Rocaglamide Derivatives Are Potent Inhibitors of NF-κB Activation in T-cells

Bernd Baumann,Frank Bohnenstengel,Daniela Siegmund,Harald Wajant,Christoph Weber,Ingrid Herr,Klaus-Michael Debatin,Peter Proksch,Thomas Wirth

doi:10.1074/jbc.m208003200

Abstract

Crude extracts from different Aglaia species are used as anti-inflammatory remedies in the traditional medicine of several countries from Southeast Asia. Because NF-kappaB transcription factors represent key regulators of genes involved in immune and inflammatory responses, we supposed that the anti-inflammatory effects of Aglaia extracts are mediated by the inhibition of NF-kappaB activity. Purified compounds of Aglaia species, namely 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one aglain congener were tested for their ability to inhibit NF-kappaB activity. We show that a group of rocaglamides represent highly potent and specific inhibitors of tumor necrosis factor-alpha (TNFalpha) and phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB-dependent reporter gene activity in Jurkat T cells with IC(50) values in the nanomolar range. Some derivatives are less effective, and others are completely inactive. Rocaglamides are able to suppress the PMA-induced expression of NF-kappaB target genes and sensitize leukemic T cells to apoptosis induced by TNFalpha, cisplatin, and gamma-irradiation. The suppression of NF-kappaB activation correlated with the inhibition of induced IkappaB(alpha) degradation and IkappaB(alpha) kinase activation. The level of interference was determined and found to be localized upstream of the IkappaB kinase complex but downstream of the TNF receptor-associated protein 2. Our data suggest that rocaglamide derivatives could serve as lead structures in the development of anti-inflammatory and tumoricidal drugs.

Highlights

In recent years Aglaia species have attracted considerable interest due to their unique 1H-cyclopenta[b]benzofuran lignans, which have been isolated from more than ten Aglaia species so far and are exclusively confined to members of this genus [1,2,3,4]
We show that a group of rocaglamides represent highly potent and specific inhibitors of tumor necrosis factor-␣ (TNF␣) and phorbol 12-myristate 13-acetate (PMA)-induced NF-␬B-dependent reporter gene activity in Jurkat T cells with IC50 values in the nanomolar range
Rocaglamides are able to suppress the PMA-induced expression of NF-␬B target genes and sensitize leukemic T cells to apoptosis induced by TNF␣, cisplatin, and ␥-irradiation

Summary

EXPERIMENTAL PROCEDURES

Cells and Cell Culture—CEM-S (acute T cell leukemia) cells die rapidly in response to ␥-irradiation, treatment with chemotherapeutic agents, or direct triggering of death receptors. Subclones of CEM-S have been selected from the parental cells by periodical triggering of CD95 with ␣APO-1 (50 ng/ml to 10 ␮g/ml) for at least 1 year [28] These subclones (CEM-R) do not die in response to ␣APO-1 and exhibit a strongly delayed percentage of apoptosis following ␥-irradiation or treatment with chemotherapeutic agents. These cell lines were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Biochrom, Hamburg, Germany), 100 units/ml penicillin, 100 ␮g/ml streptomycin, 25 mM HEPES, and 2 mM L-glutamine (all from Invitrogen, Karlsruhe, Germany). Western immunoblot analysis for monitoring I␬B␣ degradation and RelA expression were performed as described earlier [37] using I␬B␣ (Cell Signaling, #9242)- and RelA (Santa Cruz Biotechnology, sc-372)-specific antibodies. Measurement of Apoptosis—Early apoptotic changes were identified by staining of cells with fluorescein thiocyanate-conjugated annexin V and propidium iodide (Becton Dickinson, Heidelberg, Germany) and analyzed by flow cytometry (FACScan, Becton Dickinson) as described earlier [38]

RESULTS

TABLE I

DISCUSSION