Propyl Gallate Exerts an Antimigration Effect on Temozolomide-Treated Malignant Glioma Cells through Inhibition of ROS and the NF-κB Pathway

Figure 6

Inhibition of critical ROS events enhances antimigration with TMZ treatment. U-87 MG cells (1 × 10⁶) were plated in 60 mm cultured dishes for 24 h and then treated with TMZ (200 μM) alone for 48 h or pretreated with 1 nM rotenone (RO, a complex I inhibitor), 10 μM 2-thenoyltrifluoroacetone (TTFA, a complex II inhibitor), 10 μM carboxin (CAR, a complex II inhibitor), 0.01 nM antimycin A (AA, a complex III inhibitor), 1 μM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, a NADPH oxidase inhibitor), and 30 μM apocynin (APO, a NADPH oxidase inhibitor) for 1 h, followed by TMZ (200 μM) for 48 h. After treatment, the cells were stained with 2,7-dichlorfluorescein-diacetate (DCFDA) for ROS analysis and were then evaluated by flow cytometry. Data represent the fluorescence intensity within the cells. The values are presented as mean ± standard deviation (–8) of individual experiments. Significant differences for the TMZ group were and . (b) U87MG cells (1 × 10⁴) were plated in 24-well Millicell hanging cell culture inserts in an 8 μm polyethylene terephthalate membrane for 24 h and then treated with TMZ (200 μM) alone for 48 h or pretreated with 1 nM rotenone (RO, a complex I inhibitor), 10 μM 2-thenoyltrifluoroacetone (TTFA, a complex II inhibitor), 10 μM carboxin (CAR, a complex II inhibitor), 0.01 nM antimycin A (AA, a complex III inhibitor), 1 μM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, a NADPH oxidase inhibitor), and 30 μM apocynin (APO, a NADPH oxidase inhibitor) for 1 h, followed by TMZ (200 μM) for 48 h. Transwell migration assay was carried out. The migrated cells were stained using crystal violet. Random fields from each of the triplicate migration assays were counted using phase contrast microscopy (magnification 200x). (c) The dye was eluted with 33% acetic acid, and crystal violet absorbance was measured at 570 nm using a microplate reader. The values are presented as mean ± standard deviation (–8) of individual experiments. Significant differences for the TMZ group were and .

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