The Role of Peroxiredoxin III and Sulfiredoxin for Mitochondrial ROS Production to Arsenic Trioxide in Acute Promyelocytic Leukemia Cells

Backgrounds: The Arsenic trioxide (ATO) is an effective cancer therapeutic drug for acute promyelocytic leukemia (APL), but in some cases, APL cells are resistant to ATO treatment. ATO exerts its effect mainly raising oxidative stress. However, not only the mechanisms of reactive oxygen species (ROS) generation by ATO but involvement of redox enzymes including peroxiredoxin (PRX) during ATO-induced apoptosis and its resistance remain elusive. Recently, Rhee et al had reported that PRX III and sufiredoxin together protect mice from pyrazole-induced oxidative liver injury was found (Antioxid & Redox Signal, 2012:17:1351-1361). Aims of current study are to elucidate that the changes of redox enzyme could be a mechanism of anti-leukemia effect in APL-derived NB4 cells during ATO treatment and to find ways to potentiate the anti-leukemic effects of ATO on APL cells.

Methods: NB4, one of the human acute promyelocytic leukemia cell lines, was treated with 0~10 μM arsenic trioxide to induce apoptosis for 16-48 hours in RPMI-1640 medium supplemented with 10% FBS in CO₂humidified atmosphere at 37°C. Apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) with flow cytometry. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red was used to detect cellular and mitochondrial ROS. SO2 form for PRX I, PRX II, and PRX III was detected by western blot assay using PRX SO2 form-specific antibody. Sulfiredoxin (SRX) and caspase 3, 9 were also detected by western blot analysis. To evaluate the effect of SRX depletion, NB4 cells were transfected with small interfering RNA (siRNA).

Results: Intracellular ROS of NB4 cells was increased significantly after 16 hour of ATO treatment but decreased after 24 hour of ATO treatment. Mitochondrial ROS of NB4 cells was increased significantly after 39 hour of ATO treatment. Apoptosis of NB4 cell after ATO treatment was increased as time elapsed (24% on 16hr, 26% on 24hr, 48% on 39hr, and 60% on 48hr). Increased cysteine sulfinic acid (Cys–SO₂H) PRX III, inactive and oxidized form, was observed as a hyperoxidation reaction in NB4 cells after ATO treatment in concordance with mitochondrial ROS increment of NB4 cells. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during NB4 cell apoptosis by ATO treatment. Meanwhile, SRX expression was increased in NB4 cells after ATO treatment. Down regulation of SRX by siRNA promoted ROS generation and apoptosis in ATO-treated NB4 cells.

Conclusions: Our data showed inactivation of PRX III by Cys–SO₂H formation as hyperoxidation is developed during ATO-induced mitochondrial ROS generation and apoptosis process in APL cells. In addition, ATO promotes expression of SRX, which is known as reducing enzyme of Cys–SO₂H PRX and which leads to down regulation of ROS accumulation in APL cells. These findings might be due to protective effect of SRX from ATO on mitochondrial oxidative stress. These findings suggest ATO-induced anti-leukemic activity could be down regulated by an enhancing PRX III reduction after ATO-induced SRX activation. Currently, the effect of down regulation of SRX by siRNA are being investigated to amplify the apoptosis in ATO-treated NB4 cells. Our study may provide the insights for finding novel targets in the development of new therapies, which potentiate ATO-induced apoptosis in APL cells.