Celastrol and its effects. (A) Structure of triterpene, celastrol. (Bi-iii) Celastrol enhances TNF- and chemotherapeutic agent-induced cytotoxicity. In total, 10 000 cells were seeded in triplicate in 96-well plates. The cells were pretreated with 2.5 μM celastrol and then incubated with the indicated concentrations of TNF, paclitaxel, and doxorubicin for 24 hours. Cell viability was analyzed by the MTT method as described in “Materials and methods.” (C) Celastrol potentiates TNF-induced apoptosis. KBM-5 cells were pretreated with 2.5 μM celastrol for 6 hours and then incubated with 1 nM TNF for 16 hours. The cells were stained with a live/dead assay reagent for 30 minutes and then analyzed under a fluorescence microscope as described in “Materials and methods.” The results shown are representative of 3 independent experiments. (D) Cells were pretreated with 2.5 μM celastrol for 6 hours and then incubated with 1 nM TNF for 16 hours. Cells were fixed, stained with TUNEL assay reagent, and then analyzed with a flow cytometer for apoptotic effects. The results shown are representative of 2 independent experiments. (E) Cells were pretreated with 2.5 μM celastrol for 6 hours and then incubated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and analyzed by Western blotting using an anti-PARP antibody. The results shown are representative of 3 independent experiments. (F) Celastrol suppresses TNF-induced invasion activity. H1299 cells (2.5 × 104 cells) were seeded to the top chamber of a Matrigel invasion chamber system overnight in the absence of serum and then treated with celastrol. After incubation, the cells were treated with TNF in the presence of 1% serum and then assayed for invasion as described in “Materials and methods.” Results are expressed as fold activity of the untreated control.
