BACKGROUND: Double-suicide gene therapy is a promising strategy for the treatment of advanced cancer. It has become an important research line in the development of gene therapy to overcome the drawbacks of single-gene therapy. OBJECTIVE: The aim of this study was to investigate the usefulness of double-suicide gene therapy with the two suicide genes, gef and apoptin, in colon carcinoma. METHODS: gef and apoptin genes were cloned into a doxycycline-regulated retrovirus-mediated gene expression system. Expression of both genes in the DLD-1 cell line was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Cell viability was determined with the sulforhodamine B colorimetric assay, and the cell cycle was studied by propidium iodide (PI) staining. Annexin V-FITC and PI assays were used to evaluate apoptosis, and the results were confirmed by electron microscopy. The mitochondrial membrane potential was measured by JC-1 assay. RESULTS: Our results showed that the combined expression of gef and apoptin genes was strikingly more effective than the expression of either gene alone. Co-expression of gef and apoptin synergistically enhanced the decrease in cell viability, increasing necrosis and inducing apoptosis in colon cancer cells via the mitochondrial pathway, which can be deficient in advanced or metastatic colon cancer. CONCLUSIONS: Double-suicide gene therapy based on gef and apoptin genes may be a candidate for the development of new colon cancer strategies, and further studies are warranted to establish the usefulness of double-suicide gene therapy in vivo.
BACKGROUND: Double-suicide gene therapy is a promising strategy for the treatment of advanced cancer. It has become an important research line in the development of gene therapy to overcome the drawbacks of single-gene therapy. OBJECTIVE: The aim of this study was to investigate the usefulness of double-suicide gene therapy with the two suicide genes, gef and apoptin, in colon carcinoma. METHODS:gef and apoptin genes were cloned into a doxycycline-regulated retrovirus-mediated gene expression system. Expression of both genes in the DLD-1 cell line was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Cell viability was determined with the sulforhodamine B colorimetric assay, and the cell cycle was studied by propidium iodide (PI) staining. Annexin V-FITC and PI assays were used to evaluate apoptosis, and the results were confirmed by electron microscopy. The mitochondrial membrane potential was measured by JC-1 assay. RESULTS: Our results showed that the combined expression of gef and apoptin genes was strikingly more effective than the expression of either gene alone. Co-expression of gef and apoptin synergistically enhanced the decrease in cell viability, increasing necrosis and inducing apoptosis in colon cancer cells via the mitochondrial pathway, which can be deficient in advanced or metastatic colon cancer. CONCLUSIONS: Double-suicide gene therapy based on gef and apoptin genes may be a candidate for the development of new colon cancer strategies, and further studies are warranted to establish the usefulness of double-suicide gene therapy in vivo.
Authors: Alberto Ramírez; Ana Conejo-García; Carmen Griñán-Lisón; Luisa C López-Cara; Gema Jiménez; Joaquín M Campos; Juan A Marchal; Houria Boulaiz Journal: Front Pharmacol Date: 2018-07-26 Impact factor: 5.810