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Updated: Jul 14, 2026
A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells
Published on: May 6, 2018
[Suppression of osteosarcoma in vitro by coexpression of antisense VEGF165 cDNA and thymidine kinase gene]
Yan Wang1, Hui Qiao, Ting-Sheng Peng
1Department of Pathology, Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou 510089, China. wyan_sky@163.com
Objective:
To investigate the effect of VEGF expression in osteosarcoma cell line and the target killing effect of HSV1-TK/GCV system on transfected osteosarcoma cells under hypoxia conditions.
Methods:
Eukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF165 cDNA and Hygromycin phospho-transferase-thymidine kinase (HyTK) fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MG63 with lipofectin mediated gene transfer methods. PCR and RT-PCR were used to confirm the presence and expression of TK gene. The sensitivity of transfected cells to GCV and "bystander effect (BSE)" of HSV1-TK/GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed co-culture experiment. The expression of VEGF protein was detected by ELISA under hypoxia condition. Cell cycle phase distribution was determined by flow cytometry. In addition, electromicroscopy was used to document ultrastructural alterations.
Results:
The eukaryotic expression vector pBI-HRE-AsVEGF165 -HyTK was constructed successfully. The transfected cell line MG63TV was established and confirmed by PCR and RT-PCR of the presence of transgene and its mRNA expression. GCV was toxic to transfected cells in a concentration-dependent manner. The sensitivity to GCV toxicity was 100 times higher under hypoxia condition than that under normoxic condition. The mixed culture experiments showed that the "bystander effect" was enhanced significantly under hypoxia condition. VEGF expression of transgene cells under hypoxia condition decreased 50% compared to that of normal condition. Under hypoxia and GCV, DNA synthesis of MG63TV cells was inhibited along with an increase of cells at G0 approximately G1 phase, apoptosis and necrosis.
Conclusions:
Antisense VEGF expression driven by HRE promoter in combination with hypoxia can provide a target inhibition of VEGF expression in human osteosarcoma cells, with an enhanced selective killing effect and BSE of the HSV-TK/GCV system. The double-gene co-expression system in study provides experimental basis for therapy against osteosarcoma by a synchronous antiangiogenic and suicide gene approach.
Insights
Hypoxia enhances the killing effect of the herpes simplex virus 1 thymidine kinase/ganciclovir (HSV1-TK/GCV) system in osteosarcoma cells by targeting VEGF. This approach offers a promising strategy for osteosarcoma therapy.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Context:
- Osteosarcoma is a malignant bone tumor with limited treatment options.
- Vascular Endothelial Growth Factor (VEGF) plays a crucial role in tumor angiogenesis and progression.
- Hypoxia is a common characteristic of solid tumors, influencing gene expression and therapeutic response.
Purpose:
- To investigate the effect of VEGF expression in osteosarcoma cells under hypoxia.
- To evaluate the efficacy of the herpes simplex virus 1 thymidine kinase/ganciclovir (HSV1-TK/GCV) suicide gene system in targeting osteosarcoma cells.
- To assess the impact of hypoxia on the HSV1-TK/GCV system's efficacy and bystander effect.
Summary:
- A eukaryotic expression vector was constructed to express antisense VEGF165 cDNA and the HyTK fusion gene in MG63 osteosarcoma cells.
- Transfected cells showed increased sensitivity to ganciclovir (GCV) under hypoxia, with enhanced bystander effect.
- Hypoxia led to decreased VEGF protein expression and inhibited cell proliferation, inducing apoptosis and necrosis.
Impact:
- This study demonstrates that combining antisense VEGF gene therapy with hypoxia potentiates the selective killing of osteosarcoma cells.
- The enhanced efficacy of the HSV1-TK/GCV system under hypoxia provides a novel therapeutic strategy for osteosarcoma.
- The findings support a dual approach targeting both angiogenesis and tumor cells for improved osteosarcoma treatment.
