20S proteasome (IC50 = 2.5 μM)

Celastrol inhibits the purified 20S proteasome's chymotrypsin-like, PGPH-like, and trypsin-like activities by 80%, 5%, and less than 1%, respectively, at 5 μM; at 10 μM, it inhibits these three proteasomal activities by approximately 90%, 15%, and less than 1%, respectively. In PC-3 cells, celastrol significantly and concentration-dependently inhibits the activity of the proteasomal chymotrypsin. In PC-3 cells, celastrol at 2.5 μM to 5 μM increases caspase-3 activity by 4.7–5.5 fold. In cells treated with celastrol (5 μM), the levels of the proteasome target proteins, Bax and IκB-α, increase after one hour and continue to rise to their maximum for four to twelve hours. Celastrol (2.5 μM) treatment results in 40% inhibition of the proteasome, as evidenced by the reduction of chymotrypsin-like activity and the elevation of ubiquitinated protein accumulation in LNCaP cells. Increased levels of caspase-3 activity (up to 3.5-fold), PARP cleavage, and apoptotic morphology indicate that Celastrol (2.5 μM) induces apoptosis in LNCaP cells.[1] The production of TNF-alpha and IL-1beta by human monocytes and macrophages stimulated by LPS is found to be suppressed by celastrol (300 nM). Moreover, microglia's LPS-induced production of class II MHC molecules is reduced by celastrol (100 nM). In macrophage lineage cells, celastrol has an IC50 of 200 nM, which is a strong inhibitor of LPS and IFN-y-induced NO production. Celastrol has an IC50 of 200 nM in endothelial cells, which effectively suppresses the production of NO induced by TNF-α and IFN-γ.[2] Celastrol (2.5 μM) inhibits invasion and amplifies apoptosis brought on by TNF and chemotherapy drugs, both of which are controlled by NF-kappaB activation, in KBM-5 cells. The expression of gene products involved in antiapoptosis (IAP1, IAP2, Bcl-2, Bcl-XL, c-FLIP, and survivin), proliferation (cyclin D1 and COX-2), invasion (MMP-9), and angiogenesis (VEGF) in KBM-5 cells is suppressed by celastrol (2.5 μM). It is discovered that celastrol (5 μM) inhibits the activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, nuclear translocation and phosphorylation of p65, and reporter gene expression mediated by NF-kappaB when stimulated with TNF.[3] Celastrol has an IC50 of 0.52 μM, 0.54 μM, 0.76 μM, 0.69 μM, and 0.67 μM, respectively, which inhibits the proliferation of RPMI 8226, KATOIII, UM-SCC1, U251MG, and MDA-MB-231 cells. Reduced levels of cyclin D1 and cyclin E, but increased levels of p21 and p27, are the result of celastrol (1 μM) inhibiting the growth of RPMI 8226. RPMI-8226 cells undergo apoptosis when celastrol is administered, as evidenced by the downregulation of anti-apoptototic proteins and the activation of caspase-8, bid, caspase-9, caspase-3, and PARP cleavage. RPMI-8226 cells are exposed to 1 μM celastrol, which inhibits the Akt pathway and activates JNK kinase.[4]

Celastrol (3 mg/kg) significantly (up to 70%) inhibits the growth of PC-3 tumors in male nude mice, and this effect is correlated with elevated Bax and p27 levels. Celastrol (3 mg/kg) causes a greater number of apoptotic tumor cells, as evidenced by the appearance of different PARP cleavage fragments in the tumors of male naked mice carrying PC-3 tumors. In nude mice with C4-2B tumors, celastrol (3 mg/kg) results in 35% tumor inhibition, which is correlated with decreased proteasome activity and decreased expression of AR protein. (Source: ) In mice, celastrol (3 mg/kg) is found to significantly reduce joint swelling and other adjuvant arthritis symptoms. Rats' performance in tests of psychomotor activity, memory, and learning is considerably enhanced by celastrol (0.2 mg/kg).[2]

A rabbit 20S proteasome that has been purified (0.1 μg) is held in an assay buffer (20 mM Tris-HCl, pH 7.5) containing 40 μM of different fluorogenic peptide substrates for two hours at 37 °C. Celastrol is added at varying concentrations or the proteasome is dissolved in DMSO. The amount of inhibition of each proteasomal activity is then measured.

The MTT uptake method determines Celastrol's anti-proliferative effect on different human tumor cell lines. In a 96-well plate, 5×10³ cells are incubated with Celastrol in triplicate at 37 °C. After that, MTT solution is added to every well. Following a two-hour incubation period at 37 °C, cells are treated with extraction buffer (20% SDS, 50% dimethylformamide) and left to incubate for an additional night at 37 °C. The optical density is then measured at 570 nm using a Tecan plate reader.

Mice: Five-week-old male NCRNU-M nude immunodeficient mice are utilized. Day 0 involves the subcutaneous injection of human prostate cancer PC-3 or C4-2B cells (5-10×10⁶) suspended in 0.1 mL of serum-free RPMI 1640 into the right flank of each mouse (four mice per group). On day 14 following inoculation, the animals in the first PC-3 cell experiment began receiving daily intraperitoneal injections (i.p.) of either 1.0 or 3.0 mg/kg of Celastrol or 50 to 100 μL of a vehicle (10% DMSO, 70% Cremophor/ethanol (3:1), and 20% PBS). Every day, tumor sizes are measured with calipers, and their volumes are computed using a standard formula: width²×length/2. Weekly measurements are made of body weight. Once three days of treatment are up, one control and one 3.0 mg/kg Celastrol-treated mouse is sacrificed to investigate whether the proteasome is inhibited early in the experiment. Upon reaching 1,400 mm³, the control tumors, the remaining ones are killed after 16 days of treatment. In the subsequent PC-3 tumor experiment, mice are randomized into three groups 12 days post-inoculation and administered 1.5 mg/kg daily oral cetonin, control, or cetonin for the full 31-day study period. Nude mice with C4-2B tumors are given daily intraperitoneal injections (i.p.) of either the vehicle or 3.0 mg/kg Celastrol in order to investigate the effects of the drug on AR expression.
Rats: Ninety male Sprague-Dawley (SD) rats, weighing 161±9 g at six weeks of age, are randomly assigned to the high energy diet (HED) and control groups. Rats in the HED group are fed an additional high energy emulsion, while those in the control group are fed a standard chow diet. In order to create a model of type 2 diabetes, rats in the HED group receive an injection of streptozotocin (STZ; 45 mg/kg) dissolved in 0.1 mol/l citrate buffer (pH 4.5) into their caudal vein, whereas rats in the control group receive an injection of sodium citrate buffer. The rats used as the diabetes model are those whose blood glucose levels are ≥16.7 mM seven days after receiving the STZ injection. Rats injected with STZ exhibited these characteristics in 80% of cases on average. Two weeks after the STZ injection, the rats that developed diabetes successfully are split into four groups at random: the diabetes model (DM) group, the middle-dose group (1 mg/kg/day), the high-dose group (6 mg/kg/day) of Celastrol, and the diabetes model (n = 15 rats/group). When compared to the rats in the NC and DM groups, which receive an equivalent volume of distilled water (2 mL), the treatment groups' rats receive Celastrol by gavage. Rats are given an intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight) to induce anesthesia after 8 weeks of the regimen, and tissue samples are taken for examination. The paravertebral muscle is removed from the rat bodies, cut perpendicular to the longitudinal axis, and preserved in 20% formaldehyde that has been buffered with phosphate. After that, 5 μm histological paraffin-embedded sections are ready for H&E staining. The liquid nitrogen-snap-frozen paravertebral muscle sections are kept at?80°C for future examination.

[1].Cancer Res . 2006 May 1;66(9):4758-65.

[2]. Prog Neuropsychopharmacol Biol Psychiatry . 2001 Oct;25(7):1341-57.

[3]. Blood . 2007 Apr 1;109(7):2727-35.

[4]. Apoptosis . 2011 Oct;16(10):1028-41.

C29H38O4

450.61

450.28

C, 77.30; H, 8.50; O, 14.20

34157-83-0

Pristimerin;1258-84-0

Red solid powder

CC1=C(C(=O)C=C2C1=CC=C3

KQJSQWZMSAGSHN-JJWQIEBTSA-N

InChI=1S/C29H38O4/c1-17-18-7-8-21-27(4,19(18)15-20(30)23(17)31)12-14-29(6)22-16-26(3,24(32)33)10-9-25(22,2)11-13-28(21,29)5/h7-8,15,22,31H,9-14,16H2,1-6H3,(H,32,33)/t22-,25-,26-,27+,28-,29+/m1/s1

(2R,4aS,6aR,6aS,14aS,14bR)-10-hydroxy-2,4a,6a,6a,9,14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydropicene-2-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)