A novel double-enhanced suicide gene therapy in a colon cancer cell line mediated by gef and apoptin.
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.
Basic Cardiovascular Research Section, Department of Anatomy and Human Embryology, School of Medicine, University of Granada, Granada, 18012, Spain, hboulaiz@ugr.es.
Houria
H
Boulaiz
Antonia
A
Aránega
Blanca
B
Cáceres
Cáceres
C
Blanca
Pablo
P
Alvarez
Alvarez
A
Pablo
Fernando
F
Serrano-Rodríguez
Rodríguez-Serrano
RS
Fernando
Esmeralda
E
Carrillo
Carrillo
C
Esmeralda
Consolación
C
Melguizo
Melguizo
M
Consolación
Jose
J
Prados
Prados
P
Jose
D017209:Apoptosis / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics*; D036022:Capsid Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D029968:Escherichia coli Proteins / Q000235:genetics*; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics
D001427:Bacterial Toxins; D036022:Capsid Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C091717:VP3 protein, Chicken anemia virus; C071524:HokC protein, E coli