Figure 1

Celastrol mediates a ROS and JNK-independent mechanism of cell death in human glioblastoma cells. (a) In a number of different cell types, it has been proposed that celastrol generates ROS and subsequently activates stress-dependent pathways (e.g., JNK) and inhibits pro-survival signaling (e.g., decreased AKT/PKB expression). (b) U251N cells were treated with celastrol at the indicated concentrations (0.3–10 μM) and labeled with Hoechst (nuclear stain). Scale bar in top left panel (200 μm) is representative for all panels shown. (c) U251N cells were treated with celastrol at the indicated concentrations (0.3–10 μM) in serum-containing and serum-deprived media for 24 h. Cell viability was assessed with the MTT assay. LD50: 4.07±0.29 μM (Serum−) versus 3.16±0.09 μM (Serum+); P=2.67 × 10⁻¹² (see Statistical analysis in Materials and Methods section). (d), U251N cells were treated with serum-deprived media with or without 50 μM L-BSO, an inhibitor of gamma-glutamylcysteine synthetase, for 3 h, after which celastrol was added at various concentrations (0.3–10 μM) for another 24 h. Cell viability was assessed as in panel (c). L-BSO treatment did not have a significant effect (LD50: 3.65±0.24 μM without L-BSO versus 3.25±0.27 μM with L-BSO; P=1.06). (e) Intracellular GSH content was assessed as described in the Materials and Methods section, following 24 h treatment with serum-containing or serum-deprived media in the presence and absence of 50 μM L-BSO and 3 μM celastrol. L-BSO led to a significant reduction in intracellular GSH relative to untreated cells. No significant change in GSH content was noted for celastrol alone. (f) U251N cells were treated with serum-deprived media with or without SP600125, an inhibitor of JNK, at the indicated concentrations for 3 h, after which celastrol was added at various concentrations (0.1–10 μM) for another 24 h. Cell viability was assessed as in panels (c) and (d). No significant effect was seen with SP600125 (LD50: 2.5±0.2 μM without SP versus 2.2±0.58 μM with 10 μM SP and 1.89±0.71 μM with 20 μM SP; P=0.0834). Average values and S.D. are reported for triplicate measurements (N=3). Results are representative of at least three independent experiments. ***P<0.001