cells were immediately fixed in 700mL·L^-1 ethanol and stored at 4℃ in PBS in preparation for fluorescent-activated cell sorting. Flow cytometry analysis was performed on a FACStar flow cytometer (Becton Dickinson). Histograms of cell number logarithmic fluorescence intensity were recorded for 10 000 cells per sample.

Transmission electron microscope examination
The centrifugalized cells were placed in 40g·L^-1 glutaraldehyde and then post-fixed in osmium tetroxide and embedded in Epon. Routine thin sections were stained with uranyl acetate and lead citrate. Thin sections were mounted on grids and examined under a transmission electron microscope (JEM-2000EX) at 60kV.

Laser confocal microscope analysis
Indirect immunofluorescence techniques were applied in the transfected EC109 cells and the parental cells. VEGF₁₆₅ protein was detected with mouse anti-human VEGF₁₆₅ antibody and sheep anti-mouse IgG-FITC (Dako A/S Denmark). FITC was activated by light with a wavelength of 488nm. The data of laser scanning were 3%. The expression of VEGF₁₆₅ was analyzed by confocal microscope system controlled by software obtained by Bio-Rad.

Tumorigenicity assay
Athymic Balb/c nude mice were obtained from the Animal Center of Fourth Military Medical University. The mice were maintained in a laminar airflow cabinet under specific pathogen-free conditions and used at 8-12 weeks of age. Cells used for injection were grown to subconfluence, trypsinized, washed once, and resuspended in serum-free DMEM. The cell suspensions were examined microscopically to ensure that they were composed of single-cell suspensions. Mice were injected s.c. on the hind leg with 5×10⁶ single cells in 0.1mL. The mice were then separated into three groups, depending on whether they were injected with pCEP-AVEGF₁₆₅ transfected cells, pCEP4 empty vector transfected cells, or control cells. Each group contained five mice. Calipers was used for the calculation of tumor size. Microvessel density was determined under light microscopy after immunostaining of sections with anti-CD34 monoclonal antibody according to the strepto ABC kit (Dako A/S Denmark) instruction.

Statistical analysis
The data were analyzed for significance by ANOVA.

RESULTS
VEGF₁₆₅ antisense vector construction
After ligation, transformation and selection, three clones were found likely to contain the desired recombinant. These clones were digested by restriction enzymes Kpn Ⅰ/ Hind Ⅲ or Kpn Ⅰ/ Sfi Ⅰ.The 640bp or the 660bp fragment was found by using polyacrylamide gel electrophoresis. These recombinant plasmids were the eukaryotic expression vectors of antisense RNA to VEGF₁₆₅ (Figure 2).

Expression of VEGF₁₆₅ antisense RNA
Two weeks after being transfected and selected by hygromycin B, the EC109 cells transfected by pCEP-AVEGF₁₆₅ expressed antisense RNA to VEGF₁₆₅ which was confirmed by dot blot analysis, whereas the cells transfected by pCEP4 empty vector and control group cells were negative (Figure 3).

Expression of VEGF₁₆₅ in vitro
ELISA showed that a great number of VEGF₁₆₅ accumulated in the pCEP4 empty vector transfected group and control group cells, whereas in the pCEP-AVEGF₁₆₅ transfected group cells, the level of VEGF₁₆₅ was very low. The level of VEGF₁₆₅ expression was significantly lower in EC109 cells transfected by pCEP-AVEGF₁₆₅ than that in the pCEP4 empty vector transfected group and control group cells (P<0.01) determined under confocal microscope, as indicated in Figure 4.

The change of ultrastructrue and cell cycle
There was no substantial change neither in the ultrastructure examined under transmission electron microscope nor in the cell cycle determined by flow cytometer.

The change of tumorigenic capacity in vivo
The nude mice were sacrificed at week 5. Tumor volume was measured and morphological characteristics were assessed in HE stained sections. pCEP-AVEGF₁₆₅ transfected xenografts grew very slowly, pCEP4 empty vector transfected group and nontransfected control xenografts were significantly larger than pCEP-AVEGF₁₆₅ transfected xenografts (P<0.01), and the mean tumor volumes were (820±112.5)mm³，(7930±1035)mm³ and (7850±950)mm³, respectively. pCEP-AVEGF₁₆₅ transfected xenografts had a relatively large area of central necrosis. Immunohistochemical staining for CD34 was performed to evaluate tumor microvessel density. The microvessel density was expressed as the average number of the five highest areas identified within a single×200 field, for the pCEP-AVEGF₁₆₅ transfected mice, pCEP4 empty vector group and nontransfected controls were (8.5±1.2)mm^-2, (44.3±9.4) mm^-2 and (46.4±12.6)mm^-2, respectively (P<0.01).

Figure 2 Identification of recombinant clone by restriction enzyme     
1: Fragment of 640bp digested with KpnⅠ/Hind Ⅲ
2: DL2000 markers (2000,1000,750,500,250,100bp)
3: Fragment of 660bp digested with KpnⅠ/ SfiⅠ
Figure 3 Expression of antisense RNA to VEGF₁₆₅ in EC109 cell
1: Transfected by pCEP-AVEGF₁₆₅
2: Transfected by empty vector
3: Control group
Figure 4 Expression of VEGF₁₆₅ in Ec109 cell(×40): transfected by pCEP-AVEGF₁₆₅ (A), transfected by empty vector (B),
and control group (C).

DISCUSSION
Mammalian cells require oxygen and nutrients for their survival and are therefore located within 100μm -200μm blood vessels-the diffusion limit for oxygen. For multicellular organisms which grow beyond this size, they must recruit new blood vessels by angiogenesis and vasculogenesis. This process is regulated by a balance between pro- and anti-angiogenic molecules, and is derailed in various diseases, especially cancer. Without blood vessels, tumor can not grow beyond a critical size or metastasize to another organ^[25-29]. In 1971, Folkman^[30] proposed that solid tumor growth and metastasis are critically dependent on angiogenesis, the formation of new blood vessels from pre-existing vasculature, and hence, blocking angiogenesis could be a strategy to arrest tumor growth. The induction of angiogenesis is mediated by several factors released by both tumor and host cells. One of the key mediators of angiogenesis is VEGF, a multifunctional growth factor that is overexpressed and secreted by a majority of human and animal tumors. VEGF was purified by Ferrara et al ^[31] from the conditioned medium of bovine pituitary folliculo stellate cells. VEGF is a homodimeric 46ku heparin-binding glycoprotein with potent angiogenic, mitogenic, and vascular permeability-enhancing activities specific for endothelial cells. By alternative splicing of messenger RNA, VEGF may exist in at least four different homodimeric molecular species each monomer having 121, 165, 189 or 206 amino acids, respectively (VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, VEGF₂₀₆). Among this family, VEGF₁₆₅ is the most important effector. Antiangiogenic therapy targeting VEGF has been proposed as a means of inhibiting VEGF-dependent tumor growth and metastasis ^[32-40].
       It has been suggested that antisense RNA could block the translation progress of aim protein effectively and inhibit expression ^[41-45]. DeFatta et al ^[46] found that reducing eIF4E express on via antisense RNA suppressed both the tumorigenic and angiogenic properties of the head and neck squamous cell cancers, cell line FaDu, as demonstrated by lowered capacity to grow in soft agar, reduced expression of angiogenic factors, and loss of tumorigenicity in nude mice. Oku and associates^[47] transfected human SK-MEL-2 melanoma cells with antisense VEGF which resulted in substantial inhibition of intracerebral tumor growth in nude mice, and a decrease in tumor vascularity, blood flow, and permeability.
      The prognosis of human esophageal squamous cell carcinoma after curative resection is dismal. Radiotherapy and several conventionalchemotherapeutic agents have been tried to improve the prognosis, but the results are generally disappointing. In this regard, antiangiogenic therapy could be a promising and hopeful strategy for esophageal cancer^[48-51]. In this study, an antisense RNA to VEGF₁₆₅ eukaryotic expression vector pCEP-AVEGF₁₆₅ was constructed successfully. We transfected it into human esophageal squamous cell carcinoma cell line EC109. Under immunohistochemistry and confocal microscopy, it was found that the expression of VEGF₁₆₅ decreased significantly in the cells transfected with VEGF₁₆₅ antisense RNA compared with the empty vector transfected and control group. Under transmission electron microscopy and flow cytometry, we observed that the ultrastructure and cell cycle had no change among transfected and control groups. In the nude mice tumor model, the tumorigenicity, the rate of tumor growth, and microvessel density were significantly decreased for the tumors derived from antisense RNA transfected cells as compared with the empty vector transfected and parental cells. pCEP-AVEGF₁₆₅ transfected tumors had a very low initial growth rate with central necrosis. These results suggested that inhibition of tumor growth might be achieved by VEGF₁₆₅ antisense RNA's down-regulation of endogenous VEGF expression in tumor tissues. In the meantime, we found that the VEGF₁₆₅ antisense RNA therapy could slow the rate of tumor growth and not inhibit completely the tumorigenicity. This demonstrated that the process of angiogenesis and tumorigenicity is complex and involves multifactors. To the best of our knowledge, this is the first experimental report which shows that VEGF₁₆₅ antisense RNA suppresses the growth of human esophageal squamous cell carcinoma in vivo in association with decreased vessel number in the treated tumors.
      Esophageal carcinoma is still common in China^[52-58], and the treatment remains a big problem up to date^[59-65]. Our present study suggests that antisese RNA to VEGF₁₆₅ can potentially be used as an adjuvant therapy for human esophageal squamous cell carcinoma. Further studies are needed to understand the details of the mechanisms for appropriate clinical application.

ACKNOLEDGMENTS We especially thank Dr. Xiao Yan Sun for providing us the EC109 cell line, Dr. Abraham for providing the pGEM-3Zf (+) vector, we also thank Dr. Zhi Pei Zhang for his assistance with the animal experiments, and Prof. Bo Rong Pan for improving the paper.

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