Fig. 3.

Effects of ROS scavengers on p53 activation and hydroxyl radical formation during cisplatin treatment. (A–C) RTPC cells were incubated with 20 µM cisplatin for 16 h in the absence or presence of 20 µM pifithrin-α, 10 mM N-acetyl-cysteine (NAC), 10 mM DMTU, or 10 mM Tiron. Whole cell lysate was collected for immunoblot analysis of p53 and phospho-p53. The blots were also reprobed for β-actin to monitor protein loading and transferring. (A) Representative immunoblots. (B) Densitometry of p53. (C) Densitometry of p-p53. For densitometric analysis, the p53 or p-p53 signals of control cells was arbitrarily set as 1 in each blot, and the signals of experimental conditions in the same blot were normalized with the control to show their p53 or p-p53 levels. The results were from at least three immunoblots of separate experiments. (D) RPTC cells were incubated with 20 µM cisplatin for 1 h in the absence or presence of 10 mM NAC, 10 mM DMTU, 10 mM mannitol, 2 mM benzoate, or 10 mM Tiron. Hydroxyl radicals were measured by the deoxyribose degradation assay as detailed in Section 2. Data are expressed as mean ± S.D. (n ≥ 3). *Statistically significantly different from the control; ^#significantly different from the cisplatin-only group. The results show that p53 activation during cisplatin treatment is suppressed by the general antioxidant NAC and the hydroxyl radical scavenger DMTU.