Backgrounds : Bortezomib (BTZ) is the first generation proteosome inhibitor providing excellent response in newly diagnosed multiple myeloma. BTZ treatment increases oxidative stress in myeloma cells. However the roles of antioxidant enzymes during upregulation of ROS and the mechanisms of resistance from BTZ treatment are unclear. The aims of current study are to elucidate that the changes of redox enzyme could have a important roles of anti-myeloma effect during BTZ treatment.
Methods : MM.1S, MM.1R, and RPMI8226, the human myeloma cell lines, were treated with BTZ to induce apoptosis. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS) respectively. Sulfinic acid, (SO2) form of preoxiredoxin (Prx) was studied by western blot assay using Prx SO2 form-specific antibody. Monomer/dimer assay for subtypes of Prx and thioredoxin (Trx) was performed by western blot using non-reducing gel. To evaluate the effect of down regulation on sulfiredoxin (Srx), myeloma cells were transfected with small interfering RNA (siRNA) followed by Western blot analysis.
Results : Mitochondrial over cytosolic ROS of MM cells was increased significantly after 19 hour of BTZ (2.5 nM). Apoptosis of MM cell after BTZ treatment was increased in concordance with mitochondrial ROS increment of MM cells. N-acetylcystein (NAC) reversed BTZ-induced mitochondrial ROS elevation and apoptosis of MM cells as well. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during BTZ-induced MM cell apoptosis. Monomer, indicated active and reduced form, of Prx III was decreased and dimer, indicated inactive and oxidized form, of Prx III was increased in MM cells after treatment of BTZ among Prxs. Similarly, monomer of Trx 2, mitochondrial Trx was decreased in MM cells after BTZ treatment. However, increment of cysteine SO2 Prx III was not observed. Meanwhile, Srx, reducing enzyme of SO2 Prx, was induced in MM cells after BTZ treatment. Down regulation of Srx by siRNA did not promote ROS generation or apoptosis in BTZ-treated MM cells, otherwise.
Conclusions : Our results showed inactivation of Prx III by multimer formation as hyperoxidation is found during BTZ-induced mitochondrial ROS generation and apoptosis in MM cells. This Prx III oxidation was due to down regulation of reduced Trx 2 in BTZ-treated MM cells. To design of raising ROS stress and down regulation of anti-oxidants(ie, Trx), as a treatment-strategies may be worthwhile to potentiate BTZ-induced apoptosis in MM cells.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.