Tumor-specific expression of shVEGF and suicide gene as a novel strategy for esophageal cancer therapy

Abstract

Aim: To develop a potent and safe gene therapy for esophageal cancer.

Methods: An expression vector carrying fusion suicide gene (yCDglyTK) and shRNA against vascular endothelial growth factor (VEGF) was constructed and delivered into EC9706 esophageal cancer cells by calcium phosphate nanoparticles (CPNP). To achieve tumor selectivity, expression of the fusion suicide gene was driven by a tumor-specific human telomerase reverse transcriptase (hTERT) promoter. The biologic properties and therapeutic efficiency of the vector, in the presence of prodrug 5-fluorocytosine (5-FC), were evaluated in vitro and in vivo.

Results: Both in vitro and in vivo testing showed that the expression vector was efficiently introduced by CPNP into tumor cells, leading to cellular expression of yCDglyTK and decreased VEGF level. With exposure to 5-FC, it exhibited strong anti-tumor effects against esophageal cancer. Combination of VEGF shRNA with the fusion suicide gene demonstrated strong anti-tumor activity.

Conclusion: The shVEGF-hTERT-yCDglyTK/5-FC system provided a novel approach for esophageal cancer-targeted gene therapy.

Keywords: Esophageal cancer; Nanoparticles; RNA interference; Suicide gene; Vascular endothelial growth factor.

Figure 1

Human telomerase reverse transcriptase promoter activities in cancerous and normal cells. EC9706 and HLF cells were transfected with hTERT reporter vector (hTERT-pGL3 basic). pGL3-Control and pGL3 basic were transfected as positive and negative controls, respectively. hTERT: Human telomerase reverse transcriptase.

Figure 2

Transfection efficiency of calcium phosphate nanoparticles. A-C: Images of transfected EC9706 cells acquired by fluorescence microscope at magnification × 200 after 48 h of transfection. Negative control (A), CPNP-GFP complex (B), Liposome/GFP complex (C); D-F: Qualitative analysis of transfection efficiency by flow cytometric assay. Negative control (D), CPNP-GFP complex (E), Liposome/GFP complex (F). CPNP: Calcium phosphate nanoparticles; GFP: Green fluorescent protein.

Figure 3

Changes of vascular endothelial growth factor and yCDglyTK expression in established stable cell lines. A: Representative VEGF mRNA and protein expression were analyzed by RT-PCR (top panel) and western blot (bottom panel), respectively. GAPDH was used as an internal control. Lane 1, EC9706/null; lane 2, EC9706/yCDglyTK; lane 3, EC9706/shVEGF; lane 4, EC9706/shVEGF-yCDglyTK; B: Representative yCDglyTK mRNA and protein expression were analyzed by semiquantitative RT-PCR (top panel) and western blot (bottom panel), respectively. GAPDH was used as an internal control. Lane 1, EC9706/null; lane 2, EC9706/shVEGF; lane 3, EC9706/yCDglyTK; lane 4, EC9706/shVEGF-yCDglyTK. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; RT-PCR: Reverse transcription polymerase chain reaction; VEGF: Vascular endothelial growth factor.

Figure 4

Effects of shVEGF-yCDglyTK/5-FC system on EC9706 cells. A: Cell viability of parental and stable EC9706 cells were determined with MTT assay at various time points after 5-FC treatment. The results shown are representative of three independent experiments; B: Representative images of Hoechst 33258-stained nuclei at magnification × 200. Apoptotic nuclei are condensed or fragmented; parental EC9706 cells (a); EC9706/null (b); EC9706/shVEGF (c); EC9706/yCDglyTK (d); EC9706/shVEGF-yCDglyTK (e); C: Representative dot plots of flow cytometry analysis. The numbers represent the percentage (%) of cells. Upper right quadrant, late apoptosis; lower right quadrant, early apoptosis; lower left, live cells. 5-FC: 5-fluorocytosine.

Figure 5

shVEGF-yCDglyTK/5-FC system inhibited tumor growth in the EC9706 xenograft model. Twenty-five BALB/C nude mice bearing EC9706 xenografts were randomized into five groups. The CPNPs/null, CPNPs/shVEGF, CPNPs/yCDglyTK or CPNPs/shVEGF-yCDglyTK complexes were delivered by intratumoral injection every other day, and the injection was repeated three times in total. 5-FC (500 mg/kg) was administered daily for 14 consecutive days. Tumors were measured every 3 d. All of the mice were scarified at day 36 after inoculation.

Figure 6

Immunohistochemistry analysis for yCDglyTK and vascular endothelial growth factor in EC9706 xenograft sections. A: Histological expression and distribution of yCDglyTK at magnification × 200; no-treatment control group (a); CPNPs/null + 5-FC (b); CPNPs/shVEGFX (c); CPNPs/yCDglyTK+5-FC (d); CPNPs/shVEGF-yCDglyTK +5-FC (e); B: Integrated optical density (IOD) values of VEGF expression in EC9706 xenografts. Anti-VEGF antibody was used for immunohistochemistry assay; C: Quantification of angiogenesis by microvessel counts (MVC) in EC9706 xenografts. Anti-CD34 was used for microvessel staining. CPNPs: Calcium phosphate nanoparticles; VEGF: Vascular endothelial growth factor; 5-FC: 5-fluorocytosine.