Prednisolone inhibits SaOS2 osteosarcoma cell proliferation by activating inducible nitric oxide synthase

Figure 1 Prednisolone inhibits the growth of SaOS2 but not of U2OS. A: Viable SaOS2 cells were counted after 4 d in the presence of different concentrations of prednisolone (^aP < 0.05; ^bP < 0.01); B: Viable U2OS cells were treated as above and counted after 96 h (^aP < 0.05). Results are shown as the mean of three separate experiments ± SD. CTR: Control.

Figure 2 Prednisolone does not induce the formation of reactive oxygen species and antioxidants do not prevent growth inhibition. A: SaOS2 cells were treated with various concentrations of prednisolone. H₂O₂ was used as positive control. Reactive oxygen species generation was measured. Data are shown as the mean of three separate experiments ± SD. P value was calculated vs untreated cells and found not significant; B: SaOS2 cells were treated with apocynin (10 μg/mL) or trolox (40 μmol/L) in the presence of prednisolone (Prd). Viable cells were counted after 96 h. Results are shown as the mean of three separate experiments ± SD. CTR: Control.

Figure 3 Prednisolone increases the release of nitric oxide in SaOS2. A: SaOS2 were cultured in the presence of different concentrations of prednisolone. Nitric oxide was measured after 24, 48 and 96 h. Results are expressed as the mean ± SD of four different experiments (^aP < 0.05, ^bP < 0.01, ^dP < 0.001); B: U2OS cells were processed as described in (A). No statistical significance was achieved; C: SaOS2 cells were exposed to prednisolone for 12 and 24 h and then lysed. 80 μg of protein extracts were loaded on SDS-PAGE. Western blots using specific antibodies against iNOS, eNOS, p-eNOS-P-Ser1177 were performed. Actin shows that equal amounts of protein were loaded per lane. The figure shows a representative blot; D: The histogram shows the quantitative evaluation of NOS/actin ratio by densitometry. Results are expressed as the mean ± SD of three separate experiments (^bP < 0.01). CTR: Control; iNOS: Inducible nitric oxide synthase; eNOS: Endothelial nitric oxide synthase; Prd: Prednisolone.

Figure 4 Pharmacological inhibition of inducible nitric oxide synthase rescues SaOS2 cell growth. A: SaOS2 were treated with prednisolone in the presence or in the absence of L-NIL (100 μmol/L). Nitric oxide was measured as described (^bP < 0.01); B: Viable SaOS2 cells were counted after 4 d in the presence of different concentrations of prednisolone with or without L-NIL (^bP < 0.01). CTR: Control; L-NIL: l-N6-(iminoethyl)-lysine-HCl.