Tripterin (Celastrol) is a natural product, a proteasome inhibitor that inhibits the pancreatic rennet-like activity of the 20S proteasome (IC50=2.5 μM). Tripterin has anti-inflammatory, anti-infectious, and immunomodulatory properties.

20S proteasome:2.5 μM

METHODS: Human prostate cancer cells PC-3 were treated with Tripterin (0.5-5 µM) for 12 h. Proteasomal chymotrypsin-like activity was assayed using Z-GGL-AMC.
RESULTS: Tripterin significantly inhibited proteasomal chymotrypsin-like activity in PC-3 cells in a concentration-dependent manner, reaching about 55% inhibition at 2.5 µM. [1]
METHODS: Human chronic myeloid leukemia cells KBM-5 were incubated with Tripterin (2.5 µM) for 6 h, followed by treatment with TNF (1 nM) for 6-24 h. Target protein expression levels were detected using Western Blot.
RESULTS: TNF induced the expression of anti-apoptotic proteins IAP1, IAP2, Bcl-2, Bcl-XL, c-FLIP and survivin in a time-dependent manner, which was inhibited by Tripterin. [2]

METHODS: To detect anti-tumor activity in vivo, Tripterin (1-3 mg/kg, 10% DMSO+70% Cremophor/ethanol (3:1)+20% PBS) was injected intraperitoneally once daily for sixteen days into nude immunodeficient mice bearing human prostate cancer tumor PC-3.
RESULTS: Tripterin treatment significantly inhibited the growth of prostate cancer xenografts and suppressed proteasome activity and induced apoptosis in vivo. [1]
METHODS: To detect anti-tumor activity in vivo, Tripterin (1.25 mg/kg) was intraperitoneally injected into BALB/c (nu/nu) mice bearing vestibular nerve sheath tumor SC4 every three days for two weeks.
RESULTS: Tripterin significantly inhibited tumor growth without showing toxicity. [3]

Inhibition of purified 20S proteasome activity: A purified rabbit 20S proteasome (0.1 μg) is incubated with 40 μM of various fluorogenic peptide substrates in 100 μL assay buffer (20 mM Tris-HCl (pH 7.5)), in the presence of Celastrol at different concentrations or in the solvent DMSO for 2 hours at 37 ℃, followed by measurement of inhibition of each proteasomal activity.

The anti-proliferative effect of celastrol on various human tumor cell lines is determined by the MTT uptake method. Briefly, 5×103 cells are incubated with Celastrol in triplicate in a 96-well plate at 37 ℃. MTT solution is then added to each well. After a 2 hours incubation at 37 ℃, extraction buffer (20% SDS, 50% dimethylformamide) is added, cells are incubated overnight at 37 ℃, and the optical density is then measured at 570 nm using a Tecan plate reader.(Only for Reference)