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.

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Celastrol, a quinone methide triterpene derived from the medicinal plant Tripterygium wilfordii, has been used to treat chronic inflammatory and autoimmune diseases, but its mechanism is not well understood. Therefore, we investigated the effects of celastrol on cellular responses activated by TNF, a potent proinflammatory cytokine. Celastrol potentiated the apoptosis induced by TNF and chemotherapeutic agents and inhibited invasion, both regulated by NF-kappaB activation. We found that TNF induced 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) and that celastrol treatment suppressed their expression. Because these gene products are regulated by NF-kappaB, we postulated that celastrol mediates its effects by modulating the NF-kappaB pathway. We found that celastrol suppressed both inducible and constitutive NF-kappaB activation. Celastrol was found to inhibit the TNF-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 nuclear translocation and phosphorylation, and NF-kappaB-mediated reporter gene expression. Recent studies indicate that TNF-induced IKK activation requires activation of TAK1, and we indeed found that celastrol inhibited the TAK1-induced NF-kappaB activation. Overall, our results suggest that celastrol potentiates TNF-induced apoptosis and inhibits invasion through suppression of the NF-kappaB pathway.[3]

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Celastrol, a plant triterpene has attracted great interest recently, especially for its potential anti-inflammatory and anti-cancer activities. In the present report, we investigated the effect of celastrol on proliferation of various cancer cell lines. The mechanism, by which this triterpene exerts its apoptotic effects, was also examined in detail. We found that celastrol inhibited the proliferation of wide variety of human tumor cell types including multiple myeloma, hepatocellular carcinoma, gastric cancer, prostate cancer, renal cell carcinoma, head and neck carcinoma, non-small cell lung carcinoma, melanoma, glioma, and breast cancer with concentrations as low as 1 μM. Growth inhibitory effects of celastrol correlated with a decrease in the levels of cyclin D1 and cyclin E, but concomitant increase in the levels of p21 and p27. The apoptosis induced by celastrol was indicated by the activation of caspase-8, bid cleavage, caspase-9 activation, caspase-3 activation, PARP cleavage and through the down regulation of anti-apoptototic proteins. The apoptotic effects of celastrol were preceded by activation of JNK and down-regulation of Akt activation. JNK was needed for celastrol-induced apoptosis, and inhibition of JNK by pharmacological inhibitor abolished the apoptotic effects. Overall, our results indicate that celastrol can inhibit cell proliferation and induce apoptosis through the activation of JNK, suppression of Akt, and down-regulation of anti-apoptotic protein expression.[4]

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.

Celastrol is a pentacyclic triterpenoid that is 24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic acid bearing an oxo substituent at position 2, a hydroxy substituent at position 3 and two methyl groups at positions 9 and 13. An antioxidant and anti-inflammatory agent. Potently inhibits lipid peroxidation in mitochondria and inhibits TNF-alpha-induced NFkappaB activation. Also shown to inhibit topoisomerase II activity in vitro (IC50 = 7.41 muM). It has a role as an antioxidant, an anti-inflammatory drug, an EC 5.99.1.3 [DNA topoisomerase (ATP-hydrolysing)] inhibitor, an antineoplastic agent, a Hsp90 inhibitor and a metabolite. It is a pentacyclic triterpenoid and a monocarboxylic acid.
Celastrol has been reported in Celastrus paniculatus, Tripterygium wilfordii, and other organisms with data available.

C29H38O4

450.61

450.277

C, 77.30; H, 8.50; O, 14.20

34157-83-0

Pristimerin;1258-84-0; 34157-83-0 (castrol); 193957-88-9 (dihydrocelastrol)

122724

Orange to red solid powder

1.2±0.1 g/cm3

645.7±55.0 °C at 760 mmHg

185-200ºC

358.3±28.0 °C

0.0±4.4 mmHg at 25°C

1.602

7.08

2

4

1

33

1100

6

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.55 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)