Figure 3

Celastrol-mediated glioblastoma cell death is dependent on thiol reactivity and is abrogated by co-treatment or preincubation of the drug with free thiols. (a) U251N cells were pretreated with either 2 mM NAC (NAC PreTreat) or serum-deprived media (Serum− and NAC Co-Treat) for 24 h. Media was replenished with celastrol at various concentrations (0.3–10 μM) with (NAC Co-Treat) or without (Serum−, NAC PreTreat) 2 mM NAC for a subsequent 24 h. Cell viability was assessed with the MTT assay. Significant differences in cell viability are noted when NAC is co-treated with celastrol relative to cells treated with celastrol alone (LD50: 12.6±161 μM; P=1.6E-23) but not when NAC is removed following pretreatment and before the addition of celastrol (LD50: 3.38±0.11 μM without NAC versus 3.55±0.19 μM with NAC pretreatment; P=0.0856). (b) U251N cells were pretreated with either 2 mM NAC or serum-deprived media for 24 h. Media was replenished with paraquat at various concentrations (0–1 mM) for a subsequent 24 h. Cell viability was assessed with the MTT assay. Significant rescue of cell viability was noted with NAC pretreatment compared with no pretreatment for the two highest doses of paraquat. (c) U251N cells were treated with 1 mM Trolox, 50 μM DTT or serum-deprived media in combination with celastrol at various concentrations (0.1–10 μM) for 24 h. Cell viability was assessed as in panels (b) and (c). No significant effect was seen with trolox (P=0.0102), whereas the presence of DTT significantly delayed the response to celastrol (LD50: 5.65±0.08 μM without DTT versus 12.6±14.1 μM with DTT; P=5.13 × 10⁻⁸) (see Statistical analysis section in Materials and Methods). Average values and S.Ds. are reported for triplicate measurements (N=3). Results are representative of at least three independent experiments. **P<0.01. (d) Chemical structure for celastrol–NAC adduct (6-N-Acetylcysteinyldihydrocelastrol) formed upon Michael addition of NAC to C6 electrophilic center of the celastrol quinone methide moeity