IKKβ; COX2
Dehydrocostus Lactone (DHE) targets IKKβ by binding to its ATP-binding site, thereby inhibiting the NF-κB/COX-2 signaling pathway. The study does not provide IC₅₀, Ki, or EC₅₀ values for binding to IKKβ.

- Directly targets IKKβ (Inhibitor of Nuclear Factor Kappa-B Kinase subunit beta) by binding to its ATP-binding site, as demonstrated by molecular docking studies. [2]
- Inhibits the phosphorylation of IKKα/β, IκBα, and p65 (NF-κB subunit). [2]
- Downregulates COX-2 (Cyclooxygenase-2) expression at both mRNA and protein levels. [2]
- Modulates apoptosis-related proteins: decreases Bcl-2, increases BAX and cleaved caspase-3/9, and promotes cytochrome c release. [2]

Dehydrocostus lactone can inhibit PGE2 synthesis in vitro. It significantly reduces COX-2 expression but not COX-1 expression[1]. Glioblastoma (U118, U251 or U87) cells are significantly inhibited in their viability, proliferation, and migration following treatment with DHE. The release of cytochrome c into the cytosol, which activates the caspase signaling pathway, is another way that DHE triggers mitochondria-mediated apoptosis. By blocking IKK phosphorylation by aiming at the ATP-binding site and preventing NF-κB binding and p300 recruitment to the COX-2 promoter, DHE significantly reduces COX-2 expression. DHE inhibits the ability of glioblastoma cells to migrate and form colonies, and it triggers apoptosis via the mitochondrial pathway[2].

---

Dehydrocostus Lactone exhibits significant anti-glioma effects in various glioblastoma cell lines through multiple mechanisms.

1. Inhibition of Cell Viability: In U118, U251, and U87 glioblastoma cells, DHE (0-100 μM) inhibited cell viability in a dose- and time-dependent manner (12, 24, 36, 48 h). The IC₅₀ values at 48 h were 17.16 ± 2.11 μM (U118), 22.33 ± 1.93 μM (U251), and 26.42 ± 2.84 μM (U87). [2]
2. Suppression of Colony Formation: Treatment with DHE (10 and 20 μM) for 3 h significantly reduced the number of colonies formed by U87 and U251 cells in a dose-dependent manner. [2]
3. Inhibition of Cell Migration (Wound Healing Assay): In U118 and U251 cells, DHE (10 and 20 μM) treatment for 48 h significantly decreased the migration rate compared to control groups (P < 0.01). [2]
4. Induction of Mitochondrial Apoptosis: In U87 cells, DHE treatment (48 h) induced cytochrome c release from mitochondria to cytosol (confocal microscopy and Western blot). It also decreased the anti-apoptotic Bcl-2/BAX ratio and increased the cleavage of procaspase-9 and procaspase-3 in a dose-dependent manner. [2]
5. Downregulation of COX-2 Expression: DHE (10, 20, 30 μM; 48 h) dose-dependently inhibited COX-2 protein expression in U87 and U251 cells, as well as COX-2 mRNA levels, as shown by Western blot and RT-PCR. [2]
6. Inhibition of NF-κB Transcriptional Activity: ChIP and streptavidin-agarose pulldown assays in U87 cells showed that DHE dose-dependently inhibited the recruitment of p300 and the binding of p50/p65 NF-κB to the COX-2 promoter. Western blot and immunofluorescence confirmed that DHE decreased the nuclear translocation of p300, p65, and p50. [2]
7. Inhibition of IKKβ/NF-κB Signaling: Western blot analysis in U87 and U251 cells revealed that DHE (10, 20, 30 μM; 48 h) dose-dependently reduced the phosphorylation of IKKα/β, IκBα, and p65, without affecting total protein levels of IKKα, IKKβ, IκBα, and p65. [2]
8. Molecular Docking (IKKβ Binding): Molecular modeling studies predicted that DHE binds to the ATP-binding site of IKKβ, forming a hydrogen bond with Cys99 via its lactone ring, which extends into the hydrophobic cavity of the pocket. [2]

Dehydrocostus lactone inhibits colon cancer cell line colo205-transplanted colorectal cancer xenograft growth in athymic mice. The inhibition effect depends on the dose[1]. Blood-brain barrier (BBB) can be crossed by DHE. In the xenograft nude mouse model, treatment with DHE significantly reduces neoplastic weight and volume without observable adverse effects, and these effects may be mediated by inhibiting the IKKβ/NF-κB/COX-2 signaling pathway[2].

---

Dehydrocostus Lactone demonstrates significant anti-tumor efficacy in a xenograft nude mouse model without notable toxicity.

1. Blood-Brain Barrier Penetration: LC-MS/MS analysis of cerebrospinal fluid from SD rats 1 hour after intraperitoneal administration of DHE (100 mg/kg) showed a single peak, indicating that DHE can rapidly cross the blood-brain barrier. [2]
2. Inhibition of Tumor Growth in Xenograft Model: Nude mice bearing U87 subcutaneous xenografts were treated with DHE (10 or 20 mg/kg/d, i.p.) or PBS for 14 days. Both doses of DHE significantly reduced tumor volume and tumor weight compared to the control group. [2]
3. Lack of Systemic Toxicity: Body weights of mice were not significantly different between DHE-treated and control groups throughout the experiment, suggesting no obvious side effects at the tested doses. [2]
4. Inhibition of IKKβ/NF-κB/COX-2 Pathway in vivo: Immunohistochemical staining of tumor xenografts showed that DHE treatment dose-dependently decreased the expression of COX-2, phosphorylated p65 (p-p65), and phosphorylated IKKβ (p-IKKβ). [2]

No direct enzyme activity assays on purified enzymes were performed. The primary evidence for IKKβ targeting comes from molecular docking studies.

1. Molecular Docking: The study used molecular docking to simulate the interaction between DHE and IKKβ. The optimized structure of DHE was docked into the active site of IKKβ (PDB Code: 4KIK) using SurflexDock. The results predicted that DHE binds to the ATP-binding site, forming a hydrogen bond with Cys99. MOLCAD surface analysis was performed to visualize the binding mode. [2]

To obtain a 70% confluent monolayer, cells from the U118 (6×103 cells/well), U251 (8×103 cells/well), and U87 (7×103 cells/well) are counted and allowed to adhere. The cells are then given treatments with DHE at the indicated concentrations. Following 12, 24, 36, or 48 hours of incubation, 10 l of MTT (5 mg/ml) is added to each well, and the cells are then incubated for an additional 4 hours. In order to dissolve the formazan crystals, the medium is then replaced with 100 μl of DMSO. A microplate reader is used to measure the absorbance at 570 nm. At least three times each experiment is run again. By extrapolating from dose-response curves, the IC50 values are determined.

---

Multiple cell-based assays were used to characterize the effects of Dehydrocostus Lactone on glioblastoma cells.

1. Cell Viability (MTT Assay): U118, U251, and U87 cells were seeded in 96-well plates, treated with various concentrations of DHE for 12-48 h, incubated with MTT for 4 h, and absorbance was measured at 570 nm to calculate IC₅₀ values. [2]
2. Colony Formation Assay: U87 and U251 cells were seeded in 6-well plates, treated with DHE for 3 h, then cultured in fresh medium for 15 days. Colonies were fixed, stained with crystal violet, and counted. [2]
3. Wound Healing Assay: Confluent monolayers of U118 and U251 cells were scratched with a pipette tip, treated with DHE in serum-free medium for 48 h, and wound closure was imaged and quantified. [2]
4. Immunofluorescence: U87 cells grown on coverslips were treated with DHE, fixed, permeabilized, and incubated with primary antibodies against cytochrome c, p300, p65, or p50, followed by fluorescent secondary antibodies. Nuclear counterstaining with DAPI was performed, and images were captured by confocal microscopy. [2]
5. Western Blot: Cell lysates or nuclear extracts were prepared, separated by SDS-PAGE, transferred to PVDF membranes, and probed with specific antibodies against proteins of interest (e.g., cleaved caspase-3/9, COX-2, p-IKK, p-p65). Protein bands were visualized by ECL and quantified. [2]
6. RT-PCR: Total RNA was extracted from cells, reverse transcribed, and PCR was performed using specific primers for COX-2 and GAPDH. PCR products were analyzed by agarose gel electrophoresis. [2]
7. Chromatin Immunoprecipitation (ChIP): U87 cells were cross-linked with formaldehyde, sonicated, and incubated with antibodies against p300, p65, p50, or IgG. DNA was purified and analyzed by PCR using COX-2 promoter-specific primers. [2]
8. Streptavidin-Agarose Pulldown: Nuclear extracts from U87 cells were incubated with biotinylated COX-2 promoter probe and streptavidin-agarose beads. Bound proteins were analyzed by Western blot for p300, p65, and p50. [2]

The female athymic nude mice (4-6 weeks old) and Sprague Dawley rats (8-10 weeks old)
10 and 20 mg/kg
i.p.

---

Two main in vivo protocols were used: a pharmacokinetic study for BBB penetration and an efficacy/toxicity study in a xenograft model.

1. **Blood-Brain Barrier Penetration Study:**
- **Animals:** Male Sprague Dawley rats (200-220 g). [2]
- **Drug Administration:** Rats received an intraperitoneal injection of DHE (100 mg/kg). [2]
- **Sample Collection:** One hour post-injection, rats were anesthetized, and cerebrospinal fluid (CSF) was collected from the cisterna magna. [2]
- **Sample Preparation and Analysis:** CSF was mixed with acetonitrile, centrifuged, and the supernatant was dried under nitrogen. The residue was reconstituted and analyzed by LC-MS/MS to detect DHE. [2]
2. **Xenograft Tumor Model (Efficacy and Toxicity):**
- **Animals:** Female athymic nude mice (4-6 weeks old). [2]
- **Tumor Inoculation:** U87 cells (1 × 10⁷) were subcutaneously injected into the left axillary fossa of each mouse. [2]
- **Treatment Protocol:** After two weeks, mice were randomly divided into three groups (n=6/group) and received daily intraperitoneal injections of PBS (control), DHE (10 mg/kg), or DHE (20 mg/kg) for 14 days. [2]
- **Monitoring:** Body weight and tumor volume (calculated as V = 1/2 × length × width²) were recorded every 2 days. [2]
- **Terminal Procedures:** On day 30, mice were euthanized. Tumors were excised, weighed, and photographed. Tumor tissues were fixed in formalin for immunohistochemical analysis of COX-2, p-p65, and p-IKKβ. [2]

---

Two main in vivo protocols were used: a pharmacokinetic study for BBB penetration and an efficacy/toxicity study in a xenograft model.

1. Blood-Brain Barrier Penetration Study:
- Animals: Male Sprague Dawley rats (200-220 g). [2]
- Drug Administration: Rats received an intraperitoneal injection of DHE (100 mg/kg). [2]
- Sample Collection: One hour post-injection, rats were anesthetized, and cerebrospinal fluid (CSF) was collected from the cisterna magna. [2]
- Sample Preparation and Analysis: CSF was mixed with acetonitrile, centrifuged, and the supernatant was dried under nitrogen. The residue was reconstituted and analyzed by LC-MS/MS to detect DHE. [2]
2. Xenograft Tumor Model (Efficacy and Toxicity):
- Animals: Female athymic nude mice (4-6 weeks old). [2]
- Tumor Inoculation: U87 cells (1 × 10⁷) were subcutaneously injected into the left axillary fossa of each mouse. [2]
- Treatment Protocol: After two weeks, mice were randomly divided into three groups (n=6/group) and received daily intraperitoneal injections of PBS (control), DHE (10 mg/kg), or DHE (20 mg/kg) for 14 days. [2]
- Monitoring: Body weight and tumor volume (calculated as V = 1/2 × length × width²) were recorded every 2 days. [2]
- Terminal Procedures: On day 30, mice were euthanized. Tumors were excised, weighed, and photographed. Tumor tissues were fixed in formalin for immunohistochemical analysis of COX-2, p-p65, and p-IKKβ. [2]

The study provides evidence for brain penetration but does not provide detailed PK parameters.

- Blood-Brain Barrier Penetration: DHE was detected in the cerebrospinal fluid of rats 1 hour after intraperitoneal administration (100 mg/kg), demonstrating its ability to rapidly cross the blood-brain barrier. [2]

The study provides preliminary safety data from the xenograft model.

- In Vivo Toxicity (Xenograft Model): In nude mice bearing U87 xenografts, daily intraperitoneal administration of DHE at 10 and 20 mg/kg for 14 days did not cause significant changes in body weight compared to the control group, suggesting no obvious systemic toxicity or side effects at these doses. [2]

[1]. Dehydrocostus lactone as Cyclooxygenase-2 specific inhibitor.

[2]. Am J Cancer Res . 2017 Jun 1;7(6):1270-1284. eCollection 2017.

Dehydroauracene lactone is an organic heterotricyclic compound belonging to the guaiacane sesquiterpene lactone family. Its structure involves the substitution of acrylic acid at the 2-position with a 4-hydroxy-3,8-bis(methylene)decylhydroazine-5-yl group, where the hydroxyl and carboxyl groups undergo a condensation reaction to form the corresponding γ-lactone. It can be used as a metabolite, trypanosome killer, antitumor agent, cyclooxygenase 2 inhibitor, antimycobacterial drug, and apoptosis inducer. It is a sesquiterpene lactone, a guaiacane sesquiterpene compound, an organic heterotricyclic compound, and a γ-lactone. Dehydroauracene lactone has been reported to exist in Ainsliaea uniflora, Costus, and other organisms with relevant data. See also: Burdock root (partial).

---

Dehydrocostus Lactone (DHE) is a natural sesquiterpene lactone derived from medicinal plants such as Inula helenium L. and Saussurea lappa. It has been traditionally used for its anti-inflammatory, anti-ulcer, and immunomodulatory properties. This study is the first to comprehensively investigate its anti-glioma effects and underlying mechanisms. The key findings demonstrate that DHE potently inhibits the growth, proliferation, migration, and survival of glioblastoma cells in vitro and in vivo. Its primary mechanism of action involves directly targeting IKKβ at its ATP-binding site, thereby inhibiting the canonical NF-κB pathway. This leads to reduced phosphorylation and degradation of IκBα, decreased nuclear translocation of p50/p65 NF-κB, reduced recruitment of the coactivator p300 to the COX-2 promoter, and ultimately downregulation of COX-2 expression. DHE also induces mitochondrial-mediated apoptosis. Importantly, the study demonstrates for the first time that DHE can cross the blood-brain barrier, a critical requirement for a brain tumor therapeutic. In a xenograft model, DHE significantly suppressed tumor growth without observable toxicity. These findings position DHE as a promising lead compound for the treatment of glioblastoma. [2]

C15H18O2

230.307

230.13

C, 78.23; H, 7.88; O, 13.89

477-43-0

477-43-0

73174

White to off-white solid powder

1.1±0.1 g/cm3

383.7±42.0 °C at 760 mmHg

59 °C

161.2±25.3 °C

0.0±0.9 mmHg at 25°C

1.536

3.4

0

2

0

17

432

4

O1C(C(=C([H])[H])[C@]2([H])C([H])([H])C([H])([H])C(=C([H])[H])[C@]3([H])C([H])([H])C([H])([H])C(=C([H])[H])[C@]3([H])[C@@]12[H])=O

NETSQGRTUNRXEO-XUXIUFHCSA-N

InChI=1S/C15H18O2/c1-8-4-7-12-10(3)15(16)17-14(12)13-9(2)5-6-11(8)13/h11-14H,1-7H2/t11-,12-,13-,14-/m0/s1

(3aS,6aR,9aR,9bS)-3,6,9-trimethylidene-3a,4,5,6a,7,8,9a,9b-octahydroazuleno[4,5-b]furan-2-one

(-)-Dehydrocostus lactone; Dehydrocostus Lactone

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)

DMSO: 46~250 mg/mL (199.7~1085.5 mM)
Ethanol: 15~46 mg/mL (65.1 mM~199.7)

Solubility in Formulation 1: ≥ 2.08 mg/mL (9.03 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 20.8 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (9.03 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 20.8 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (9.03 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 20.8 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.)