In vitro CO2-induced ROS production impairs cell cycle in SH-SY5Y neuroblastoma cells.

PURPOSE

We evaluated in vitro the role of CO(2)-induced oxidative stress on the expression of proteins involved in cell-cycle regulation of neuroblastoma cells.

METHODS

SH-SY5Y cells were exposed to CO(2) at 15 mmHg pressure (100 %) for 4 h and then moved to normal condition for 24 h. Control cells were maintained in 5 % CO(2) for the same time. ROS production was determined by fluorescent staining with H2DCF-DA. DNA damage was measured by COMET assay. p53 protein expression was analyzed by western blot and confocal laser scanning microscopy was used to evaluate its sub-cellular localization. Cyclin expression was quantified by real-time PCR and western blot. Cell-cycle analysis was performed by FACS.

RESULTS

CO(2) incubation was associated with an increase in ROS production (p < 0.01), cell DNA damage mainly after 24 h (12 % increase of tail DNA content and 4-fold increase of tail length) and a significant up-regulation in p53 expression at 24 h with an intense nuclear staining. In CO(2)-treated cells, we observed an S-phase arrest in correlation with a reduction of cyclin B1 expression.

CONCLUSIONS

In vitro-simulated pneumoperitoneum environment with CO(2) induces oxidative stress and cell DNA damage, leading to p53 up-regulation involved in cell-cycle arrest of neuroblastoma cells.