Background & objective: Gap junctional intercellular communication (GJIC) is the main mechanism mediating the bystander effect in herpes simplex virus thymidine kinase (HSV-tk) gene therapy. All-trans retinoic acid (ATRA) may augment the therapeutic efficacy of HSV-tk gene therapy by both upregulating GJIC in glioma cells and inhibiting tumor growth. This study was designed to investigate the synergistic effect of ATRA and HSV-tk gene therapy in treatment of glioma.
Methods: Rat C6 glioma cells were exposed to ATRA at concentrations of 1 mumol/L, 10 mumol/L, or 100 mumol/L, respectively. The effects of ATRA on C6 glioma cell differentiation, proliferation, GJIC, and connexin 43 (Cx 43) gene transcription were studied. C6 cells and C6tk cells, a stable transfectant of HSV-tk gene, mixed at various ratios. The mixtures were treated with GCV with ATRA at various concentrations or without ATRA, and the bystander effect was measured with MTT assay in 7 days after treatment.
Results: A morphological change of differentiation was observed in C6 glioma cells after exposure to each concentration of ATRA. The proliferation of C6 cells was also significantly inhibited by ATRA, majority of living cells being arrested at G1 phase, especially at the concentration of 100 mumol/L. At the concentration of 100 mumol/L, ATRA also induced significant apoptosis in C6 glioma cells. The GJIC was significantly enhanced in C6 cells after ARTA treatment at each concentration, while there was no ATRA induced effect observed upon Cx43 transcription. The results of bystander effect assay revealed that ATRA at each concentration exerted a significant augmentation effect on bystander effect.
Conclusion: The combination of two clinically safe protocols, ATRA and HSV-tk/GCV gene therapy, resulted in a synergistic effect in glioma treatment, and would become a promising strategy for clinical administration.