Figure 3.

The anticancer effect of Rh1 is associated with the PI3K/Akt pathway and ROS production. (A) MCF-7 cells were treated with Rh1 (25 to 100 µM) for 24 h. ROS production was determined using the DCF-DA assay. (B,C) MCF-7 cells were pretreated with NAC (20 mM), APO (500 µM), or DPI (250 nM) for 1 h, followed by the treatment with Rh1 (50 µM) for 24 h. ROS production was determined using the DCF-DA assay (B). Cell viability was measured by the SRB assay (C). (D,E) MCF-7 cells were treated with Rh1 at various concentrations for 30 min. The whole-cell lysates were then used to analyze the kinase activity of ERK1/2 and Akt by Western blotting (D). The relative quantification of the protein levels was analyzed using Image J software (E). (F,G) MCF-7 cells were pretreated with NAC (20 mM) for 3 h, followed by the treatment with Rh1 (25 and 50 µM) for 30 min or 24 h. Total cell lysates were subjected to Western blotting. The relative quantification of the protein levels in the experiment with a 24-h treatment of Rh1 was analyzed using Image J software (G). (H) The cells were pretreated with 5-µM LY294002 for 3 h, followed by the treatment with Rh1 for 24 h. Total cell lysates were subjected to Western blotting. (A–C,E,G) Data are expressed as the mean ± SD (n = 5), * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with the control and # p < 0.05, ## p < 0.01, and ### p < 0.001 as compared with the Rh1-treated samples. NAC: N-acetylcysteine, LY294002: PI3K inhibitor, APO: apocynin, and DPI: diphenyleneiodonium.