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• W2005672276 abstract "Tumor necrosis factor (TNF) is a potent activator of the nuclear factor-κB (NF-κB) pathway that leads to up-regulation of anti-apoptotic proteins. Hence, TNF induces apoptosis in the presence of inhibitors of protein or RNA synthesis. We report that a novel triterpenoid, 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) inhibits NF-κB-mediated gene expression at a step after translocation of activated NF-κB to the nucleus. This effect appears specific for the NF-κB pathway as CDDO does not inhibit gene expression induced by the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). CDDO in combination with TNF caused a dramatic increase in apoptosis in ML-1 leukemia cells that was associated with activation of caspase-8, cleavage of Bid, translocation of Bax, cytochrome crelease, and caspase-3 activation. Experiments with caspase inhibitors demonstrated that caspase-8 was an initiator of this pathway. TNF also induced a transient activation of c-Jun N-terminal kinase (JNK), which upon addition of CDDO was converted to a sustained activation. The activation of JNK was also dependent on caspase-8. Sustained activation of JNK is frequently pro-apoptotic, yet inhibition of JNK did not prevent Bax translocation or cytochromec release, demonstrating its lack of involvement in CDDO/TNF-induced apoptosis. Apoptosis was acutely induced by CDDO/TNF in every leukemia cell line tested including those that overexpress Bcl-xL, suggesting that the mitochondrial pathway is not required for apoptosis by this combination. These results suggest that the apoptotic potency of the CDDO/TNF combination occurs through selective inhibition of NF-κB-dependent anti-apoptotic proteins, bypassing potential mitochondrial resistance mechanisms, and thus may provide a basis for the development of novel approaches to the treatment of leukemia. Tumor necrosis factor (TNF) is a potent activator of the nuclear factor-κB (NF-κB) pathway that leads to up-regulation of anti-apoptotic proteins. Hence, TNF induces apoptosis in the presence of inhibitors of protein or RNA synthesis. We report that a novel triterpenoid, 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) inhibits NF-κB-mediated gene expression at a step after translocation of activated NF-κB to the nucleus. This effect appears specific for the NF-κB pathway as CDDO does not inhibit gene expression induced by the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). CDDO in combination with TNF caused a dramatic increase in apoptosis in ML-1 leukemia cells that was associated with activation of caspase-8, cleavage of Bid, translocation of Bax, cytochrome crelease, and caspase-3 activation. Experiments with caspase inhibitors demonstrated that caspase-8 was an initiator of this pathway. TNF also induced a transient activation of c-Jun N-terminal kinase (JNK), which upon addition of CDDO was converted to a sustained activation. The activation of JNK was also dependent on caspase-8. Sustained activation of JNK is frequently pro-apoptotic, yet inhibition of JNK did not prevent Bax translocation or cytochromec release, demonstrating its lack of involvement in CDDO/TNF-induced apoptosis. Apoptosis was acutely induced by CDDO/TNF in every leukemia cell line tested including those that overexpress Bcl-xL, suggesting that the mitochondrial pathway is not required for apoptosis by this combination. These results suggest that the apoptotic potency of the CDDO/TNF combination occurs through selective inhibition of NF-κB-dependent anti-apoptotic proteins, bypassing potential mitochondrial resistance mechanisms, and thus may provide a basis for the development of novel approaches to the treatment of leukemia. Tumor necrosis factor (TNF) 1The abbreviations used are: TNFtumor necrosis factoract Dactinomycin DCDDO2-cyano-3,12-dioxooleana-1,9-dien-28-oic acidCHXcycloheximideERKextracellular signal-regulated kinaseIκBαinhibitor of NF-κBJNKc-Jun N-terminal kinaseNF-κBnuclear factor-κBMAPKmitogen-activated protein kinaseMEKMAPK/ERK kinasezbenzyloxycarbonylfmkfluoromethylketoneTPA12-0-tetradecanoylphorbol-13-acetateCOX-2cyclooxygenase-2iNOSinducible nitric-oxide synthaseRelArel family member p65MKK4MAP kinase kinase 4MEKK1MAP/ERK kinase kinase 11The abbreviations used are: TNFtumor necrosis factoract Dactinomycin DCDDO2-cyano-3,12-dioxooleana-1,9-dien-28-oic acidCHXcycloheximideERKextracellular signal-regulated kinaseIκBαinhibitor of NF-κBJNKc-Jun N-terminal kinaseNF-κBnuclear factor-κBMAPKmitogen-activated protein kinaseMEKMAPK/ERK kinasezbenzyloxycarbonylfmkfluoromethylketoneTPA12-0-tetradecanoylphorbol-13-acetateCOX-2cyclooxygenase-2iNOSinducible nitric-oxide synthaseRelArel family member p65MKK4MAP kinase kinase 4MEKK1MAP/ERK kinase kinase 1 induces a broad range of cellular effects including inflammatory responses, NF-κB activation, and apoptosis (for review, see Ref. 1.Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Abstract Full Text Full Text PDF PubMed Scopus (1374) Google Scholar). In many systems, the apoptotic potential of TNF is only realized when cells are co-treated with the protein synthesis inhibitor cycloheximide (CHX). This effect of CHX may be caused by inhibition of the translation of NF-κB-dependent anti-apoptotic proteins such as TRAF1/2 and c-IAP1/2 (2.Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2573) Google Scholar). Apoptosis induced by TNF is initiated at the membrane where engagement of the tumor necrosis factor receptor results in the recruitment of TRADD and then FADD. A conserved sequence in FADD called the death effector domain serves as a docking site for procaspase-8, which upon activation initiates apoptosis by either of two distinct routes. The first pathway involves caspase-8-directed cleavage of Bid to its active form, tBid, resulting in translocation to the mitochondria and release of cytochrome c into the cytoplasm (3.Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3076) Google Scholar, 4.Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3344) Google Scholar). The released cytoplasmic cytochrome cinteracts with caspase-9 and Apaf-1 in the presence of dATP to create the “apoptosome” that serves to cleave and activate caspase-9 (5.Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6219) Google Scholar). Caspase-9 then activates caspase-3, which carries out the execution phase of apoptosis. The second pathway of apoptosis involves the direct proteolysis and activation of caspase-3 by caspase-8. tumor necrosis factor actinomycin D 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid cycloheximide extracellular signal-regulated kinase inhibitor of NF-κB c-Jun N-terminal kinase nuclear factor-κB mitogen-activated protein kinase MAPK/ERK kinase benzyloxycarbonyl fluoromethylketone 12-0-tetradecanoylphorbol-13-acetate cyclooxygenase-2 inducible nitric-oxide synthase rel family member p65 MAP kinase kinase 4 MAP/ERK kinase kinase 1 tumor necrosis factor actinomycin D 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid cycloheximide extracellular signal-regulated kinase inhibitor of NF-κB c-Jun N-terminal kinase nuclear factor-κB mitogen-activated protein kinase MAPK/ERK kinase benzyloxycarbonyl fluoromethylketone 12-0-tetradecanoylphorbol-13-acetate cyclooxygenase-2 inducible nitric-oxide synthase rel family member p65 MAP kinase kinase 4 MAP/ERK kinase kinase 1 CDDO is a novel oleanane triterpenoid with promising clinical potential as a chemopreventive agent and as a therapeutic agent for the treatment of cancer. In vitro studies have shown that nanomolar levels of CDDO induce differentiation or inhibit the proliferative capacity of human leukemia and breast cancer cell lines in culture (6.Suh N. Wang Y. Honda T. Gribble G.W. Dmitrovsky E. Hickey W.F. Maue R.A. Place A.E. Porter D.M. Spinella M.J. Williams C.R. Wu G. Dannenberg A.J. Flanders K.C. Letterio J.J. Mangelsdorf D.J. Nathan C.F. Nguyen L. Porter W.W. Ren R.F. Roberts A.B. Roche N.S. Subbaramaiah K. Sporn M.B. Cancer Res. 1999; 59: 336-341PubMed Google Scholar). At these concentrations, CDDO also binds as a partial agonist to peroxisome proliferator-activated receptor-γ (7.Wang Y. Porter W.W. Suh N. Honda T. Gribble G.W. Leesnitzer L.M. Plunket K.D. Mangelsdorf D.J. Blanchard S.G. Willson T.M. Sporn M.B. Mol. Endocrinol. 2000; 14: 1550-1556Crossref PubMed Google Scholar). Concentrations of CDDO near 1 μm completely inhibit cytokine-induced COX-2 and iNOS mRNA (6.Suh N. Wang Y. Honda T. Gribble G.W. Dmitrovsky E. Hickey W.F. Maue R.A. Place A.E. Porter D.M. Spinella M.J. Williams C.R. Wu G. Dannenberg A.J. Flanders K.C. Letterio J.J. Mangelsdorf D.J. Nathan C.F. Nguyen L. Porter W.W. Ren R.F. Roberts A.B. Roche N.S. Subbaramaiah K. Sporn M.B. Cancer Res. 1999; 59: 336-341PubMed Google Scholar), whereas 5-fold higher concentrations cause apoptosis through a caspase-8-dependent mechanism in human leukemia (8.Ito Y. Pandey P. Place A. Sporn M.B. Gribble G.W. Honda T. Kharbanda S. Kufe D. Cell Growth & Differ. 2000; 11: 261-267PubMed Google Scholar) and osteosarcoma (9.Ito Y. Pandey P. Sporn M.B. Datta R. Kharbanda S. Kufe D. Mol. Pharmacol. 2001; 59: 1094-1099Crossref PubMed Scopus (130) Google Scholar) cell lines. The c-Jun N-terminal kinase (JNK) cascade is activated in response to a wide array of cellular stresses including environmental damage, chemotherapeutic agents, and cytokines such as TNF (10.Saleem A. Datta R. Yuan Z. Kharbanda S. Kufe D. Cell Growth & Differ. 1995; 6: 1651-1658PubMed Google Scholar, 11.Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1379) Google Scholar, 12.Stadheim T.A. Saluta G.R. Kucera G.L. Biochem. Pharmacol. 2000; 59: 407-418Crossref PubMed Scopus (26) Google Scholar). The kinetics of JNK activation are also stimulus dependent with cytokines inducing a rapid transient increase in JNK activity and chemical stresses causing a delayed but sustained activation of JNK. Sustained JNK activation has been implicated as an upstream signal for the initiation of apoptosis (13.Chen Y.R. Wang X.P. Templeton D. Davis R.J. Tan T.H. J. Biol. Chem. 1996; 271: 31929-31936Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 14.Stadheim T.A. Xiao H. Eastman A. Cancer Res. 2001; 61: 1533-1540PubMed Google Scholar). Genetic studies have confirmed the importance of JNK in apoptosis whereby JNK-deficient mouse embryonic fibroblasts resist chemical and UV radiation-induced cytochromec release and apoptosis (15.Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1540) Google Scholar). However, these cells retain apoptotic sensitivity to the Fas ligand. The observation that cells deficient in JNK are sensitive to death receptor-induced apoptosis lends support to other studies that have concluded that JNK is dispensable for TNF-induced apoptosis (16.Liu Z.G. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1780) Google Scholar). Conversely, recent studies have demonstrated that JNK signaling is required for TNF-induced death (17.De Smaele E. Zazzeroni F. Papa S. Nguyen D.U. Jin R. Jones J. Cong R. Franzoso G. Nature. 2001; 414: 308-313Crossref PubMed Scopus (656) Google Scholar, 18.Tang G. Minemoto Y. Dibling B. Purcell N.H. Li Z. Karin M. Lin A. Nature. 2001; 414: 313-317Crossref PubMed Scopus (661) Google Scholar). It was reported that TNF-induced JNK activation is inhibited by NF-κB-inducible genes and that suppression of these genes by inhibition of the NF-κB signaling pathway causes a sustained increase in JNK activation. Moreover, inhibition of JNK was found to suppress TNF-induced apoptosis. Here, we investigated the effect of CDDO on NF-κB signaling and apoptosis in response to TNF treatment. We found that whereas CDDO did not inhibit the initial TNF-induced phosphorylation and degradation of IκBα, NF-κB-dependent resynthesis of IκBα was blocked. The inhibition of IκBα resynthesis was followed by rapid apoptosis that was much greater than additive when compared with cells treated with TNF or CDDO alone. Moreover, CDDO converted TNF-induced JNK activation from a transient signal to a sustained induction that was sensitive to caspase-8 inhibition. However, inhibition of JNK activity did not prevent CDDO/TNF-induced apoptosis. The combination of CDDO plus TNF was effective at inducing apoptosis in a variety of human leukemia cell lines including those overexpressing Bcl-xL. Hence, this combination may represent an effective therapeutic strategy in the treatment of human leukemia. Stock solutions of CDDO (10 mm) were prepared in dimethyl sulfoxide (Me2SO) and stored at −20 °C. TNF, CHX, and actinomycin D (act D) were purchased from Sigma and prepared in Me2SO at stock concentrations of 10 μg/ml, 15 mg/ml, and 1 mg/ml, respectively. The general caspase inhibitor zVAD-fmk and the caspase-8 selective inhibitor zIETD-fmk (Enzyme Systems, Livermore, CA) were dissolved in Me2SO at stock concentrations of 20 mm and 10 mm, respectively, and then stored at −20 °C. SP600125 (Biomol, Plymouth Meeting, PA) was dissolved in Me2SO at a stock concentration of 5 mm and stored at −20 °C. Antibodies were obtained from the following sources: IκBα (9242) polyclonal, phospho-IκBα (9246) monoclonal, and phospho−JNK (9251) polyclonal,Cell Signaling (Beverly, MA); JNK1 (SC-474) (also detects JNK2) polyclonal and p65RelA (SC-109), Santa Cruz Biotechnology (Santa Cruz, CA); cytochrome c (clone 7H8.2C12) monoclonal, BD PharMingen (San Diego, CA); Bax (clone 2D2) monoclonal, Zymed Laboratories Inc. (San Francisco, CA); caspase-8 (AAP-118) polyclonal, StressGen Biotechnologies Corporation (Victoria, BC, Canada); p21WAF1(clone EA10) monoclonal, Oncogene Research Products (Boston, MA); the D4-GDI polyclonal antibody was developed in this laboratory (19.Krieser R.J. Eastman A. Cell Death Differ. 1999; 6: 412-419Crossref PubMed Scopus (63) Google Scholar); the Mcl-1 monoclonal antibody and the Bid monoclonal antibody were generously provided by Dr. R. Craig (Dartmouth Medical School, Hanover, NH) and Dr. X. Wang (HHMI, Dallas, TX), respectively. Unless otherwise specified, all other reagents were purchased from Sigma. ML-1 (kindly provided by Dr. R. Craig Dartmouth Medical School, Hanover, NH), HL-60 (American Type Culture Collection, Manassas, VA), HL-60/Bcl-xL (kindly provided by Dr. K. Bhalla, USF, Tampa, FL), U937, U937/Bcl-xL (kindly provided by Dr. S. Grant, MCV, Richmond, VA), THP-1 (kindly provided by Dr. R. Perez, DHMC, Lebanon, NH) and Jurkat cells were passaged in RPMI 1640 plus 10% fetal calf serum and incubated at 37 °C in 5% CO2/95% humidified air. Cells were treated according to the schedules described in the results. In studies utilizing CDDO, cells were treated for 1 h with CDDO prior to addition of TNF. Cells were incubated with 2 μg/ml Hoechst 33342 for 20 min at 37 °C. An aliquot of cells was transferred to a microscope slide, fitted with a coverslip, and DNA staining was visualized with an inverted Nikon Diaphot microscope. Cells exhibiting condensed chromatin and fragmented nuclei were scored as apoptotic. At least 200 cells were scored in each group, and data were expressed as the percentage of cells with condensed chromatin. The preparation of nuclear and cytosolic extracts is a modification of a previously reported procedure (20.Barchowsky A. Munro S.R. Morana S.J. Vincenti M.P. Treadwell M. Am. J. Physiol. 1995; 269: L829-L836PubMed Google Scholar). Briefly, cells (5 × 106) were washed in ice-cold phosphate-buffered saline pH 7.2, resuspended in 250 μl buffer A (10 mm HEPES, pH 7.9, 0.1 mm EDTA, 0.1 mm EGTA, 10 mm KCl, 1.0 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride) plus 1% Nonidet P-40 and incubated on ice for 15 min. Samples were centrifuged (15,800 × g for 2 min), and the supernatant (cytosolic fraction) was reserved. The pellet (nuclear fraction) was washed in Buffer A plus 1% Nonidet P-40 and resuspended in boiling Laemmli sample buffer. Samples were obtained using the digitonin permeabilization method (21.Single B. Leist M. Nicotera P. Cell Death Differ. 1998; 5: 1001-1003Crossref PubMed Scopus (97) Google Scholar). Briefly, cells were permeabilized on ice with 8.75 μg of digitonin/106 cells in 33 μl of buffer containing 75 mm NaCl, 1 mmNaH2PO4, 8 mmNa2HPO4, and 250 mm sucrose. Cells were incubated for 30 s in ice-cold buffer followed by centrifugation for 1 min at 14,600 × g. The supernatant was then harvested as the cytosolic fraction, and the pellet was resuspended in the same volume of buffer not containing digitonin. Whole cell lysates were prepared by boiling samples in Laemmli sample buffer. Cells were pelleted (200 ×g for 5 min), washed in ice-cold phosphate-buffered saline (pH 7.2), resuspended in boiling Laemmli sample buffer, and boiled for 5 min. Samples were then sonicated and stored at −20 °C until assayed. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%), with the exception of blots probed for cytochrome c and Bax where 15% SDS-PAGE gels were used, and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). Membranes were subsequently blocked in 5% nonfat milk/Tris-buffered saline (pH 7.4) and 0.05% Tween-20. They were then incubated with appropriate antibody overnight at 4 °C. Membranes were washed in Tris-buffered saline (pH 7.4) and 0.05% Tween-20. They were then incubated for 45 min with either goat anti-rabbit or goat anti-mouse antibody conjugated to horseradish peroxidase (Bio-Rad). Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences). We initially characterized the response of ML-1 human myelocytic leukemia cells to TNF. Since TNF is known to activate NF-κB signaling we performed a time course with TNF and measured IκBα phosphorylation and degradation, a requisite sequence of events in the activation of NF-κB (22.Karin M. J. Biol. Chem. 1999; 274: 27339-27342Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). In agreement with a previous report (23.Mudipalli A. Li Z. Hromchak R. Bloch A. Leukemia. 2001; 15: 808-813Crossref PubMed Scopus (16) Google Scholar), ML-1 cells exhibited rapid IκBα phosphorylation and degradation in response to TNF (Fig. 1 A). This response was followed by IκBα resynthesis and phosphorylation, both of which depend on activation of the NF-κB signaling pathway (Fig. 1 A). In addition to NF-κB activation, TNF transiently stimulates JNK signaling (24.Guo Y.L. Baysal K. Kang B. Yang L.J. Williamson J.R. J. Biol. Chem. 1998; 273: 4027-4034Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). We observed TNF to induce a rapid but transient increase in JNK activity with peak activation occurring at 10 min and then declining by 30 min as assessed with a phospho-specific antibody that recognizes the activated form of JNK (Fig. 1 A). The kinetics of JNK dephosphorylation were strongly associated with the kinetics of IκBα resynthesis. This finding is in agreement with recent reports suggesting the involvement of NF-κB-dependent gene transcription in the suppression of JNK activation (17.De Smaele E. Zazzeroni F. Papa S. Nguyen D.U. Jin R. Jones J. Cong R. Franzoso G. Nature. 2001; 414: 308-313Crossref PubMed Scopus (656) Google Scholar, 18.Tang G. Minemoto Y. Dibling B. Purcell N.H. Li Z. Karin M. Lin A. Nature. 2001; 414: 313-317Crossref PubMed Scopus (661) Google Scholar). To assess the capacity of ML-1 cells to undergo apoptosis in response to TNF, cells were exposed to TNF in combination with either CHX or act D for 3 h. As an index of apoptotic activity, lysates were immunoblotted for D4-GDI cleavage, a marker of caspase-3 activation (19.Krieser R.J. Eastman A. Cell Death Differ. 1999; 6: 412-419Crossref PubMed Scopus (63) Google Scholar). A 3-h treatment with either TNF, CHX, or act D alone exhibited no evidence of apoptosis as measured by D4-GDI cleavage (Fig. 1 B). However, apoptosis was markedly increased by TNF in combination with either CHX or act D (Fig. 1 B). We and others have shown that stress-induced apoptosis is associated with a sustained activation of the JNK pathway (13.Chen Y.R. Wang X.P. Templeton D. Davis R.J. Tan T.H. J. Biol. Chem. 1996; 271: 31929-31936Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 14.Stadheim T.A. Xiao H. Eastman A. Cancer Res. 2001; 61: 1533-1540PubMed Google Scholar, 25.Stadheim T.A. Kucera G.L. Leuk. Res. 2002; 26: 55-65Crossref PubMed Scopus (97) Google Scholar). Therefore, we tested whether apoptosis induced by TNF was also associated with a sustained increase in JNK activation. Whereas treatment with either TNF, CHX, or act D alone produced little increase in JNK phosphorylation by 3 h, TNF in combination with CHX or act D increased JNK phosphorylation with the intensity of JNK phosphorylation correlating with D4-GDI cleavage (Fig. 1 B). Moreover, we observed cleavage of the 54-kDa phospho-JNK isoform that correlated with D4-GDI cleavage. Cleavage of JNK has previously been reported to occur in a caspase-dependent manner after treatment with microtubule-interfering agents (14.Stadheim T.A. Xiao H. Eastman A. Cancer Res. 2001; 61: 1533-1540PubMed Google Scholar). The correlation of sustained JNK activation and cell death induced by CHX/TNF or act D/TNF is consistent with the findings of others (24.Guo Y.L. Baysal K. Kang B. Yang L.J. Williamson J.R. J. Biol. Chem. 1998; 273: 4027-4034Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). CHX or act D inhibited TNF-induced IκBα resynthesis (Fig. 1 B). The inhibition of IκBα resynthesis correlated with sustained phospho-JNK levels and D4-GDI cleavage suggesting that the synthesis of NF-κB-dependent proteins may be important to maintaining the anti-apoptotic phenotype in TNF-treated cells. Taken together, these results show that apoptosis induced by TNF in combination with CHX or act D is characterized by a sustained activation of JNK and an inhibition of IκBα resynthesis. TNF induces COX-2 expression in a NF-κB-dependent manner (26.Yamamoto K. Arakawa T. Ueda N. Yamamoto S. J. Biol. Chem. 1995; 270: 31315-31320Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar), whereas CDDO has been reported to inhibit cytokine-induced COX-2 mRNA and protein (6.Suh N. Wang Y. Honda T. Gribble G.W. Dmitrovsky E. Hickey W.F. Maue R.A. Place A.E. Porter D.M. Spinella M.J. Williams C.R. Wu G. Dannenberg A.J. Flanders K.C. Letterio J.J. Mangelsdorf D.J. Nathan C.F. Nguyen L. Porter W.W. Ren R.F. Roberts A.B. Roche N.S. Subbaramaiah K. Sporn M.B. Cancer Res. 1999; 59: 336-341PubMed Google Scholar). This suggests that CDDO may negatively regulate NF-κB signaling. Since NF-κB has been implicated in the protection of TNF-treated cells from apoptosis (27.Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2447) Google Scholar), we tested whether CDDO would inhibit NF-κB signaling and increase apoptosis in TNF-treated ML-1 cells. Cells were treated with CDDO alone or in combination with TNF for 3 h and then assayed for JNK phosphorylation and apoptosis as determined by D4-GDI cleavage. CDDO concentrations of as much as 1 μm did not activate JNK and caused no cytotoxicity as assessed by D4-GDI cleavage (Fig. 2). However, JNK activation and apoptosis were detected at a CDDO concentration of 10 μm, the highest dose tested. In contrast, CDDO and TNF co-treatment resulted in a marked increase in JNK phosphorylation and apoptosis at lower concentrations of CDDO (1 μm) (Fig. 2). ERK activation was also measured and found to be unaffected by 1 μm CDDO treatment (data not shown). These data indicate that whereas short term exposure to CDDO or TNF separately are not acutely toxic, their use in combination results in a dramatic increase in the incidence of apoptosis. To investigate where CDDO inhibits the NF-κB signal transduction cascade, we measured TNF-induced IκBα phosphorylation and degradation over a 10-min timeframe in the presence or absence of CDDO. CDDO had no effect on TNF-induced phosphorylation or degradation of IκBα, suggesting that CDDO may be inhibiting NF-κB signaling at a level downstream of IκBα degradation (Fig. 3 A). The impact of CDDO on TNF-induced JNK activation was also measured. CDDO did not affect the kinetics of JNK phosphorylation by TNF over this short time period (Fig. 3 A). As mentioned previously, TNF causes the phosphorylation and degradation of IκBα followed by NF-κB-dependent resynthesis of IκBα. Thus, we tested whether CDDO affects IκBα resynthesis. IκBα was degraded and then resynthesized in response to TNF (Fig. 3 B). However, when cells were exposed to TNF in combination with CDDO no resynthesis of IκBα was observed (Fig. 3 B). p65RelA is a member of the NF-κB family and heterodimerizes with other family members and binds to IκBα in an inactive complex in unstimulated cells. Upon incubation with TNF, IκBα is phosphorylated and degraded by a ubiquitination-dependent process allowing NF-κB translocation into the nucleus where it can modulate gene expression. Because of our finding that resynthesis of the NF-κB-dependent protein IκBα was inhibited by CDDO, we next tested whether CDDO might be interfering with the translocation of NF-κB from the cytosol to the nucleus. ML-1 cells were pretreated with CHX or CDDO for 1 h followed by TNF for 15 min. Nuclear and cytosolic fractions were prepared and immunoblotted for p65RelA expression. TNF treatment caused the translocation of p65RelA from the cytosol to the nucleus, whereas treatment with either CDDO or CHX alone had no affect on p65RelA translocation (Fig. 3 C). In cells treated with CDDO or CHX in combination with TNF, there was no inhibition of p65RelAtranslocation. We also measured IκBα protein levels, which should be predominantly cytosolic. Indeed, we found IκBα to be exclusively cytosolic and, as expected, was degraded in all samples treated with TNF. Taken together these results indicate that CDDO inhibits IκBα resynthesis at a level downstream of p65RelAaccumulation in the nucleus. The finding that CDDO did not inhibit TNF-induced p65RelA translocation into the nucleus suggested that CDDO might be having an effect at the level of NF-κB binding to DNA or on subsequent transcriptional activity. Alternatively, CDDO could be acting in a nonselective manner thereby suppressing the synthesis of all proteins. We measured the capacity of CDDO to inhibit protein expression induced by the phorbol ester TPA. Cells were treated with either CDDO or CHX for 1 h followed by TPA or TNF for 3 h. Lysates were immunoblotted using antibodies to two proteins known to be induced by TPA, p21WAF1 and Mcl-1 (28.Akashi M. Osawa Y. Koeffler H.P. Hachiya M. Biochem. J. 1999; 337: 607-616Crossref PubMed Scopus (74) Google Scholar, 29.Yang T. Buchan H.L. Townsend K.J. Craig R.W. J. Cell. Physiol. 1996; 166: 523-536Crossref PubMed Scopus (160) Google Scholar, 30.Townsend K.J. Trusty J.L. Traupman M.A. Eastman A. Craig R.W. Oncogene. 1998; 17: 1223-1234Crossref PubMed Scopus (97) Google Scholar). CHX caused a reduction in basal levels of p21WAF1 and Mcl-1 as well as an inhibition of IκBα resynthesis after treatment with TNF (Fig. 4 A). However, CDDO did not inhibit TPA-induced p21WAF1 or Mcl-1. These results suggest that CDDO does not suppress protein synthesis in general but instead exerts an inhibitory effect on protein synthesis that is selective in nature. Presumably, the target of CDDO is at the level of transcription because 1 μm CDDO has been previously shown to inhibit cytokine-induced iNOS and Cox-2 mRNA (6.Suh N. Wang Y. Honda T. Gribble G.W. Dmitrovsky E. Hickey W.F. Maue R.A. Place A.E. Porter D.M. Spinella M.J. Williams C.R. Wu G. Dannenberg A.J. Flanders K.C. Letterio J.J. Mangelsdorf D.J. Nathan C.F. Nguyen L. Porter W.W. Ren R.F. Roberts A.B. Roche N.S. Subbaramaiah K. Sporn M.B. Cancer Res. 1999; 59: 336-341PubMed Google Scholar). However, TPA can also cause mRNA stabilization in addition to enhancing transcription (28.Akashi M. Osawa Y. Koeffler H.P. Hachiya M. Biochem. J. 1999; 337: 607-616Crossref PubMed Scopus (74) Google Scholar). To confirm that TPA was indeed inducing transcription in this model, cells were incubated with act D. We found that whereas CDDO pretreatment had no effect on TPA-induced p21WAF1 and Mcl-1 levels, act D completely blocked the induction of these proteins (Fig. 4 B). These results are consistent with the idea that CDDO is a selective inhibitor of transcription. CDDO at a concentration of 5 μm has been shown to induce apoptosis after 24 h in cell culture through a caspase-8-dependent mechanism (8.Ito Y. Pandey P. Place A. Sporn M.B. Gribble G.W. Honda T. Kharbanda S. Kufe D. Cell Growth & Differ. 2000; 11: 261-267PubMed Google Scholar). Therefore, we examined whether nontoxic concentrations of CDDO that sensitize ML-1 cells to apoptosis in the presence of TNF also activated caspase-8. ML-1 cells were treated with TNF in the presence or absence of CDDO, and cells were scored for apoptosis. Whereas CDDO or TNF treatment alone displayed no apoptosis after 3 h, the combination of CDDO and TNF caused a rapid induction of apoptosis in 100% of the cell population by 3 h (Fig. 5 A). We also observed the conversion of TNF-induced JNK phosphorylation from a weak transient signal to a strong sustained activation (Fig. 5 B). This increase in JNK phosphorylation preceded the cleavage of D4-GDI, the activation of caspa" @default.
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• W2005672276 title "The Novel Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) Potently Enhances Apoptosis Induced by Tumor Necrosis Factor in Human Leukemia Cells" @default.
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