Fluorescence polarization competitive binding assay

Fluorescence polarization experiments were performed in Dynex 96-well, black, round-bottom plates. A 5 μL sample of Embelin dilutions in DMSO, and preincubated XIAP BIR3 protein (0.06 μM) and the N terminus of a Smac peptide (SM7F) (0.01 μM) in the assay buffer were added to 96-well plates to produce a final volume of 125 μL. For each assay, the bound peptide control containing XIAP BIR3 protein and SM7F (equivalent to 0% inhibition) and free peptide control containing only free SM7F (equivalent to 100% inhibition) were included. The plates were mixed and incubated at room temperature for 3 hrs to reach equilibrium.

Cell lines

PC-3, LNCap, PrEC and WI-38 cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 ℃ for several months.

Reaction Conditions

1 ~ 64 μM; 4 ~ 5 days

Applications

Embelin dose-dependently inhibited cell growth of both PC-3 and LNCap cells, with the IC50 values of 3.7 and 5.7 μM, respectively. In normal PrEC and WI-38 cells, the toxicity of Embelin was much lower, with the IC50 values of 20.1 μM and 19.3 μM, respectively.

Animal models

AOM/DSS-induced colitis-associated cancer (CAC) model

Dosage form

50 mg/kg/day; p.o.

Applications

In a CAC model, Embelin reduced both incidence and tumor size in mice by inhibiting proliferation of tumor epithelial cells and suppressing IL6 expression and IL6-activated STAT3.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

Embelin, a natural benzoquinone from plants of the genus Embelia, is an inhibitor of X-linked inhibitor of apoptosis protein (XIAP) with IC50 of 4.1 μM.
XIAP is a member of the inhibitor of apoptosis family of proteins. This protein modulates signaling pathways involved in cell apoptosis and stops cell apoptosis induced by viral infection or overproduction of caspases.
Embelin was shown to exhibit anti-tumor and anti-inflammatory activity in cells. For instance, embelin inhibits cell growth and activates caspases to promote apoptosis in cancer cells with high expression of XIAP [1]. Additionally, embelin was reported to prevent NF-κB activation by inhibiting IKK and thus resulted in suppression of NF-κB-regulated anti-apoptotic and metastatic gene expression [2].
Embelin has also been used extensively in various animal models to study the role of XIAP. In the azoxymethane/dextran sulfate sodium (AOM/DSS) induced colitis-associated cancer (CAC) model, embelin reduced both incidence and tumor size in mice by inhibiting proliferation of tumor epithelial cells and suppressing IL6 expression and IL6-activated STAT3 in vivo [3].
References
1.Nikolovska-Coleska Z, Xu L, Hu Z, Tomita Y, Li P, Roller PP, et al. Discovery of embelin as a cell-permeable, small-molecular weight inhibitor of XIAP through structure-based computational screening of a traditional herbal medicine three-dimensional structure database. J Med Chem 2004,47:2430-2440.
2.Ahn KS, Sethi G, Aggarwal BB. Embelin, an inhibitor of X chromosome-linked inhibitor-of-apoptosis protein, blocks nuclear factor-kappaB (NF-kappaB) signaling pathway leading to suppression of NF-kappaB-regulated antiapoptotic and metastatic gene products. Mol Pharmacol 2007,71:209-219.
3.Dai Y, Jiao H, Teng G, Wang W, Zhang R, Wang Y, et al. Embelin reduces colitis-associated tumorigenesis through limiting IL-6/STAT3 signaling. Mol Cancer Ther 2014,13:1206-1216.