Figure 2. Artocarpin-induced activation of NADPH oxidase and generation of ROS triggers apoptosis in tumor cells.

Time dependence of artocarpin-induced ROS generation and p47^phox activation are shown. (A) A549 cells and HPAEpiCs were stained with DCF-DA (10 μM) and treated with artocarpin for different periods of time. The fluorescence intensity was then determined. (B) A549 and H1299 cells were pretreated with DPI (10 μM), APO (100 μM), or NAC (100 μM) for 1 h and then stimulated with 10 μM artocarpin for 120 min. ROS generation was determined via fluorescent plate reader and flow cytometry. (C) Cells were stained with DCF-DA (10 μM) and TMRM (10 μM) and then pretreated with APO or NAC for 1 h before artocarpin administration. The ROS generation (green color) and mitochondrial membrane potential (red color) were detected via confocal microscope.(D–F)A549 cells were pretreated with APO or NAC for 1h, and then incubated with 10 μM artocarpin for 24h. Cytotoxicity was determined using MTT assay, flow cytometry(cells stained with Annexin-V and PI) and real-time cytotoxicity assay. (G) The membrane (ME) and cytosolic (CE) fractions were collected and subjected to Western blot analysis with an anti-phospho-p47^phox antibody. (H)A549 cells were transfected with siRNAs for scrambled, Nox2 (gp91^phox) or p47^phox, and then treated with 10 μM artocarpin for 120 min. ROS generation (open bars) and Nox activity (shaded bars) were determined via ELISA reader. (I) A549 cells were transfected with siRNAs of Nox2 (gp91^phox) or p47^phox, and then treated with 10 μM artocarpin for 24 hr. Cytotoxicity was determined using MTT assay. (A–I) are means ± SEM. *P < 0.05, ^#P < 0.01, compared with the control group.