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

Crude extracts from different Aglaiaspecies are used as anti-inflammatory remedies in the traditional medicine of several countries from Southeast Asia. Because NF-κB 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-κB activity. Purified compounds ofAglaia 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-κB activity. We show that a group of rocaglamides represent highly potent and specific inhibitors of tumor necrosis factor-α (TNFα) and phorbol 12-myristate 13-acetate (PMA)-induced NF-κB-dependent reporter gene activity in Jurkat T cells with IC50 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-κB target genes and sensitize leukemic T cells to apoptosis induced by TNFα, cisplatin, and γ-irradiation. The suppression of NF-κB activation correlated with the inhibition of induced IκBα degradation and IκBα kinase activation. The level of interference was determined and found to be localized upstream of the IκB 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. Crude extracts from different Aglaiaspecies are used as anti-inflammatory remedies in the traditional medicine of several countries from Southeast Asia. Because NF-κB 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-κB activity. Purified compounds ofAglaia 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-κB activity. We show that a group of rocaglamides represent highly potent and specific inhibitors of tumor necrosis factor-α (TNFα) and phorbol 12-myristate 13-acetate (PMA)-induced NF-κB-dependent reporter gene activity in Jurkat T cells with IC50 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-κB target genes and sensitize leukemic T cells to apoptosis induced by TNFα, cisplatin, and γ-irradiation. The suppression of NF-κB activation correlated with the inhibition of induced IκBα degradation and IκBα kinase activation. The level of interference was determined and found to be localized upstream of the IκB 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. 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 (1Chaidir Hiort J. Nugroho B.W. Bohnenstengel F.I. Wray V. Witte L. Hung P.D. Kiet L.C. Sumaryono W. Proksch P. Phytochemistry. 1999; 52: 837-842Google Scholar, 2Hiort J. Chaidir Bohnenstengel F.I. Nugroho B.W. Schneider C. Wray V. Witte L. Hung P.D. Kiet L.C. Proksch P. J. Nat. Prod. 1999; 62: 1632-1635Google Scholar, 3Nugroho B.W. Edrada R.A. Wray V. Witte L. Bringmann G. Gehling M. Proksch P. Phytochemistry. 1999; 51: 367-376Google Scholar, 4Brader G. Vajrodaya S. Greger H. Bacher M. Kalchhauser H. Hofer O. J. Nat. Prod. 1998; 61: 1482-1490Google Scholar). 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The members of the Rel/NF-κB transcription factor family bind to DNA as homo- and/or heterodimers. They are critically involved in the regulation of genes mediating inflammatory responses and cellular processes such as cell survival, apoptosis, development, differentiation, cell growth, and neoplastic transformation (reviewed in Ref. 10Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar). In unstimulated cells, NF-κB dimers are sequestered in the cytoplasm due to the interaction with proteins of the IκB family. Stimulation of cells, e.g. by pro-inflammatory agents results in the rapid activation of the IκB kinase (IKK) 1The abbreviations used are: IKK, IκB kinase; EMSA, electrophoretic mobility shift; GST, glutathioneS-transferase; IκBα, inhibitor κBα; LT-β, lymphotoxin-β; NEMO, NF-κB essential modulator; NF-κB, nuclear factor-κB; PMA, phorbol 12-myristate 13-acetate; TNF, tumor necrosis factor; TRAF2, TNF receptor-associated protein 2; TRAIL, TNF-related apoptosis-inducing ligand; RocB, didesmethyl-rocaglamide B; NIK, NF-κB-inducing kinase; AP-1, activator protein. MEKK1, mitogen-activated protein kinase kinase kinase I; RPA, RNase protection assay; CEM-S, CEM T cells sensitive to therapy-induced apoptosis; CEM-R, CEM T cells resistant to therapy-induced apoptosis. complex. This complex consists of two kinases IKK1/α and IKK2/β as well as a regulatory component called NEMO/IKK-γ (10Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 11Ghosh S. Karin M. Cell. 2002; 109 (suppl.): S81-S96Google Scholar). Its activation results in thede novo phosphorylation of conserved serine residues in the N-terminal domain of the IκB proteins marking them for ubiquitination and subsequent degradation by the proteasome. This allows nuclear translocation of NF-κB and binding to cognate DNA motifs in the promoter region of target genes, which subsequently initiates transcription of these genes and finally starts a genetic program responsible for e.g. inflammatory responses (12Denk A. Wirth T. Baumann B. Cytokine Growth Factor Rev. 2000; 11: 303-320Google Scholar). Numerous efforts have been initiated to develop or to identify specific low molecular weight compounds to inhibit this pathway (13Epinat J.C. Gilmore T.D. Oncogene. 1999; 18: 6896-6909Google Scholar, 14Garg A. Aggarwal B.B. Leukemia. 2002; 16: 1053-1068Google Scholar). Substances that inhibit the proteasome as well as radical scavengers have been shown to block NF-κB activation. 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The rationale for studying a series of rocaglamide derivatives for their influence on NF-κB activity was based on the observation that leaves and flowers ofAglaia duperreana and Aglaia odorata are used in the traditional medicine of several countries from Southeast Asia (e.g. Vietnam) for the treatment of asthma and inflammatory skin diseases. Here we show that certain rocaglamide derivatives are efficient inhibitors of NF-κB activation and NF-κB target gene expression, which could explain the anti-inflammatory function of these herbal remedies. Interestingly, they show a high degree of cell type specificity being much more active in T lymphocytes than in other cell types. Furthermore, inhibition of NF-κB by rocaglamides is responsible for sensitization of resistant leukemic T cells toward cancer therapy induced apoptosis. The mechanism of NF-κB inhibition by rocaglamides is predominantly localized upstream of the IκB kinase complex. CEM-S (acute T cell leukemia) cells die rapidly in response to γ-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 αAPO-1 (50 ng/ml to 10 μg/ml) for at least 1 year (28Friesen C. Herr I. Krammer P.H. Debatin K.M. Nat. Med. 1996; 2: 574-577Google Scholar). These subclones (CEM-R) do not die in response to αAPO-1 and exhibit a strongly delayed percentage of apoptosis following γ-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 μg/ml streptomycin, 25 mm HEPES, and 2 mm l-glutamine (all from Invitrogen, Karlsruhe, Germany). Jurkat T and Jurkat T cells deficient in IKKγ/NEMO expression (kindly provided by S.-C. Sun, Pennsylvania State University), EL-4, HeLa, S107, NIH3T3, and A549 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Paisley, Scotland) containing 10% fetal calf serum (PAN Systems, Aidenbach, Germany), 50 μm β-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Karlsruhe, Germany). PC12 cells were cultured in the same medium but containing 10% horse serum (PAN Systems, Aidenbach, Germany), 5% fetal calf serum, and no β-mercaptoethanol. Cisplatin (Sigma, Deisenhofen, Germany) was dissolved in Me2SO at a concentration of 10 μg/μl, stored in aliquots at −80 °C and further diluted in medium to the working solution of 1 μm. PMA (Sigma, Deisenhofen, Germany), Ionomycin (Calbiochem, Germany) and the various rocaglamide derivatives were also dissolved in Me2SO and used at the indicated concentrations. Human recombinant TNFα (a gift of Dr. G. Adolf, Boehringer Ingelheim, Vienna, Austria) was dissolved in a buffer containing 10 mm sodium phosphate, pH 7, 200 mm sodium chloride, and 2 mg/ml bovine serum albumin and used at the indicated concentrations. Cells were γ-irradiated (10 Gy) in their flasks using a cesium radiator. For transient transfections, 3 μg of the NF-κB-dependent or the AP-1-dependent luciferase reporter and 50 ng ofRenilla luciferase reporter (under control of the ubiquitin promoter) or 1 μg of Rous sarcoma virus LacZ were cotransfected with 20 μg of empty vector (pcDNA3) or expression vectors for TRAF2, NIK, MEKK1, and IKK2-EE (29Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Google Scholar, 30Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Google Scholar, 31Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Google Scholar, 32Flory E. Weber C.K. Chen P. Hoffmeyer A. Jassoy C. Rapp U.R. J. Virol. 1998; 72: 2788-2794Google Scholar, 33Hsu H. Shu H.-B. Pan M.-G. Goeddel D.V. Cell. 1996; 84: 299-308Google Scholar). Cells (1 × 107) were transfected in 300 μl of culture medium by electroporation using a Bio-Rad gene pulser (Germany) at 975 microfarads (μFa) and 250 V (for Jurkat, A401, and EL-4) and at 250 μFa and 450 V (for HeLa). After electroporation cells were immediately resuspended in medium, seeded in six-well plates, and treated 16 h later as indicated. Cells were harvested 7 h later, and luciferase enzyme activity was measured. Renilla luciferase activity or β-galactosidase activity were used to normalize for differences in transfection efficiencies. For generation of stable transfectants of the Jurkat T and PC12 cell lines, cells were electroporated with 20 μg of the 3xκB.luc reporter plasmid together with 1 μg of a pSV.puro vector at 975 μFa and 250 V and at 250 μFa and 450 V, respectively. Cell clones with an integrated reporter gene were selected in medium containing 2 μg/ml (Jurkat) and 2.5 μg/ml (PC12) puromycin. Preparation of whole cell extracts was performed by the freeze-thaw method, and EMSAs were performed as described earlier (34Lernbecher T. Müller U. Wirth T. Nature. 1993; 365: 767-770Google Scholar). In all cases, whole cell extracts were incubated with radiolabeled double-stranded oligonucleotides containing a Ig-κ enhancer consensus NF-κB site, a octamer-specific site (35Wirth T. Staudt L. Baltimore D. Nature. 1987; 329: 174-178Google Scholar), or an Sp-1-specific site (5′-attcgatcggggcggggcgagc-3′) for 20 min at room temperature, and the DNA-protein complexes formed were then separated from free oligonucleotides on a native 4% polyacrylamide gel. For IKK activity assays, cells were lysed in Nonidet P-40 lysis buffer (36Daub M. Jockel J. Quack T. Weber C.K. Schmitz F. Rapp U.R. Wittinghofer A. Block C. Mol. Cell. Biol. 1998; 18: 6698-6710Google Scholar), and the endogenous IKK complex was immunoprecipitated from 1 mg of extract using IKK1/2-specific antibodies (Santa Cruz Biotechnology, sc-7607). The precipitated IKK complex was incubated in kinase assay mixture containing 25 mm HEPES (pH 7.5), 150 mmNaCl, 25 mm β-glycerophosphate, 10 mm MgCl, 1 mm dithiothreitol, 10 μCi of [γ-32]ATP and 600 ng of GST-IκBα substrate. Coupled in vitrokinase assays were performed in kinase assay mixture supplemented with 1 μm unlabeled ATP. After 20 min (30 °C) the reaction was terminated by boiling with SDS sample puffer, and the proteins were separated on 10% polyacrylamide gels. Finally, the gel was either dried or the proteins were electrotransferred to a polyvinylidene difluoride membrane, and radioactive bands were visualized by phosphorimaging or autoradiography. Kinase activities were quantified by PhosphorImager (Amersham Biosciences) analysis. The membranes were used for immunoblot analysis as described elsewhere (37Denk A. Goebeler M. Schmid S. Berberich I. Ritz O. Lindemann D. Ludwig S. Wirth T. J. Biol. Chem. 2001; 276: 28451-28458Google Scholar) and were labeled with antibodies specific for IKK1 (Santa Cruz Biotechnology, sc-7183) to determine total amounts of immunoprecipitated IKK1. Western immunoblot analysis for monitoring IκBα degradation and RelA expression were performed as described earlier (37Denk A. Goebeler M. Schmid S. Berberich I. Ritz O. Lindemann D. Ludwig S. Wirth T. J. Biol. Chem. 2001; 276: 28451-28458Google Scholar) using IκBα (Cell Signaling, #9242)- and RelA (Santa Cruz Biotechnology, sc-372)-specific antibodies. Jurkat cells (10 × 106) were treated with PMA (50 ng/ml) and RocB (200 nm) as indicated. Total RNAs were extracted with the RNA INSTAPURE kit (Eurogentech, Belgium). The presence of the indicated transcripts was detected using RPA analysis using the Multi-Probe template sets (Pharmingen, Hamburg, Germany) hAPO-2b (Bcl family members), hAPO-3c (death receptor-related proteins), and hCK-3 (ligands). Probe synthesis, hybridization, and RNase treatment were performed with the RiboQuant Multi-Probe RNase Protection Assay system (Pharmingen). After RNase treatment, protected transcripts were resolved by electrophoresis on 5% urea-polyacrylamide-bis-acrylamide gels and visualized on a PhosphorImager with ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA). 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 (38Herr I. Wilhelm D. Bohler T. Angel P. Debatin K.M. EMBO J. 1997; 16: 6200-6208Google Scholar). Sixteen naturally occurring 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one naturally occurring aglain congener were tested for their ability to inhibit NF-κB function. The different substances were isolated from various Aglaiaspecies as described earlier (3Nugroho B.W. Edrada R.A. Wray V. Witte L. Bringmann G. Gehling M. Proksch P. Phytochemistry. 1999; 51: 367-376Google Scholar). All compounds were prepared to at least 98% purity as assayed by high-performance liquid chromatography. These compounds (named 1–16) differed mainly in their amide side chains and/or in the substitution patterns of aromatic rings A and B (Fig. 1). Jurkat T cells bearing an integrated NF-κB-dependent luciferase reporter (3xκB.luc) served as a test system for NF-κB activity. Jurkat T cells were pre-treated with the different rocaglamide compounds at a concentration of 100 nm. One hour later the cells were stimulated with TNF, and at 6 h post-stimulation luciferase activities were determined. TNF stimulation resulted in a potent activation of NF-κB-dependent transcription (>30-fold). Interestingly we found that pretreatment of the Jurkat T cells with several of the different rocaglamide compounds led to inhibition of TNF-induced NF-κB-dependent luciferase activity (Fig. 2 Aand data not shown). The most efficient inhibitors wereN,N-didesmethyl-N-4-hydroxybutyl-rocaglamide (compound 8), didesmethyl-rocaglamide (synonym with RocB, compound 1), and compounds 3 and 4. These substances show an inhibition activity ranging from 70% up to complete inhibition. Compounds 2, 5–7, 9–12, 14, and 15 are less powerful inhibitors that repress TNF-induced NF-κB- dependent luciferase activity to levels less than 50% (Fig. 2 A and data not shown). The aglain derivative (compound 17), 8β-methoxyrocaglaol (compound 13), and compound 16 were completely inactive even at concentrations up to 2 mm(Fig. 2 A and data not shown). We next investigated the dose dependence of the inhibition and determined the IC50 values for several rocaglamides following TNF treatment. All investigated active compounds showed a dose-dependent inhibition of TNF-induced NF-κB-driven gene expression. An example of the dose-dependent inhibition of the transcription factor NF-κB by RocB is shown in Fig.2 B. A complete inhibition of the transcription factor NF-κB-dependent gene expression in Jurkat T cells was observed, when the T-cells were treated with RocB at a concentration of 200 nm. The IC50 value was determined to be 58 nm, and several other compounds showed IC50values in the range of 200 nm (TableI).Table IIC50 values of rocaglamides for the inhibition of TNF-α and PMA-induced NF-κB activity in Jurkat T cellsRocaglamidesTNF-α nMPMA[1]58.443.6[9]161.3197.4[11]208.0499.0[14]205.0319.4[15]159.9243.4 Open table in a new tab A wide variety of stimuli has been shown to induce NF-κB activity. We therefore asked whether rocaglamides would specifically inhibit TNFα-induced NF-κB or whether they might also interfere with PMA-induced NF-κB activation. RocB (compound 1) was able to inhibit PMA stimulation in the same concentration range as shown for TNFα stimulation. Other rocaglamide congeners were somewhat less efficient in their inhibitory action for PMA-induced NF-κB activity. IC50 values of five rocaglamide derivatives were determined for their inhibition of PMA stimulation (Table I). The aglain derivative and 8β-methoxyrocaglaol were again completely inactive up to the final concentration tested. Our results demonstrate that, depending on their chemical structure, rocaglamide derivatives represent potent inhibitors of the NF-κB pathway. We next asked whether rocaglamide derivatives specifically interfere with the NF-κB pathway or whether they affect the activity of other transcription factors, too. For this purpose we analyzed the influence of rocaglamides on AP-1 activity. AP-1 is a dimeric transcription factor, which regulates gene expression in response to a great variety of stimuli, including TNF and PMA (39Angel P. Karin M. Biochim. Biophys. Acta. 1991; 1072: 129-157Google Scholar). Jurkat T cells transiently transfected with an AP-1-dependent reporter gene (5xTRE-tk-luc) showed a strong basal activity already (in comparison to tk-luc). This activity was only marginally induced by TNF or PMA. We investigated the influence of rocaglamides on this basal AP-1 activity in Jurkat T cells. We found that AP-1 activity was not altered in the presence of active rocaglamide derivatives 1–5 and the previously inactive aglain derivative (compound 17). Interestingly, compound 8 strongly reduced reporter gene activity (Fig. 2 C). Similar results were obtained when we investigated the PMA/ionomycin-induced activity of the Oct-coactivator BOB.1/OBF.1 (40Zwilling S. Dieckmann A. Pfisterer P. Angel P. Wirth T. Science. 1997; 277: 221-225Google Scholar, 41Pfisterer P. Annweiler A. Ullmer C. Corcoran L. Wirth T. EMBO J. 1994; 13: 1654-1663Google Scholar). PMA/ionomycin-induced Oct-dependent activity was not inhibited by rocaglamides (data not shown), but again compound 8 showed a dramatic reduction of Oct-dependent activity. From this and several other experiments we concluded that the effects of compound 8 were unspecific. This rocaglamide derivative also showed strong cytotoxic effects (data not shown) and was therefore excluded from further experiments. In conclusion, rocaglamide derivatives are specific inhibitors of the NF-κB system and do not block the activity of the AP-1 and Oct transcription factors. We next tested the inhibition properties of rocaglamides in other cell lines to address the question whether the inhibition of the NF-κB-dependent gene expression might be due to a cell-type-specific mechanism. First we analyzed the A301 (human) and EL-4 (mouse) T-cell lines, which were transiently transfected with an NF-κB-dependent reporter construct. Similar to the results obtained with Jurkat T cells, the TNF (Fig.3 A) or PMA (Fig.3 B) stimulated activation of the NF-κB-driven reporter gene could be inhibited by RocB in EL-4 cells and in A301 cells (data not shown). The concentrations of RocB were in the same range as those needed for the inhibition of the NF-κB activation in Jurkat T cells. Again, the inactive compounds (the aglain derivative and 8β-methoxyrocaglaol) showed no inhibitory activity. Thus, we conclude that rocaglamides inhibit NF-κB induction in mouse and human T cell lines. Surprisingly, a remarkable resistance of several other cell lines toward inhibition of NF-κB by rocaglamide congeners was observed. In PC-12 cells bearing an integrated NF-κB-dependent reporter gene and transiently transfected human HeLa cells, rocaglamide derivatives were not able to block NF-κB activation. Even RocB concentrations up to 800 nm failed to inhibit NF-κB activation by PMA (Fig. 4 A) or TNF in PC-12 cells (data not shown). In HeLa cells several rocaglamides used at a concentration of 200 nm showed no inhibitory effect on TNF and PMA-induced NF-κB activity (Fig. 4, Band C). In contrast, when PC-12 and HeLa cells were treated with highly active rocaglamide derivatives, an increase of the NF-κB-dependent gene expression was found. The aglain derivative and 8β-methoxyrocaglaol showed no signs of inhibition or induction on the NF-κB system in these cells. Similar results were obtained in NIH3T3 fibroblasts and A549 human lung epithelial cells bearing an integrated NF-κB-dependent reporter gene (data not shown). We therefore conclude that rocaglamides do not block NF-κB in these non-T cells. All the cell lines that exhibited no rocaglamide-dependent inhibition of NF-κB were of non-lymphoid origin. We therefore asked whether B lymphocytes might respond to rocaglamides. For this purpose we used the variant plasmacytoma cell line S107, which lacks constitutive NF-κB activity typical for mature B cells but is still highly responsive to TNF (42Baumann B. Kistler B. Kirillov A. Bergman Y. Wirth T. J. Biol. Chem. 1998; 273: 11448-11455Google Scholar). NF-κB-dependent gene expression was investigated using S107 cells with an integrated reporter gene. Although the required rocaglamide concentrations for inhibiting TNF-induced NF-κB activity in S107 cells were ∼5 to 7 times higher than in Jurkat T cells, inhibition could be observed (Fig. 4 D). Again, 8β-methoxyrocaglaol showed no inhibitory activity. From these results we conclude that NF-κB activation in lymphocytes, but not in several other cell types, is controlled by a rocaglamide-sensitive pathway. All of the NF-κB inhibitory effects of rocaglamides described here were observed in reporter gene assays. Therefore, we next investigated whether rocaglamides also influence the activation of endogenous NF-κB target genes. Jurkat T cells were pretreated with RocB, a highly active rocaglamide derivative and subsequently exposed to PMA. Total RNAs were then prepared and analyzed by RNase protection assay (RPA) analysis for the expression of known NF-κB target genes. To prove the NF-κB dependence of the respective genes, Jurkat cells deficient in IKKγ (43Harhaj E.W. Good L. Xiao G. 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