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Triptonide (NSC-165677; PG-492), a natural product isolated from Tripterygium wilfordii Hook, a novel and potent Wnt/β-catenin inhibitor that inhibited the proliferation of mouse splenocytes induced by suboptimal concentration of concanavalin A or lipopolysaccharide at concentrations of 0.02, 0.1, and 0.5 mg/ml. Triptonide can effectively inhibit canonical Wnt/β-catenin signaling by targeting the downstream C-terminal transcription domain of β-catenin or a nuclear component associated with β-catenin. It also effectively induced apoptosis of Wnt-dependent cancer cells, supporting the therapeutic potential of triptonide.
Physicochemical Properties
| Molecular Formula | C20H22O6 |
| Molecular Weight | 358.3851 |
| Exact Mass | 358.141 |
| CAS # | 38647-11-9 |
| PubChem CID | 65411 |
| Appearance | White to off-white solid powder |
| Density | 1.5±0.1 g/cm3 |
| Boiling Point | 581.1±50.0 °C at 760 mmHg |
| Flash Point | 257.8±30.2 °C |
| Vapour Pressure | 0.0±1.6 mmHg at 25°C |
| Index of Refraction | 1.638 |
| LogP | 1.49 |
| Hydrogen Bond Donor Count | 0 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 1 |
| Heavy Atom Count | 26 |
| Complexity | 860 |
| Defined Atom Stereocenter Count | 8 |
| SMILES | CC(C)[C@@]12[C@@H](O1)[C@H]3[C@@]4(O3)[C@]5(CCC6=C([C@@H]5C[C@H]7[C@]4(C2=O)O7)COC6=O)C |
| InChi Key | SWOVVKGLGOOUKI-ZHGGVEMFSA-N |
| InChi Code | InChI=1S/C20H22O6/c1-8(2)18-13(25-18)14-20(26-14)17(3)5-4-9-10(7-23-15(9)21)11(17)6-12-19(20,24-12)16(18)22/h8,11-14H,4-7H2,1-3H3/t11-,12-,13-,14-,17-,18-,19+,20+/m0/s1 |
| Chemical Name | (5bS,6aS,7aS,8aS,9aS,9bS,10aS,10bS)-8a-isopropyl-10b-methyl-2,5,5b,6,6a,9a,9b,10b-octahydrotris(oxireno)[2',3':4b,5;2'',3'':6,7;2''',3''':8a,9]phenanthro[1,2-c]furan-3,8(1H,8aH)-dione |
| Synonyms | NSC165677; PG492; NSC-165677; PG-492; NSC 165677; PG 492; |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
Triptonide presumptively inhibits canonical Wnt/β-catenin signaling by targeting the C-terminal transactivation domain of β-catenin (3CTA) or a nuclear component associated with it. [1] |
| ln Vitro |
Triptonide stimulates Wnt-dependent cancer cell apoptosis and inhibits Wnt/β-catenin signaling via the β-catenin C-terminal transactivation domain [1]. Triptonide, with an IC50 of 5.7 nM and 4.8 nM, respectively, potently suppresses the growth of human B lymphoma Raji and T lymphoma Jurkat cells [2]. The capacity of B lymphoma cells to form colonies is considerably inhibited by triptolide (2.5-10 nM; 6 days) [2]. Repunide (20 nM; 3 days) decreases BCL2 protein levels in lymphoma cells while increasing apoptosis by activating PARP and caspase 3 [2]. Significant reductions in total and phosphorylated Lyn protein, as well as in Lyn downstream ERK and ATK signaling pathways, are observed with triptolide (5-10 nM; 72 hours) [2]. Triptonide effectively inhibited Wnt3a-conditioned media-induced TOPFlash luciferase activity in STF293 cells in a dose-dependent manner with an IC50 of approximately 0.3 nM. [1] Triptonide dramatically attenuated the gene expression of Wnt target genes Axin2 and Cyclin D1 induced by the GSK-3β inhibitor CHIR 99021 (Chir) in STF293 cells, as determined by RT-PCR. [1] Triptonide did not downregulate Wnt3a-conditioned media-induced levels of active (non-phosphorylated) β-catenin protein in HEK293 cells, as shown by Western blotting. [1] Triptonide inhibited Chir-induced TOPFlash luciferase activity in STF293 cells in a dose-dependent manner but did not affect Chir-induced β-catenin protein levels in HEK293 cells. [1] Triptonide did not block BIO-induced nuclear translocation of β-catenin in human colon carcinoma RKO cells, as assessed by immunostaining. [1] Triptonide effectively abrogated TOPFlash luciferase activity induced by overexpression of a constitutively active β-catenin mutant (S33Y) in STF293 cells. [1] Triptonide did not block the interaction between TCF4 and β-catenin in co-immunoprecipitation assays using HEK293 cells treated with BIO. [1] Triptonide effectively attenuated TOPFlash luciferase activity induced by overexpression of the LEFΔN-3CTA fusion protein (containing the β-catenin C-terminal transactivation domain) in STF293 cells in a dose-dependent manner. [1] Triptonide selectively reduced the viability of Wnt signaling-dependent cancer cell lines. After 72 hours of treatment, the IC50 values were approximately 16.8 nM for SW480 cells and 14.6 nM for RKO cells. Treatment with 50 nM Triptonide for 72 hours decreased PC3 cell viability by approximately 40%. It displayed minimal killing activity in control HEK293 cells under the same conditions. [1] Triptonide induced apoptosis in SW480 colon cancer cells, as detected by activated caspase-3/7 green detection reagent after 24 hours of treatment with 20 nM, but not in control HEK293 cells. [1] |
| ln Vivo |
Mice treated with 5 mg/kg of rapunide intraperitoneally once a day for 34 days show significant anti-lymphoma effects [2]. Triptonide reproducibly rescued the loss of eyes ("eyeless" phenotype) in zebrafish embryos induced by the GSK-3β inhibitor BIO (0.3 µM). Embryos were co-treated with 100 nM Triptonide and BIO from 6 hours post-fertilization (hpf, shield stage), and eye development was assessed at 30 hpf. [1] |
| Cell Assay |
Cell proliferation assay [2] Cell Types: B lymphoma Raji cells, T lymphoma Jurkat cells Tested Concentrations: 0-80 nM Incubation Duration: 3 days, 6 days Experimental Results: Inhibited the tumorigenic ability of lymphoma cells in a dose-dependent manner. Apoptosis analysis [2] Cell Types: Raji cells Tested Concentrations: 5 nM, 10 nM, 20 nM Incubation Duration: 3 days Experimental Results: No significant induction of apoptosis under effective tumor growth inhibition (2.5-10 nM); Moderately induces apoptosis in lymphoma cells (20 nM). Western Blot Analysis[2] Cell Types: Raji Cell Tested Concentrations: 5 nM, 10 nM, 20 nM Incubation Duration: 3 days Experimental Results: Proapoptotic proteins PARP and caspase 3 (5-10 nM) in lymphoma cells are not strongly activated ; Dramatically activates PARP and caspase 3 (20 nM); Dramatically reduces anti-apoptotic BCL2 levels. RT-PCR[2] Cell Types: Raji cells Tested Concentrations: 5 nM, 10 nM Incubation Duration: 72 hrs (hours) Experimental Results: Lyn mRNA levels were Dramatically diminished in lymphoma cells. Luciferase Reporter Assay: STF293 cells (stably transfected with the TOPFlash-firefly luciferase reporter) were seeded in 96-well plates. For experiments with transfection, cells were transfected with relevant plasmids (e.g., pcDNA3, constitutive active β-catenin S33Y, LEFΔN-3CTA) along with a Renilla luciferase control plasmid using a transfection reagent. After transfection, cells were treated with Triptonide or vehicle at indicated concentrations for 18-24 hours. Cell lysates were then prepared and subjected to dual-luciferase assay. Firefly luciferase activity was normalized to Renilla luciferase activity. For experiments without transfection, STF293 cells were treated with compounds, and luciferase activity in cell lysates was measured using a steady-glo assay and normalized to cell titer. [1] Western Blotting: Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined, and equal amounts of lysates were separated by SDS-PAGE, then transferred to a membrane. The membrane was blocked and incubated with primary antibodies (e.g., anti-active β-catenin, anti-α-tubulin) overnight at 4°C, followed by incubation with fluorescent dye-conjugated secondary antibodies. Protein bands were visualized and quantified using an infrared imaging system. [1] RT-PCR: Total RNA was extracted from cells using a lysis buffer and purification kit. cDNA was synthesized using a reverse transcription kit. Quantitative PCR was performed using a SYBR Green master mix on a real-time PCR cycler. Gene expression levels (e.g., Axin2, Cyclin D1) were normalized to GAPDH. Primer sequences for target genes are provided in the manuscript. [1] Immunofluorescence/Immunostaining: Cells (e.g., RKO) were cultured in chamber slides and treated with compounds (e.g., BIO with or without Triptonide). After treatment, cells were fixed with formaldehyde, permeabilized, and blocked. Cells were then incubated with primary antibody (e.g., anti-active β-catenin) overnight at 4°C, followed by incubation with a fluorophore-conjugated secondary antibody. Nuclei were counterstained with DAPI. Images were captured using a fluorescence microscope. [1] Cell Viability Assay: Cancer cells (SW480, RKO, PC3) and control HEK293 cells were seeded in 96-well plates. After overnight incubation, cells were treated with varying concentrations of Triptonide for 72 hours. Cell viability was then assessed using a luminescent cell viability assay reagent according to the manufacturer's protocol. Luminescence was measured and normalized to vehicle-treated controls. [1] Apoptosis Assay: Cells were cultured in chamber slides and treated with Triptonide or vehicle for 24 hours. The culture medium was removed, and adherent cells were incubated with a fluorogenic substrate for activated caspases-3/7 in a buffer containing low serum. After incubation, cells were fixed, permeabilized, and nuclei were counterstained with DAPI. Apoptotic cells (caspase-3/7 positive) were visualized and imaged using a fluorescence microscope. [1] Co-Immunoprecipitation: HEK293 cells were treated with BIO and Triptonide overnight. Cells were lysed in RIPA buffer with inhibitors. Cell lysates were incubated with an anti-TCF4 antibody at 4°C for 2 hours, followed by the addition of Protein A agarose beads and incubation overnight at 4°C. Beads were washed, and bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blotting using antibodies against β-catenin and TCF4. [1] |
| Animal Protocol |
Animal/Disease Models: Eightweeks old female NOD/SCID (severe combined immunodeficient) mouse (18-22 g) with 3 x 107 Raji cell xenografts [2] Doses: 5 mg/kg Route of Administration: intraperitoneal (ip) injection daily for 34 Experimental Results: Effectively inhibit the growth and tumorigenic ability of lymphoma cells. Zebrafish Embryo Rescue Assay: Zebrafish embryos of the AB/TL wild-type strain were used. Embryos at the shield stage (6 hours post-fertilization, hpf) were exposed to embryo water containing 0.3 µM BIO (a GSK-3β inhibitor) alone or in combination with 100 nM Triptonide. Treatment continued until 24 hpf. Embryos were then transferred to fresh embryo water and allowed to develop until 30 hpf. Embryos were anesthetized and mounted for imaging under a microscope. The presence or absence of eyes was scored. The vehicle control (DMSO) was used for comparison. [1] |
| References |
[1]. Triptonide Effectively Inhibits Wnt/β-Catenin Signaling via C-terminal Transactivation Domain of β-catenin. Sci Rep. 2016; 6: 32779. [2]. Triptonide acts as a novel potent anti-lymphoma agent with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn signal pathway. Toxicol LettPing Yang. 2017 Aug 15;278:9-17. |
| Additional Infomation |
Triptonide is a diterpene triepoxide that is triptobenzene K in which the acylhydroquinone moiety has undergone oxidation to the corresponding triepoxyketone derivative. It has been isolated from the roots of Tripterygium wilfordii. It has a role as an antineoplastic agent, an anti-inflammatory agent and an immunosuppressive agent. It is a cyclic ketone, an organic heteroheptacyclic compound, a diterpene triepoxide and a butenolide. Triptonide has been reported in Tripterygium wilfordii and Tripterygium hypoglaucum with data available. Triptonide is a key bioactive small molecule identified in the traditional Chinese medicine Tripterygium wilfordii Hook F. (Thunder God Vine). [1] Triptonide is structurally very similar to triptolide, differing only at position 14 where triptonide has a carbonyl group and triptolide has a hydroxyl group. This slight difference leads to distinct molecular mechanisms of action against the Wnt/β-catenin pathway. [1] The proposed mechanism of action for Triptonide is the inhibition of canonical Wnt/β-catenin signaling by targeting the C-terminal transactivation domain of β-catenin or an associated nuclear co-factor, thereby disrupting downstream transcriptional activation. This mechanism is different from triptolide, which can reduce β-catenin protein levels. [1] Due to its ability to inhibit aberrant Wnt signaling and induce apoptosis in Wnt-dependent cancer cells (while sparing normal cells in vitro), Triptonide may represent a potential therapeutic agent for cancers driven by Wnt/β-catenin signaling, such as colorectal cancer. Its in vivo efficacy was demonstrated in a zebrafish model of Wnt pathway hyperactivation. [1] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~100 mg/mL (~279.03 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.98 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.98 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7903 mL | 13.9513 mL | 27.9026 mL | |
| 5 mM | 0.5581 mL | 2.7903 mL | 5.5805 mL | |
| 10 mM | 0.2790 mL | 1.3951 mL | 2.7903 mL |