Antitumor efficiency of the cytosine deaminase/5-fluorocytosine suicide gene therapy system on malignant gliomas: an in vivo study
Sheng-Qing Lv, Ke-Bin Zhang, Eric E. Zhang, Fang-You Gao, Chang-Lin Yin, Chun-Ji Huang, Jia-Quan He, Hui Yang
Med Sci Monit 2009; 15(1): BR13-20
Suicide gene therapy, particularly that utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, represents a novel and attractive methodology of cancer research. Mechanistically, the CD enzyme can convert the antifungal agent 5-FC into the antimetabolite agent 5-fluorouracil (5-FU), thereby killing tumor cells. The purpose of this study was to investigate the antitumor efficiency of the CD/5-FC system in malignant gliomas using a nude mouse model.
Material and Method: The eukaryotic expression plasmid pCMV-CD was transfected into U251 malignant glioma cells. Resistant clones (labeled U251/CD cells) were subsequently isolated and further confirmed by reverse transcription polymerase chain reaction (RT-PCR), immunofluoroscence, and immunoblot. Then U251/CD cells were incubated with 5-FC at various concentrations to measure viability ratios as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. 5-FU concentrations in the media were measured by high-performance liquid chromatography (HPLC). Finally, the volumes and weights of tumors from glioma-bearing nude mice after 5-FC intervention were evaluated.
Results: The results revealed that the untreated U251 cells were insensitive to 5-FC whereas the U251/CD cells were highly sensitive. Apoptosis and cell death were observed on the U251/CD cells after 5-FC administration. HPLC analysis showed that 5-FU was detected in the U251/CD cell media. These in vivo animal data showed that the volumes and weights of the implanted tumors were dramatically decreased due to cell apoptosis and tumor necrosis.
Conclusions: The in vivo results encourage a further investigation in a controlled trial on the treatment of malignant gliomas via the CD/5-FC gene therapy system.
Keywords: Reverse Transcriptase Polymerase Chain Reaction, Mice, Mice, Nude, Thiazoles, Tetrazolium Salts, Immunoblotting, Glioma - therapy, Genetic Therapy - methods, Fluorouracil - therapeutic use, Flucytosine - metabolism, Fluorescent Antibody Technique, Escherichia coli, DNA Primers - genetics, Cytosine Deaminase - metabolism, Chromatography, High Pressure Liquid, Cell Line, Tumor, Animals