FIGURE 3.
Red-Br-nos-triggered ROS generation in PC-3 cells. Panel A, i, histogram profiles of control and drug-treated cells that were read flow cytometrically upon DCFDA staining (an indicator of ROS generation). Panel ii, bar graph representation of the quantitation of the increase in the mean fluorescence intensity (DCF-positive cells) in PC-3 cultures treated with DMSO (control) or Red-Br-nos for 24 h. Columns, mean ± S.D. (*, p < 0.05, compared with controls). Panel iii, microscopic visualization of DCF fluorescence in control and drug-treated PC-3 cells. Panel iv, fluorimetric data showing an increase in DCF fluorescence upon drug treatment compared with controls. Panel B, i, flow cytometric histogram profiles of control (blue profile) and drug-treated (red profile) cells upon DHE staining (another indicator of ROS generation). Panel ii, bar graph representation of the quantitation of the increase in the mean fluorescence intensity (EtBr-positive cells) in PC-3 cultures treated with DMSO (control) or Red-Br-nos for 24 h. Panel iii, microscopic visualization of EtBr (EB) fluorescence in control and drug-treated PC-3 cells. Panel iv, fluorimetric data showing an increase in EtBr fluorescence upon drug treatment compared with controls. Panel C, i, attenuation of ROS by tiron reduced the autophagic response as seen by a decrease in the number of red acidic compartments (AVOs), a characteristic feature of autophagic vacuoles. Panel ii, abrogation of ROS by tiron also decreased beclin-1 expression levels upon Red-Br-nos treatment for 24 h, suggesting a clear involvement of ROS in induction of autophagy. Panel iii, ROS plays a role in inducing Red-Br-nos-induced apoptosis. Attenuating ROS levels by tiron reduced the sub-G1 population as shown in the three-dimensional disposition of cell-cycle profiles. Panel iv, bar graph representation of the quantitation of the sub-G1 population upon drug treatment in the presence or absence of tiron. Panel v shows an immunoblot analysis for total and cleaved caspase-3 at the noted time points. β-Actin was used as a loading control. Panel vi is a bar graph representation for caspase-3/7 activity measured at 24 h upon treatment with vehicle control, Red-Br-nos and docetaxel. Columns, mean ± S.D. (*, p < 0.05, compared with controls).