Butein, a Tetrahydroxychalcone, Inhibits Nuclear Factor (NF)-κB and NF-κB-regulated Gene Expression through Direct Inhibition of IκBα Kinase β on Cysteine 179 Residue
2007; Elsevier BV; Volume: 282; Issue: 24 Linguagem: Inglês
10.1074/jbc.m700890200
ISSN1083-351X
AutoresManoj K. Pandey, Santosh K. Sandur, Bokyung Sung, Gautam Sethi, Ajaikumar B. Kunnumakkara, Bharat B. Aggarwal,
Tópico(s)Natural product bioactivities and synthesis
ResumoAlthough butein (3,4,2′,4′-tetrahydroxychalcone) is known to exhibit anti-inflammatory, anti-cancer, and anti-fibrogenic activities, very little is known about its mechanism of action. Because numerous effects modulated by butein can be linked to interference with the NF-κB pathway, we investigated in detail the effect of this chalcone on NF-κB activity. As examined by DNA binding, we found that butein suppressed tumor necrosis factor (TNF)-induced NF-κB activation in a dose- and time-dependent manner; suppressed the NF-κB activation induced by various inflammatory agents and carcinogens; and inhibited the NF-κB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-β. We also found that butein blocked the phosphorylation and degradation of IκBα by inhibiting IκBα kinase (IKK) activation. We found the inactivation of IKK by butein was direct and involved cysteine residue 179. This correlated with the suppression of phosphorylation and the nuclear translocation of p65. In this study, butein also inhibited the expression of the NF-κB-regulated gene products involved in anti-apoptosis (IAP2, Bcl-2, and Bcl-xL), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). Suppression of these gene products correlated with enhancement of the apoptosis induced by TNF and chemotherapeutic agents; and inhibition of cytokine-induced cellular invasion. Overall, our results indicated that antitumor and anti-inflammatory activities previously assigned to butein may be mediated in part through the direct inhibition of IKK, leading to the suppression of the NF-κB activation pathway. Although butein (3,4,2′,4′-tetrahydroxychalcone) is known to exhibit anti-inflammatory, anti-cancer, and anti-fibrogenic activities, very little is known about its mechanism of action. Because numerous effects modulated by butein can be linked to interference with the NF-κB pathway, we investigated in detail the effect of this chalcone on NF-κB activity. As examined by DNA binding, we found that butein suppressed tumor necrosis factor (TNF)-induced NF-κB activation in a dose- and time-dependent manner; suppressed the NF-κB activation induced by various inflammatory agents and carcinogens; and inhibited the NF-κB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-β. We also found that butein blocked the phosphorylation and degradation of IκBα by inhibiting IκBα kinase (IKK) activation. We found the inactivation of IKK by butein was direct and involved cysteine residue 179. This correlated with the suppression of phosphorylation and the nuclear translocation of p65. In this study, butein also inhibited the expression of the NF-κB-regulated gene products involved in anti-apoptosis (IAP2, Bcl-2, and Bcl-xL), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). Suppression of these gene products correlated with enhancement of the apoptosis induced by TNF and chemotherapeutic agents; and inhibition of cytokine-induced cellular invasion. Overall, our results indicated that antitumor and anti-inflammatory activities previously assigned to butein may be mediated in part through the direct inhibition of IKK, leading to the suppression of the NF-κB activation pathway. Most of the currently available targeted therapies for age-associated chronic illnesses are not very effective, exhibit numerous side effects, and for more than 80% of the population of the world, are too expensive. Thus alternates, which are less expensive, more efficacious, and exhibit minimum toxicity, are needed. Numerous epidemiological, clinical, and experimental evidence suggest that fruits and vegetables can lower the incidence of most diseases, including cancer (1Willett W.C. Science. 2002; 296: 695-698Crossref PubMed Scopus (671) Google Scholar). Neither the active principle in fruits and vegetables nor its mechanism of action is fully understood. 3,4,2′,4′-Tetrahydroxychalcone (butein) 2The abbreviations used are: butein, 3,4,2′,4′-tetrahydroxychalcone; NF-κB, nuclear factor-κB; IκB, inhibitory subunit of NF-κB; SEAP, secretory alkaline phosphatase; IKK, IκBα kinase; COX-2, cyclooxygenase-2; MMP-9, matrix metalloproteinase-9; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TRADD, tumor necrosis factor receptor-associated death domain; TRAF2, tumor necrosis factor receptor-associated factor; NIK, NF-κB-inducing kinase; IAP, inhibitor-of-apoptosis protein; PMA, phorbol myristate acetate; EMSA, electrophoretic mobility shift assay; ICAM-1, intercellular adhesion molecule 1; iNOS, intercellular nitric-oxide synthase; GST, glutathione S-transferase; LPS, lipopolysaccharide; PARP, poly(ADP-ribose) polymerase; VEGF, vascular epidermal growth factor; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; TAK1, transforming growth factor-β-activated kinase; TAB1, TAK1-binding protein; DTT, dithiothreitol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CSC, cigarette smoke condensate; BCl-2, B cell lymphoma-2; BCl-xL, BCl 2 like 1; c-myc, cellular myelocytomatosis oncogene; RIP, receptor interacting protein. is one such agent that has been identified from numerous plants including stem-bark of cashews (Semecarpus anacardium), the heartwood of Dalbergia odorifera, and the traditional Chinese and Tibetan medicinal herbs Caragana jubata and Rhus verniciflua Stokes. Evidence suggests that in rodents, butein suppressed phorbol ester-induced skin tumor formation (2Aizu E. Nakadate T. Yamamoto S. Kato R. Carcinogenesis. 1986; 7: 1809-1812Crossref PubMed Scopus (43) Google Scholar), abrogated carrageenan-induced rat paw edema (3Selvam C. Jachak S.M. Bhutani K.K. Phytother. Res. 2004; 18: 582-584Crossref PubMed Scopus (45) Google Scholar), exhibited hypotensive effects (4Kang D.G. Kim Y.C. Sohn E.J. Lee Y.M. Lee A.S. Yin M.H. Lee H.S. Biol. Pharm. Bull. 2003; 26: 1345-1347Crossref PubMed Scopus (44) Google Scholar), inhibited diabetic complications (5Lim S.S. Jung S.H. Ji J. Shin K.H. Keum S.R. J. Pharm. Pharmacol. 2001; 53: 653-668Crossref PubMed Scopus (109) Google Scholar), reduced antibody-associated glomerulonephritis (6Hayashi K. Nagamatsu T. Honda S. Suzuki Y. Eur. J. Pharmacol. 1996; 316: 297-306Crossref PubMed Scopus (20) Google Scholar), suppressed liver fibrosis induced by carbon tetrachloride (7Lee S.H. Nan J.X. Zhao Y.Z. Woo S.W. Park E.J. Kang T.H. Seo G.S. Kim Y.C. Sohn D.H. Planta Med. 2003; 69: 990-994Crossref PubMed Scopus (64) Google Scholar), and ameliorated renal concentrating ability in cisplatin-induced acute renal failure (8Kang D.G. Lee A.S. Mun Y.J. Woo W.H. Kim Y.C. Sohn E.J. Moon M.K. Lee H.S. Biol. Pharm. Bull. 2004; 27: 366-370Crossref PubMed Scopus (73) Google Scholar). In vitro, butein suppressed the proliferation of a wide variety of human tumor cells, including breast carcinoma (9Wang Y. Chan F.L. Chen S. Leung L.K. Life Sci. 2005; 77: 39-51Crossref PubMed Scopus (95) Google Scholar, 10Samoszuk M. Tan J. Chorn G. BMC Complement Altern. Med. 2005; 5: 1-5Crossref PubMed Scopus (74) Google Scholar), colon carcinoma (11Kang H.M. Kim J.H. Lee M.Y. Son K.H. Yang D.C. Baek N.I. Kwon B.M. Nat. Prod. Res. 2004; 18: 349-356Crossref PubMed Scopus (25) Google Scholar, 12Yit C.C. Das N.P. Cancer Lett. 1994; 82: 65-72Crossref PubMed Scopus (120) Google Scholar), osteosarcoma (13Jang H.S. Kook S.H. Son Y.O. Kim J.G. Jeon Y.M. Jang Y.S. Choi K.C. Kim J. Han S.K. Lee K.Y. Park B.K. Cho N.P. Lee J.C. Biochim. Biophys. Acta. 2005; 1726: 309-316Crossref PubMed Scopus (110) Google Scholar), lymphoma (14Lee J.C. Lee K.Y. Kim J. Na C.S. Jung N.C. Chung G.H. Jang Y.S. Food Chem. Toxicol. 2004; 42: 1383-1388Crossref PubMed Scopus (121) Google Scholar, 15Ramanathan R. Tan C.H. Das N.P. Cancer Lett. 1992; 62: 217-224Crossref PubMed Scopus (61) Google Scholar), acute myelogenous leukemia (16Kim N.Y. Pae H.O. Oh G.S. Kang T.H. Kim Y.C. Rhew H.Y. Chung H.T. Pharmacol. Toxicol. 2001; 88: 261-266Crossref PubMed Scopus (63) Google Scholar), melanoma (17Iwashita K. Kobori M. Yamaki K. Tsushida T. Biosci. Biotechnol. Biochem. 2000; 64: 1813-1820Crossref PubMed Scopus (248) Google Scholar), and hepatic stellate cells (18Lee S.H. Seo G.S. Kim H.S. Woo S.W. Ko G. Sohn D.H. Biochem. Pharmacol. 2006; 72: 1322-1333Crossref PubMed Scopus (41) Google Scholar). It is not fully understood how butein mediates the anti-inflammatory and anticellular effects. However, investigators have reported that butein modulated the expression of epidermal growth factor receptor tyrosine kinase (19Zhu L. Chen L. Xu X. Anal. Chem. 2003; 75: 6381-6387Crossref PubMed Scopus (49) Google Scholar, 20Yang E.B. Zhang K. Cheng L.Y. Mack P. Biochem. Biophys. Res. Commun. 1998; 245: 435-438Crossref PubMed Scopus (108) Google Scholar, 21Yang E.B. Guo Y.J. Zhang K. Chen Y.Z. Mack P. Biochim. Biophys. Acta. 2001; 1550: 144-152Crossref PubMed Scopus (91) Google Scholar), adhesion molecules (6Hayashi K. Nagamatsu T. Honda S. Suzuki Y. Eur. J. Pharmacol. 1996; 316: 297-306Crossref PubMed Scopus (20) Google Scholar, 22Takano-Ishikawa Y. Goto M. Yamaki K. Phytother. Res. 2003; 17: 1224-1227Crossref PubMed Scopus (42) Google Scholar), Bcl-2 (16Kim N.Y. Pae H.O. Oh G.S. Kang T.H. Kim Y.C. Rhew H.Y. Chung H.T. Pharmacol. Toxicol. 2001; 88: 261-266Crossref PubMed Scopus (63) Google Scholar, 17Iwashita K. Kobori M. Yamaki K. Tsushida T. Biosci. Biotechnol. Biochem. 2000; 64: 1813-1820Crossref PubMed Scopus (248) Google Scholar), iNOS (23Lee S.H. Seo G.S. Sohn D.H. Biochem. Biophys. Res. Commun. 2004; 323: 125-132Crossref PubMed Scopus (78) Google Scholar), phosphodiesterase IV (24Yu S.M. Cheng Z.J. Kuo S.C. Eur. J. Pharmacol. 1995; 280: 69-77Crossref PubMed Scopus (51) Google Scholar, 25Kuppusamy U.R. Das N.P. Biochem. Pharmacol. 1992; 44: 1307-1315Crossref PubMed Scopus (161) Google Scholar), glutathione S-transferase (GST) (26Zhang K. Das N.P. Biochem. Pharmacol. 1994; 47: 2063-2068Crossref PubMed Scopus (80) Google Scholar), hemeoxygenase-1 (18Lee S.H. Seo G.S. Kim H.S. Woo S.W. Ko G. Sohn D.H. Biochem. Pharmacol. 2006; 72: 1322-1333Crossref PubMed Scopus (41) Google Scholar), the uptake of Ca2+ (27Thiyagarajah P. Kuttan S.C. Lim S.C. Teo T.S. Das N.P. Biochem. Pharmacol. 1991; 41: 669-675Crossref PubMed Scopus (45) Google Scholar), 12-lipooxygenase, ornithine decarboxylase (2Aizu E. Nakadate T. Yamamoto S. Kato R. Carcinogenesis. 1986; 7: 1809-1812Crossref PubMed Scopus (43) Google Scholar), angiotensin-converting enzyme (4Kang D.G. Kim Y.C. Sohn E.J. Lee Y.M. Lee A.S. Yin M.H. Lee H.S. Biol. Pharm. Bull. 2003; 26: 1345-1347Crossref PubMed Scopus (44) Google Scholar), and tissue inhibitor of metalloproteinase-1 (7Lee S.H. Nan J.X. Zhao Y.Z. Woo S.W. Park E.J. Kang T.H. Seo G.S. Kim Y.C. Sohn D.H. Planta Med. 2003; 69: 990-994Crossref PubMed Scopus (64) Google Scholar, 28Woo S.W. Lee S.H. Kang H.C. Park E.J. Zhao Y.Z. Kim Y.C. Sohn D.H. J. Pharm. Pharmacol. 2003; 55: 347-352Crossref PubMed Scopus (25) Google Scholar). Additionally, butein has been shown to suppress the activity of aldolase reductase (5Lim S.S. Jung S.H. Ji J. Shin K.H. Keum S.R. J. Pharm. Pharmacol. 2001; 53: 653-668Crossref PubMed Scopus (109) Google Scholar), glutathione reductase (29Zhang K. Yang E.B. Tang W.Y. Wong K.P. Mack P. Biochem. Pharmacol. 1997; 54: 1047-1053Crossref PubMed Scopus (87) Google Scholar), tyrosinase (30Khatib S. Nerya O. Musa R. Shmuel M. Tamir S. Vaya J. Bioorg. Med. Chem. 2005; 13: 433-441Crossref PubMed Scopus (324) Google Scholar), COX-1 (3Selvam C. Jachak S.M. Bhutani K.K. Phytother. Res. 2004; 18: 582-584Crossref PubMed Scopus (45) Google Scholar), farnesyl protein transferase (11Kang H.M. Kim J.H. Lee M.Y. Son K.H. Yang D.C. Baek N.I. Kwon B.M. Nat. Prod. Res. 2004; 18: 349-356Crossref PubMed Scopus (25) Google Scholar), and aromatase (9Wang Y. Chan F.L. Chen S. Leung L.K. Life Sci. 2005; 77: 39-51Crossref PubMed Scopus (95) Google Scholar); mediate antioxidant effects (31Sogawa S. Nihro Y. Ueda H. Miki T. Matsumoto H. Satoh T. Biol. Pharm. Bull. 1994; 17: 251-256Crossref PubMed Scopus (78) Google Scholar, 32Lee J.C. Lim K.T. Jang Y.S. Biochim. Biophys. Acta. 2002; 1570: 181-191Crossref PubMed Scopus (164) Google Scholar); inhibit drug export (33Zhang K. Wong K.P. Biochem. Pharmacol. 1996; 52: 1631-1638Crossref PubMed Scopus (30) Google Scholar); suppress the GST-mediated conjugation of chlorambucil with GSH (34Zhang K. Wong K.P. Biochem. J. 1997; 325: 417-422Crossref PubMed Scopus (30) Google Scholar, 35Zhang K. Wong K.P. Chow P. Life Sci. 2003; 72: 2629-2640Crossref PubMed Scopus (21) Google Scholar); and chelate iron and copper (36Cheng Z.J. Kuo S.C. Chan S.C. Ko F.N. Teng C.M. Biochim. Biophys. Acta. 1998; 1392: 291-299Crossref PubMed Scopus (112) Google Scholar). Because butein exhibits anti-inflammatory and anti-proliferative effects and suppresses the expression of adhesion molecules, iNOS, Bcl-2, tissue inhibitor of metalloproteinase-1, and 12-lipooxygenase, all of which are known to be regulated by the transcription factor NF-κB, we postulated that butein must mediate its effects by modulating the NF-κB activation pathway, which has been closely linked to inflammation, tumorigenesis, proliferation, invasion, angiogenesis, and metastasis, and is activated in response to various inflammatory agents, carcinogens, tumor promoters, and growth factors (37Aggarwal B.B. Cancer Cell. 2004; 6: 203-208Abstract Full Text Full Text PDF PubMed Scopus (1377) Google Scholar, 38Aggarwal B.B. Shishodia S. Sandur S.K. Pandey M.K. Sethi G. Biochem. Pharmacol. 2006; 72: 1605-1621Crossref PubMed Scopus (1104) Google Scholar, 39Karin M. Nature. 2006; 441: 431-436Crossref PubMed Scopus (3013) Google Scholar). Therefore, the effect of butein on the regulation of this pathway was investigated in detail. We found that butein suppressed NF-κB activation pathways activated by a variety of agents through the direct inhibition of IκBα kinase (IKK), which led to the suppression of NF-κB-regulated gene products and the enhancement of apoptosis induced by inflammatory cytokines. Reagents—Butein, with chemical structure as shown in Fig. 1A was obtained from Alexis (San Diego, CA). A 50 mm solution of butein was prepared in dimethyl sulfoxide, stored as small aliquots at –20 °C, and then diluted as needed in cell culture medium. Bacteria-derived human recombinant human TNF, purified to homogeneity with a specific activity of 5 × 107 units/mg, was kindly provided by Genentech (South San Francisco, CA). Cigarette smoke condensate, prepared as previously described (40Anto R.J. Mukhopadhyay A. Shishodia S. Gairola C.G. Aggarwal B.B. Carcinogenesis. 2002; 23: 1511-1518Crossref PubMed Google Scholar), was supplied by Dr. C. Gary Gairola (University of Kentucky, Lexington, KY). Penicillin, streptomycin, RPMI 1640 medium, and fetal bovine serum were obtained from Invitrogen. Phorbol myristate acetate (PMA), lipopolysaccharide (LPS), okadaic acid, and anti-β-actin antibody were obtained from Sigma. Antibodies against p65, p50, IκBα, cyclin D1, pro-caspase-8, pro-caspase-3, JNK1, p38 MAPK, MMP-9, c-Myc, ICAM-1, IAP2, Bcl-2, Bcl-xL, RIP, TRADD, and poly-(ADP-ribose) polymerase (PARP) and the annexin V staining kits were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-VEGF was obtained from Neomarkers (Fremont, CA). Anti-COX-2 antibody was obtained from BD Biosciences. Phosphospecific anti-IκBα (Ser-32/36) and phospho-specific anti-p65 antibodies (Ser-536), cleaved caspase-8, cleaved caspase-3, and phospho-p38 MAPK were purchased from Cell Signaling (Beverly, MA). Anti-IKK-α and anti-IKK-β antibodies were kindly provided by Imgenex (San Diego, CA). Expression vector plasmids for transforming growth factor-β-activated kinase (TAK1) and TAK1 binding protein (TAB1) have been described previously (41Blonska M. Shambharkar P.B. Kobayashi M. Zhang D. Sakurai H. Su B. Lin X. J. Biol. Chem. 2005; 280: 43056-43063Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). IKK plasmids with mutated cysteine residue 179 (Cys-179) were kindly provided by Dr. T. D. Gilmore of Boston University (Boston, MA). Cell Lines—Cell lines H1299 (human lung adenocarcinoma), KBM-5 (human myeloid), Jurkat (human T cell leukemia) cells and A293 (human embryonic kidney) were obtained from American Type Culture Collection (Manassas, VA). The H1299 and Jurkat cells were cultured in RPMI 1640 medium, KBM-5 cells were cultured in Iscove's modified Dulbecco's medium with 15% fetal bovine serum, and A293 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. All culture media were supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin. Electrophoretic Mobility Shift Assay—To determine NF-κB activation, we prepared nuclear extracts and performed electrophoretic mobility shift assay (EMSA) as described previously (42Chaturvedi M.M. Mukhopadhyay A. Aggarwal B.B. Methods Enzymol. 2000; 319: 585-602Crossref PubMed Google Scholar). For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with antibodies against either p50 or p65 of NF-κB for 30 min at 37 °C before the complex was analyzed by EMSA. Preimmune serum was included as the negative control. The dried gels were visualized, and the radioactive bands were quantitated with a Storm 820 imaging system running ImageQuant software (Amersham Biosciences). Western Blot Analysis—To determine the effect of butein on TNF-dependent IκBα phosphorylation, IκBα degradation, p65 translocation, and p65 phosphorylation, cytoplasmic and nuclear extracts were prepared as previously described (43Reddy S.A. Chaturvedi M.M. Darnay B.G. Chan H. Higuchi M. Aggarwal B.B. J. Biol. Chem. 1994; 269: 25369-25372Abstract Full Text PDF PubMed Google Scholar, 44Shishodia S. Majumdar S. Banerjee S. Aggarwal B.B. Cancer Res. 2003; 63: 4375-4383PubMed Google Scholar). For the detection of cleavage products of PARP, caspases, antiapoptotic, and angiogenesis markers, whole cell extracts were prepared by subjecting TNF and TNF plus butein-treated cells to lysis in lysis buffer (20 mmol/liter Tris (pH 7.4), 250 mmol/liter NaCl, 2 mmol/liter EDTA (pH 8.0), 0.1% Triton X-100, 0.01 μg/ml aprotinin, 0.005 μg/ml leupeptin, 0.4 mmol/liter phenylmethylsulfonyl fluoride, and 4 mmol/liter NaVO4). Lysates were spun at 14,000 × g for 10 min to remove insoluble material. Supernatants were collected and kept at –80 °C. Either cytosolic, or nuclear extracts and whole cell lysates were resolved by SDS-PAGE. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blotted with the relevant antibodies, and detected by an enhanced chemiluminescence reagent (Amersham Biosciences ECL™). The bands obtained were quantified using NIH Image analyzer (NIH, Bethesda, MD). For detection of precapases and cleaved caspases, specific antibodies against each were combined for detection. Kinase Assay—To determine the effect of butein on TNF-induced IKK activation, we performed an immunocomplex kinase assay using GST-IκBα as the substrate as described previously (45Manna S.K. Mukhopadhyay A. Aggarwal B.B. J. Immunol. 2000; 165: 4927-4934Crossref PubMed Scopus (96) Google Scholar). Briefly, the IKK complex from whole cell extracts was precipitated with antibody against IKK-α and treated with protein A/G-Sepharose beads (Pierce). After 2 h, the beads were washed with whole cell extract buffer and then resuspended in a kinase assay mixture containing 50 mm HEPES (pH 7.4), 20 mm MgCl2, 2 mm DTT, 20 μCi of [α-32P]ATP, 10 μm unlabeled ATP, and 2 μg of substrate GST-IκBα (amino acids 1–54). After incubation at 30 °C for 30 min, the reaction was terminated by boiling with SDS sample buffer for 5 min. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized with the Storm 820 imaging system. To determine the total amounts of IKK-α and IKK-β in each sample, 30 μg of whole cell proteins were resolved on 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and then blotted with either anti-IKK-α or anti-IKK-β antibody. For JNK assay, whole cell extracts were precipitated with antibody against JNK1, and performed kinase assay using GST-c-Jun (amino acids 1–79) as described (46Takada Y. Kobayashi Y. Aggarwal B.B. J. Biol. Chem. 2005; 280: 17203-17212Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). To determine the amount of JNK1 in each sample, Western blotting was performed against JNK1 antibody. NF-κB-dependent Reporter Gene Expression Assay—To determine the effect of butein on TNF-, TNF receptor (TNFR-), TNFR-associated death domain (TRADD-), TNFR-associated factor 2 (TRAF2-), NF-κB-inducing kinase (NIK), TAK1/TAB1-, and IKK-NF-κB-dependent reporter gene transcription, we performed the secretory alkaline phosphatase (SEAP) assay as previously described (47Darnay B.G. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1999; 274: 7724-7731Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), with the following exceptions. Briefly, A293 cells (5 × 105 cells/well) were plated in 6-well plates and transiently transfected by the calcium phosphate method with pNF-κB-SEAP (0.5 μg). To examine TNF-induced reporter gene expression, we transfected the cells with 0.5 μg of the SEAP expression plasmid and 1.5 μg of the control plasmid pCMV-FLAG1 DNA for 24 h. We then treated the cells for 4 h with butein and stimulated them with 1 nm TNF. The cell culture medium was harvested after 24 h of TNF treatment. To examine reporter gene expression induced by various genes, A293 cells were transfected with 0.5 μg of pNF-κB-SEAP plasmid with 0.5 μg of an expressing plasmid and 1.5 μgofthe control plasmid pCMV-FLAG1 for 24 h, treated with butein, and then harvested from cell culture medium after an additional 24 h of incubation. The culture medium was analyzed for SEAP activity as recommended by the manufacturer (Clontech) using a Victor 3 microplate reader (Perkin-Elmer Life Sciences). Immunocytochemistry for NF-κB p65 Localization—Immunocytochemistry was used to examine the effect of butein on the nuclear pools of p65 as previously described (48Bharti A.C. Donato N. Singh S. Aggarwal B.B. Blood. 2003; 101: 1053-1062Crossref PubMed Scopus (638) Google Scholar). Briefly, treated cells were plated on a poly-l-lysine-coated glass slide by centrifugation (Cytospin 4; Thermoshendon), air dried, and fixed with 4% paraformaldehyde. After being washed in phosphate-buffered saline, the slides were blocked with 5% normal goat serum for 1 h and incubated with rabbit polyclonal anti-human p65 antibody at a 1/200 dilution. After overnight incubation at 4 °C, the slides were washed, incubated with goat anti-rabbit IgG-Alexa Fluor 594 antibody (Molecular Probes, Eugene, OR) at a 1/200 dilution for 1 h, and counterstained for nuclei with Hoechst 33342 (50 ng/ml) for 5 min. Stained slides were mounted with mounting medium (Sigma) and analyzed under a fluorescence microscope (Labophot-2; Nikon). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon) and MetaMorph version 4.6.5 software (Universal Imaging). Live/Dead Assay—To measure apoptosis, we also used the Live/Dead assay (Molecular Probes), which determines intracellular esterase activity and plasma membrane integrity (49Takada Y. Singh S. Aggarwal B.B. J. Biol. Chem. 2004; 279: 15096-15104Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Calcein-AM, a nonfluorescent polyanionic dye, is retained by live cells, in which it produces intense green fluorescence through enzymatic (esterase) conversion. In addition, the ethidium homodimer enters cells with damaged membranes and binds to nucleic acids, thereby producing a bright red fluorescence in dead cells. Briefly, 2 × 105 cells were incubated with 25 μm butein for 4 h and treated with 1 nm TNF up to 24 h at 37 °C. Cells were stained with the Live/Dead reagent (5 μm ethidium homodimer and 5 μm calcein-AM) and incubated at 37 °C for 30 min. Cells were analyzed under a fluorescence microscope (Labophot-2; Nikon). Cytotoxicity Assay—The effects of butein on the cytotoxic effects of TNF and other chemotherapeutic agents were determined by the MTT uptake method as previously described (48Bharti A.C. Donato N. Singh S. Aggarwal B.B. Blood. 2003; 101: 1053-1062Crossref PubMed Scopus (638) Google Scholar). Briefly, 5,000 cells were incubated with 25 μm butein in triplicate in a 96-well plate and treated with the indicated concentrations of 1 nm TNF, 0.1 μm 5-fluorouracil (5-FU), and 0.1 μm doxorubicin for 24 h at 37 °C. An MTT solution was added to each well and incubated for 2 h at 37 °C. An extraction buffer (20% SDS, 50% dimethyl formamide) was added, and the cells were incubated overnight at 37 °C. Then the optical density was measured at 570 nm using a 96-well multiscanner (MRX Revelation, Dynex Technologies, Chantilly, VA). Annexin V Assay—The annexin V assay uses the binding properties of annexin V to detect the rapid translocation and accumulation of the membrane phospholipid phosphatidyl-serine from the cytoplasmic membrane interface to the extracellular surface, an indicator of early apoptosis. We detected this loss of membrane asymmetry using an annexin V antibody conjugated with the fluorescein isothiocyanate fluorescence dye. Briefly, 5 × 105 cells were pretreated with butein (25 μm), treated with 1 nm TNF up to 16 h at 37 °C, and subjected to annexin V staining. The cells were washed in phosphate-buffered saline, resuspended in 100 μl of binding buffer containing a fluorescein isothiocyanate-conjugated anti-annexin V antibody, and analyzed with a flow cytometer (FACSCalibur, BD Biosciences). Invasion Assay—This assay was performed mostly as previously described (50Albini A. Iwamoto Y. Kleinman H.K. Martin G.R. Aaronson S.A. Kozlowski J.M. McEwan R.N. Cancer Res. 1987; 47: 3239-3245PubMed Google Scholar). Because invasion through the extracellular matrix is a crucial step in tumor metastasis, a membrane invasion culture system was used to assess cell invasion. The BD BioCoat tumor invasion system consists of chambers with a lightproof polyethylene terephthalate membrane coated with a reconstituted basement membrane gel with 8-μm diameter pores (BD Biosciences). We suspended 2.5 × 104 non-small cell adenocarcinoma H1299 cells in serum-free medium and seeded the upper wells with them. After incubation overnight, the cells were treated with the indicated concentration of butein for 4 h and stimulated with 1 nm TNF for an additional 24 h in the presence of 1% fetal bovine serum. The cells that invaded the lower chamber by migrating through the Matrigel during incubation were stained with 4 μg/ml calcein-AM in phosphate-buffered saline for 30 min at 37 °C and scanned for fluorescence with a Victor 3 multiplate reader (PerkinElmer); fluorescent cells were counted. We investigated the effects of butein on the NF-κB activation pathway induced by various carcinogens and inflammatory stimuli, on NF-κB-regulated gene expression, and on apoptosis induced by cytokines and chemotherapeutic agents. The concentration of butein used and the duration of exposure had minimal effect on the viability of different cell lines studied as determined by the trypan blue dye exclusion test (data not shown). We focused on TNF-induced NF-κB activation because the NF-κB activation pathway activated by TNF has been relatively well characterized (51Garg A. Aggarwal B.B. Leukemia. 2002; 16: 1053-1068Crossref PubMed Scopus (423) Google Scholar). Butein Suppresses TNF-induced NF-κB Activation in a Dose- and Time-dependent Manner—We first determined the dose and time of exposure to butein required to suppress TNF-induced NF-κB activation. For this cells were first pretreated with butein, then exposed to TNF for NF-κB activation. EMSA showed that butein alone had no effect on NF-κB activation, but it inhibited TNF-mediated NF-κB activation in a dose- and time-dependent manner (Fig. 1, B and C, respectively). Whether butein can suppress NF-κB in cells pre-activated with TNF was also determined. We found that butein down-regulated quite effectively TNF induced NF-κB even when cells were treated with the inhibitor after NF-κB activation (see lane 3 versus lane 5 in Fig. 1D). NF-κB is a complex of proteins, in which various combinations of Rel/NF-κB proteins constitute active NF-κB heterodimers that bind to a specific DNA sequence. Thus, to show that the band visualized by EMSA in TNF-treated cells was indeed NF-κB, nuclear extracts from TNF-activated cells were incubated with antibodies to the p50 (NF-κB) and p65 (RelA) subunits of NF-κB and analyzed by EMSA. The results in Fig. 1E, showed the bands had shifted to higher molecular masses suggesting that the TNF-activated complex consisted of p50 and p65. Preimmune serum had no effect on DNA binding. The addition of excess unlabeled NF-κB (cold oligonucleotide; 100-fold) caused a complete disappearance of the band, whereas the addition of mutated oligonucleotide had no effect on the DNA binding. Butein Inhibits Constitutive NF-κB Activation—Whether butein alone could inhibit constitutive NF-κB in tumor cells was also investigated. Multiple myeloma U266 cells are known to express constitutive NF-κB activation (52Bharti A.C. Shishodia S. Reuben J.M. Weber D. Alexanian R. RajVadhan S. Estrov Z. Talpaz M. Aggarwal B.B. Blood. 2004; 103: 3175-3184Crossref PubMed Scopus (293) Google Scholar). U266 cells were treated with different concentrations of butein for 4 h and then analyzed for NF-κB activation. Butein inhibited the constitutive NF-κB activation in MM cells (Fig. 1F). These results indicate that butein can suppress not only inducible but also constitutively active NF-κB in tumor cells. Butein Inhibits NF-κB Activation Induced by Carcinogens and Inflammatory Stimuli—Cigarette smoke condensate
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