NOX and metal chelation are involved in MA/gp120-mediated oxidative stress and cell death
The NOX family of enzymes are responsible for the transfer of electrons across biological membranes and generation of ROS.31 In addition, various NOXs also serve as essential coenzymes coupled with CYP2E1-mediated electron transfer in the generation of ROS.32 To examine the involvement of NOX in gp120±MA-mediated oxidative stress, we treated the SVGA astrocytes with various concentrations of diphenyleneiodonium (DPI), an inhibitor for NOX. DPI significantly reduced the ROS production observed with either MA or gp120 alone and MA+gp120 (Figure 5a) in a dose-dependent manner (P for trend=0.01). Furthermore, 25 nM DPI rescued the cell death induced by MA and/or gp120 (Figure 5b), thereby confirming the role of NOX in MA/gp120-mediated cell toxicity. Among the NOX family of enzymes, NOX2 and NOX4 isozymes are predominantly responsible for NOX-derived ROS in astrocytes.33, 34 Therefore, we knocked down NOX2 and NOX4 using siRNA, which also abrogated the ROS produced by MA and gp120, either alone or in combination (MA±gp120) (Figures 5c and d). Furthermore, control siRNA-transfected cells did not show any significant change in the ROS production, when compared with no-siRNA control for the respective treatment groups (P>0.3 when measured using two-way ANOVA).
Superoxides (O2•−) generated via NOX are converted into H2O2, which is further converted into tertiary effector species such as hydroxyl radical (•OH) via the Fenton–Weiss–Haber (FWH) reaction.35, 36 We therefore hypothesized that FWH reaction is a downstream mechanism of NOX-mediated ROS production. As increased expression of ferritin is an indicator of oxidative stress,37 we measured the expression of ferritin heavy chain in the cells treated with MA and/or gp120. We observed higher ferritin expressions in astrocytes treated with MA and/or gp120 than untreated controls (Figure 6a), which suggested the involvement of Fe+2↔Fe+3 cycle. Therefore, we treated astrocytes with various concentrations of deferoxamine (DFO), an inhibitor of FWH reaction, 1 h before the treatment with either MA or gp120 alone and MA+gp120. Among various doses used, 50 nM DFO was found to reduce the ROS generated by gp120 alone as well as gp120+MA (Figure 6b). Furthermore, 50 nM DFO also rescued the astrocytes from cell death induced by oxidative stress (Figure 6c). The involvement of iron cycle can also lead to increased protein carbonylation, which can further lead to apoptotic cell death.38, 39 Therefore, we measured the levels of carbonylated protein in the astrocytes treated with MA and/or gp120. As expected, we observed increased levels of protein carbonylation (Figure 6d) upon treatments. Overall, these data suggested that NOX2 and NOX4 produced superoxides, which further underwent FWH chemistry to produce peroxides, leading to increased protein carbonylation. Taken together, these resulted in increased oxidative stress and consequently cell death.