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Activation of Transcription Factor NF-κB Is Suppressed by Curcumin (Diferuloylmethane)

Activation of Transcription Factor NF-κB Is Suppressed by Curcumin (Diferuloylmethane) THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 42, Issue of October 20, pp. 24995–25000, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Activation of Transcription Factor NF-kB Is Suppressed by Curcumin (Diferulolylmethane)* (Received for publication, July 13, 1995, and in revised form, August 11, 1995) Sanjaya Singh and Bharat B. Aggarwal‡ From the Cytokine Research Laboratory, Department of Molecular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 other members of the Rel family reside in the cytoplasm in an When activated, NF-kB, a ubiquitous transcription factor, binds DNA as a heterodimeric complex composed inactive state but upon activation, they are translocated to the of members of the Rel/NF-kB family of polypeptides. Be- nucleus. The nuclear translocation of Rel proteins is induced by cause of its intimate involvement in host defense against many agents, including inflammatory cytokines (e.g. tumor disease, this transcription factor is an important target necrosis factor (TNF), lymphotoxin, and interleukin-1), mito- for therapeutic intervention. In the present report we gens, bacterial products, protein synthesis inhibitors, oxidative demonstrate that curcumin (diferuloylmethane), a stress (H O ), ultraviolet light, and phorbol esters (3, 4). Upon 2 2 known anti-inflammatory and anticarcinogenic agent, activation of NF-kB, a large number of genes are induced is a potent inhibitor of NF-kB activation. Treatment of including various inflammatory cytokines, adhesion molecules, human myeloid ML-1a cells with tumor necrosis factor and Rel proteins (for review, see Refs. 3 and 4). (TNF) rapidly activated NF-kB, which consists of p50 Curcumin (diferuloylmethane) has been shown to block and p65 subunits, and this activation was inhibited by many reactions in which NF-kB plays a major role. This agent curcumin. AP-1 binding factors were also found to be is a major active component of turmeric (Curcuma longa) and it down-modulated by curcumin, whereas the Sp1 binding gives specific flavor and yellow color to curry. The compound factor was unaffected. has been shown to display anticarcinogenic properties in ani- Besides TNF, curcumin also blocked phorbol ester- mals as indicated by its ability to inhibit both tumor initiation and hydrogen peroxide-mediated activation of NF-kB. induced by benz(a)pyrene and 7,12-dimethylbenz(a)anth- The TNF-dependent phosphorylation and degradation racene (5–8) and tumor promotion induced by phorbol esters (9, of IkBa was not observed in curcumin-treated cells; the 10), which are known to activate NF-kB. Curcumin has also translocation of p65 subunit to the nucleus was inhib- been shown to inhibit type 1 human immunodeficiency virus ited at the same time. The mechanism of action of cur- cumin was found to be different from that of protein long terminal repeat (HIV-LTR) directed gene expression and tyrosine phosphatase inhibitors. Our results indicate virus replication stimulated by TNF and phorbol ester (11), that curcumin inhibits NF-kB activation pathway at a which likewise require NF-kB activation. The anti-inflamma- step before IkBa phosphorylation but after the conver- tory and antioxidant properties of curcumin have been well gence of various stimuli. documented (12–14). How these inhibitory responses are mod- ulated by curcumin is not understood. In the present report we show that curcumin is a potent Members of the transcription factor NF-kB family play a inhibitor of NF-kB activation induced by various agents. The central role in various responses leading to host defense, acti- results also indicate that curcumin inhibits at a step in the vating a rapid progression of gene expression. These transcrip- signal transduction cascade of NF-kB activation that occurs tion factors are dimeric complexes composed of different mem- before IkBa phosphorylation but after the point at which var- bers of the Rel/NF-kB family of polypeptides. This family is ious signals transduced by different stimuli converge. This distinguished by the presence of a Rel homology domain of study shows that curcumin is a potential candidate for modu- about 300 amino acids that displays a 35 to 61% identity lation of NF-kB-dependent pathological conditions. between various family members (for review, see Ref. 1). Al- EXPERIMENTAL PROCEDURES though NF-kB is a ubiquitous transcription factor, it plays a critical role in the cells of the immune system, where it controls Materials—Penicillin, streptomycin, RPMI 1640 medium, and fetal calf serum were obtained from Life Technologies, Inc. Curcumin, gly- the expression of various cytokines and the major histocompat- cine, NaCl, and bovine serum albumin were obtained from Sigma, and ibility complex genes. The inappropriate regulation of NF-kB phenylarsine oxide from Aldrich. Bacteria-derived recombinant human and its dependent genes have been associated with various TNF, purified to homogeneity with a specific activity of 5 3 10 units/ pathological conditions including toxic/septic shock, graft ver- mg, was kindly provided by Genentech, Inc. (South San Francisco, CA). sus host reaction, acute inflammatory conditions, acute-phase Antibody against IkBa, cyclin D1, and NF-kB subunits p50 and p65 response, viral replication, radiation damage, atherosclerosis, and double-stranded oligonucleotides having AP-1 and Sp1 consensus sequences were obtained from Santa Cruz Biotechnology (Santa and cancer (1, 2). No wonder NF-kB is an important target for Cruz, CA). therapeutic intervention. Cell Lines—The cell line employed in this study was ML-1a, a human Unlike other transcription factors, the NF-kB proteins and myelomonoblastic leukemia cell line kindly provided by Dr. Ken Takeda * This research was supported by a grant from The Foundation for Research. The costs of publication of this article were defrayed in part The abbreviations used are: TNF, tumor necrosis factor; DTT, di- by the payment of page charges. This article must therefore be hereby thiothreitol; DMP, 2,3-dimercaptopropanol; HIV-LTR, human immuno- marked “advertisement” in accordance with 18 U.S.C. Section 1734 deficiency virus-1 long terminal repeat; PMA, phorbol 12-myristate solely to indicate this fact. 13-acetate; EMSA, electrophoretic mobility shift assay; TPCK, L-1-to- ‡ To whom correspondence should be addressed. Tel.: 713-792-3503/ sylamido-2-phenylethyl chloromethyl ketone; ROI, reactive oxygen 6459; Fax: 713-794-1613. intermediates. This is an Open Access article under the CC BY license. 24996 Inhibition of NF-kB Activation by Curcumin of Showa University, Japan. Cells were routinely grown in RPMI 1640 medium supplemented with glutamine (2 mM), gentamicin (50 mg/ml), and fetal bovine serum (10%). The cells were seeded at a density of 1 3 10 cells/ml in T25 flasks (Falcon 3013, Becton Dickinson Labware, Lincoln Park, NJ) containing 10 ml of medium and grown at 37 °C in an atmosphere of 95% air and 5% CO . Cell cultures were split every 3 or 4 days. Occasionally, cells were tested for mycoplasma contamination using the DNA-based assay kit purchased from Gen-Probe (San Diego, CA). Electrophoretic Mobility Shift Assays—ML-1a cells (2 3 10 cells/ml) were treated separately with different concentrations of an activator at 37 °C. Nuclear extracts were then prepared according to Schreiber et al. (15). Briefly, 2 3 10 cells were washed with cold phosphate-buffered saline and suspended in 0.4 ml of lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmeth- ylsulfonyl fluoride, 2.0 mg/ml leupeptin, 2.0 mg/ml aprotinin, and 0.5 mg/ml benzamidine). The cells were allowed to swell on ice for 15 min, after which 12.5 ml of 10% Nonidet P-40 was added. The tube was then vigorously mixed on a vortex machine for 10 s, and the homogenate was centrifuged for 30 s in a Microfuge E. The nuclear pellet was resus- pended in 25 ml of ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM phenyl- methylsulfonyl fluoride, 2.0 mg/ml leupeptin, 2.0 mg/ml aprotinin, and 0.5 mg/ml benzamidine), and the tube was incubated on ice for 30 min with intermittent mixing. The tube was then centrifuged for 5 min in a Microfuge E at 4 °C, and the supernatant (nuclear extract) was either used immediately or stored at 270 °C for later use. The protein content was measured by the method of Bradford (16). Electrophoretic mobility shift assays (EMSA) were performed by incubating 4 mg of nuclear extract (NE), with 16 fmol of P-end-labeled 45-mer double-stranded NF-kB oligonucleotide from the HIV-LTR, 59-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCG- TGG-39 (17), for 15 min at 37 °C. The incubation mixture included 2–3 mg of poly(dI-dC) in a binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM DTT, 1% Nonidet P-40, 5% glycerol, and 50 mM NaCl) (18, 19). The DNA-protein complex formed was separated from free oligonucleotide on 4.5% native polyacrylamide gel using buffer contain- ing 50 mM Tris, 200 mM glycine, pH 8.5, and 1 mM EDTA (20), and then the gel was dried. A double-stranded mutated oligonucleotide, 59-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCG- TGG-39, was used to examine the specificity of binding of NF-kBtothe DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with the antibodies against either p50 or p65 subunits of NF-kB for 30 min at room temperature before the complex was analyzed by EMSA (21). Antibodies against cyclin D1 and preim- mune serum were included as negative controls. The EMSAs for AP-1 and Sp1 were performed as described for NF-kB FIG.1. Dose response and kinetics of inhibition of TNF- using P-end-labeled double-stranded oligonucleotides. Specificity of dependent NF-kB activation by curcumin. a, ML-1a cells (2 3 binding was determined routinely by using an excess of unlabeled 10 /ml) were preincubated at 37 °C for 60 min with different concen- oligonucleotide for competition as described earlier (21). trations (2–60 mM) of curcumin followed by 30 min incubation with 0.1 Visualization and quantitation of radioactive bands was carried out nM TNF. b, ML-1a cells (2 3 10 /ml) were preincubated at 37 °C with 20 by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using mM curcumin for different times and then tested for NF-kB activation at “Image-quant” software. 37 °C for 30 min either with or without 0.1 nM TNF. 2 indicates time Western Blotting for IkBa and p65—After the NF-kB activation re- curcumin was present before the addition of TNF; 0 indicates co-incu- action described above, postnuclear extracts were resolved on 10% bation with TNF; and 1 indicates time curcumin was added after TNF. SDS-polyacrylamide gels for IkBa. To determine p65 levels, nuclear and 6 For panel c, ML-1a cells (2 3 10 /ml) were incubated at 37 °C with 50 postnuclear (cytoplasmic) extracts were resolved on 8% SDS-polyacryl- mM curcumin for 60 min followed by treatment with 10 nM TNF for amide gels. After the gels, the proteins were electrotransferred to ni- different times. After these treatments nuclear extracts were prepared trocellulose filters, probed with a rabbit polyclonal antibody against and then assayed for NF-kB as described under “Experimental Proce- IkBa or against p65, and detected by chemiluminescence (ECL, Amer- dures.” The arbitrary units represent the relative amounts of the ra- sham) (22). The bands obtained were quantitated using Personal Den- dioactivity present in respective bands. sitometer Scan v1.30 using Image Quanta software version 3.3 (Molec- ular Dynamics, Sunnyvale, CA). RESULTS trations of curcumin followed by treatment with TNF (0.1 nM) In this report we examined the effect of curcumin on the for 30 min at 37 °C. They were then examined for NF-kB activation of transcription factor NF-kB. We used human activation by electrophoretic mobility shift assay. The results ML-1a cells for these studies because their response to NF-kB in Fig. 1a indicate that 40–60 mM curcumin inhibited most of activation by various stimuli has been well characterized (21– the TNF response. Curcumin by itself did not activate NF-kB. 23). The time of incubation and the concentration of the drugs We next tested the kinetics of inhibition, incubating the cells used in our studies had no effect on the cell viability (data not with curcumin for 60, 30, and 10 min prior to the addition of shown). TNF, at the same time as the addition of TNF, or 10 min after Curcumin Inhibits TNF-dependent NF-kB Activation— the addition of TNF. The cells were treated with TNF for 30 ML-1a cells were preincubated for 1 h with different concen- min. TNF response was inhibited only when cells were pre- Inhibition of NF-kB Activation by Curcumin 24997 FIG.3. Effect of curcumin on PMA- and H O -mediated activa- 2 2 tion of NF-kB. ML-1a cells (2 3 10 /ml) were preincubated for 60 min at 37 °C with curcumin (50 mM) followed by PMA (25 ng/ml) or H O (0.5 2 2 mM) or the indicated combinations for 30 min and then tested for NF-kB activation as described under “Experimental Procedures.” Curcumin Also Blocks Phorbol Ester- and Hydrogen Perox- ide-mediated Activation of NF-kB—Besides TNF, NF-kB acti- vation is also induced by phorbol ester (PMA), and hydrogen peroxide (49). However, the initial signal transduction pathway induced by these agents that leads to the NF-kB activation differs. Therefore we examined the effect of curcumin on acti- vation of the transcription factor by these various agents. The results shown in Fig. 3 indicate that curcumin completely FIG.2. Supershift analysis and specificity of the effect of cur- blocked PMA and hydrogen peroxide-induced activation of NF- cumin on the NF-kB activation. For panel a, nuclear extracts were kB. Thus these results suggest that curcumin is a general prepared from untreated or TNF (0.1 nM)-treated ML-1a cells (2 3 10 /ml), incubated for 30 min with antibodies and then assayed for suppressor of NF-kB activation. NF-kB as described under “Experimental Procedures.” For panel b, Curcumin Down-modulates AP-1 but Not Sp1 Transcription nuclear extract prepared from TNF pretreated cells were incubated Factors—Whether curcumin specifically blocks the activation with different concentrations of curcumin for 15 min and then ana- of NF-kB or also affects other transcription factors was inves- lyzed for NF-kB by EMSA. DMSO, dimethyl sulfoxide; PIS, preimmune serum. tigated. Curcumin had no effect on the Sp1 transcription factor (Fig. 4); however, DNA binding of AP-1 transcription factors treated with curcumin (Fig. 1b). Cotreatment of cells with TNF was found to be down-modulated. This result is in agreement and curcumin was not effective. with an earlier report which showed that curcumin not only Previous studies from our laboratory have shown that a high inhibits the DNA binding activity of c-Jun/AP-1 binding factors concentration of TNF (10 nM) can activate NF-kB within 5 min but also down-modulates the level of these factors (24). and this induction is higher in its intensity than that obtained Reducing Agents Do Not Reverse the Effect of Curcumin—It with cells using 100-fold lower concentration of TNF for longer has been shown that agents like TPCK that modify the sulfhy- time (23). To determine the effect of curcumin on NF-kB acti- dryl group in NF-kB inhibit its activation but this inhibition is vation at higher TNF concentration and its effect on kinetics of prevented in the presence of DTT and DMP (25, 31). DTT and TNF-mediated activation of NF-kB, curcumin-pretreated cells DMP can also reverse the inhibitory effect of phenylarsine were exposed to 10 nM TNF for various times (Fig. 1c). In oxide (a potent protein tyrosine phosphatase inhibitor) on agreement with our previous results, the induction of NF-kBby NF-kB activation (21). To determine if the inhibitory effect of 10 nM TNF was very high and occurred within 5 min. Curcumin curcumin on NF-kB was reversed by these reducing agents, could completely inhibit the activation of NF-kB induced by 10 ML-1a cells were treated with curcumin in the presence and nM as efficiently as it did with 0.1 nM TNF. This suggests that absence of either DTT or DMP and then examined for the curcumin is a very potent inhibitor of NF-kB activation. activation of NF-kB by TNF. As shown in Fig. 5, DTT and DMP To show that the retarded band observed by EMSA in TNF- did not reverse the inhibition caused by curcumin but com- treated cells was indeed NF-kB we incubated the nuclear ex- pletely reversed the phenylarsine oxide-mediated inhibition. tracts with antibody to either p50 (NF-kB1) or p65 (Rel A) These results thus suggest that the mechanism of action of subunits and then carried out EMSA. The results from this curcumin is different from that of protein tyrosine phosphatase experiment (Fig. 2a) show that antibodies to either subunit of inhibitors. NF-kB shifted the band to higher molecular weight, thus sug- Curcumin Inhibits TNF-dependent Phosphorylation and gesting that the TNF-activated complex consisted of p50 and Degradation of IkBa and Hence Translocation of p65 Subunit of p65 subunits. Neither preimmune serum nor irrelevant anti- NF-kB to the Nucleus—The translocation of NF-kB to the nu- body against cyclin Di had any affect on the mobility of NF-kB. cleus is preceded by the phosphorylation and proteolytic deg- Both TPCK and herbimycin A have been shown to interfere radation of IkBa (for review, see Ref. 26). To determine with the binding of NF-kB to the DNA (25, 52). To determine whether the inhibitory action of curcumin was due to its effect the effect of curcumin on the binding of NF-kB to the DNA, the on IkBa degradation, the cytoplasmic levels of IkBa protein nuclear extracts from TNF-preactivated cells were incubated were examined by Western blot analysis. IkBa was phospho- with curcumin and then EMSA was performed. The results of rylated within 5 min of TNF treatment of ML-1a cells and then this experiment (Fig. 2b) show that curcumin did not modify disappeared within 15 min. However, curcumin abolished both the ability of NF-kB to bind to the DNA. the phosphorylation (as indicated by absence of the slow mi- 24998 Inhibition of NF-kB Activation by Curcumin FIG.4. Effect of curcumin on AP-1 and Sp1 transcription fac- tors. Cells were treated with different concentrations of curcumin for 60 min at 37 °C, and nuclear extract were then prepared and used for EMSA of AP-1 and Sp1 transcription factors as described. FIG.5. Effect of DTT and DMP on the curcumin and phenyl- arsine oxide (PAO)-induced inhibition of NF-kB activation. FIG.6. Effect of curcumin on TNF-induced phosphorylation ML-1a (2 3 10 /ml) were incubated at 37 °C for 60 min with DTT (100 and degradation of IkBa and on level of p65 in cytoplasm and mM) or DMP (100 mM) in the presence of curcumin (50 mM) or phenyl- 6 nucleus. ML-1a (2 3 10 /ml) pretreated (for 60 min at 37 °C) with or arsine oxide (2.4 mM) or the indicated combinations followed by 30 min without curcumin (50 mM) were incubated for different times with TNF incubation with 0.1 nM TNF and then assayed for NF-kB activation as (0.1 nM), and then assayed for IkBa (panel A) and for p65 (panel B)in described under “Experimental Procedures.” cytosolic fractions by Western blot analysis as described under “Exper- imental Procedures.” For panel C, ML-1a (2 3 10 /ml) pretreated (for 60 min at 37 °C) with curcumin were incubated with TNF (0.1 nM) for 30 min. Nuclear and cytoplasmic extracts were assayed by Western blot grating band) and degradation of IkBa induced by TNF analysis for p65. The arbitrary units represent the relative amounts of (Fig. 6A). the respective proteins as described under “Experimental Procedures.” We also measured the level of p65 in the cytoplasm and nucleus. As expected upon TNF treatment, the level of p65 declined in the cytoplasm with a concurrent increase in the ious other agents including phorbol ester and H O was also 2 2 nucleus (Fig. 6, B and C). The treatment of cells with curcumin inhibited by curcumin. As has been shown with other inhibi- abolished the TNF-dependent change in the nuclear and cyto- tors, the effect of curcumin was not due to the chemical modi- plasmic p65 levels. These results show that curcumin inhibits fication of NF-kB proteins (25, 31, 52). The inhibition of NF-kB the TNF-induced translocation of p65 to the nucleus and this is activation was accompanied by the inhibition of p65 transloca- consistent with the inhibition of TNF-dependent degrdation of tion to the nucleus and of IkBa degradation. IkBa by curcumin. Identifying how curcumin blocks the activation of NF-kB DISCUSSION requires an understanding of the mechanism by which various Curcumin is a pharmacologically safe compound with known inducers activate this important transcription factor. The role anti-inflammatory, anticarcinogenic, and free radical scav- of different TNF-activated signals including acidic and neutral enger properties (6, 10, 27–30). However, how curcumin carries sphingomyelinase-generated ceramides, proteases, serine/thre- out these functions is not very clear. We investigated cur- onine protein kinase, protein tyrosine kinase, protein tyrosine cumin’s effect on NF-kB activation because NF-kB is involved phosphatase, and superoxide radicals in the activation of in so many of the activities that curcumin is known to block. NF-kB have been implicated (1, 21, 22, 32–35). Whether these NF-kB plays a pivotal role in cells of the immune system signals are generated by TNF sequentially or independently of because it is rapidly activated by a wide variety of pathogenic each other, however, is not understood. signals and functions as a potent and pleiotropic transcrip- All three inducers of NF-kB used in our studies are known to tional activator. Intervention in NF-kB activation may be ben- produce reactive oxygen intermediates (ROI). Therefore, it is eficial in suppressing toxic/septic shock, graft versus host reac- possible that the effect of curcumin is through quenching of tions, acute inflammatory reactions, HIV replication, acute- ROI production. The inhibitors of mitochondrial electron trans- phase response, and radiation damage. port have been shown to impair the TNF-induced activation of Our results show that curcumin completely blocked the TNF- NF-kB (36), thus also suggesting the role of ROI. Several ad- dependent activation of NF-kB. The activation induced by var- ditional, indirect lines of evidence suggest a role for ROI as a Inhibition of NF-kB Activation by Curcumin 24999 shown that TPCK chemically modifies NF-kB, thus altering its release from IkBa (25). Curcumin, however, does not chemi- cally modify the DNA binding properties of NF-kB. Another level of modification that could prevent formation of p50/p65 heterodimer is down-modulation of the cytoplasmic pool of p65 subunit of NF-kB. Our results, however, show that p65 was not down-modulated by curcumin but its translocation to the nucleus was inhibited, most likely through inhibition of degradation of IkBa. The observation that TNF-induced phosphorylation and deg- radation of IkBa is abolished by curcumin indicate that the step in the signal transduction pathway of NF-kB activation inhibited by this agent is at or before the phosphorylation step of NF-kB (Fig. 7). That it can inhibit NF-kB activation by diverse agents indicate that this step is after or at the step where the diverse signals converge. Overall we conclude that because of its very low pharmacological toxicity and its ability FIG.7. Possible site of action of curcumin on TNF- PMA- and to modulate activation of NF-kB by various agents, curcumin H O -induced NF-kB activation. 2 2 has a high potential for use in modulating expression of genes regulated by NF-kB. Acknowledgment—We are thankful to Dr. B. G. Darnay for his common and critical denominator (40, 41), including evidence thoughtful criticism of the manuscript. that cellular levels of ROI increase in response to TNF, inter- leukin-1, PMA, lipopolysaccharide, UV light, and g-irradiation REFERENCES (for review, see Ref. 1). But among the various ROI adminis- 1. Siebenlist, U., Franzo, G., and Brown, K. (1994) Annu. Rev. Cell Biol. 10, tered to cells in culture, only hydrogen peroxide was found to be 405–455 2. Baeuerle, P. 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Activation of Transcription Factor NF-κB Is Suppressed by Curcumin (Diferuloylmethane)

Singh, Sanjaya; Aggarwal, Bharat B.
Journal of Biological Chemistry – Oct 1, 1995
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10.1074/jbc.270.42.24995
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 42, Issue of October 20, pp. 24995–25000, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Activation of Transcription Factor NF-kB Is Suppressed by Curcumin (Diferulolylmethane)* (Received for publication, July 13, 1995, and in revised form, August 11, 1995) Sanjaya Singh and Bharat B. Aggarwal‡ From the Cytokine Research Laboratory, Department of Molecular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 other members of the Rel family reside in the cytoplasm in an When activated, NF-kB, a ubiquitous transcription factor, binds DNA as a heterodimeric complex composed inactive state but upon activation, they are translocated to the of members of the Rel/NF-kB family of polypeptides. Be- nucleus. The nuclear translocation of Rel proteins is induced by cause of its intimate involvement in host defense against many agents, including inflammatory cytokines (e.g. tumor disease, this transcription factor is an important target necrosis factor (TNF), lymphotoxin, and interleukin-1), mito- for therapeutic intervention. In the present report we gens, bacterial products, protein synthesis inhibitors, oxidative demonstrate that curcumin (diferuloylmethane), a stress (H O ), ultraviolet light, and phorbol esters (3, 4). Upon 2 2 known anti-inflammatory and anticarcinogenic agent, activation of NF-kB, a large number of genes are induced is a potent inhibitor of NF-kB activation. Treatment of including various inflammatory cytokines, adhesion molecules, human myeloid ML-1a cells with tumor necrosis factor and Rel proteins (for review, see Refs. 3 and 4). (TNF) rapidly activated NF-kB, which consists of p50 Curcumin (diferuloylmethane) has been shown to block and p65 subunits, and this activation was inhibited by many reactions in which NF-kB plays a major role. This agent curcumin. AP-1 binding factors were also found to be is a major active component of turmeric (Curcuma longa) and it down-modulated by curcumin, whereas the Sp1 binding gives specific flavor and yellow color to curry. The compound factor was unaffected. has been shown to display anticarcinogenic properties in ani- Besides TNF, curcumin also blocked phorbol ester- mals as indicated by its ability to inhibit both tumor initiation and hydrogen peroxide-mediated activation of NF-kB. induced by benz(a)pyrene and 7,12-dimethylbenz(a)anth- The TNF-dependent phosphorylation and degradation racene (5–8) and tumor promotion induced by phorbol esters (9, of IkBa was not observed in curcumin-treated cells; the 10), which are known to activate NF-kB. Curcumin has also translocation of p65 subunit to the nucleus was inhib- been shown to inhibit type 1 human immunodeficiency virus ited at the same time. The mechanism of action of cur- cumin was found to be different from that of protein long terminal repeat (HIV-LTR) directed gene expression and tyrosine phosphatase inhibitors. Our results indicate virus replication stimulated by TNF and phorbol ester (11), that curcumin inhibits NF-kB activation pathway at a which likewise require NF-kB activation. The anti-inflamma- step before IkBa phosphorylation but after the conver- tory and antioxidant properties of curcumin have been well gence of various stimuli. documented (12–14). How these inhibitory responses are mod- ulated by curcumin is not understood. In the present report we show that curcumin is a potent Members of the transcription factor NF-kB family play a inhibitor of NF-kB activation induced by various agents. The central role in various responses leading to host defense, acti- results also indicate that curcumin inhibits at a step in the vating a rapid progression of gene expression. These transcrip- signal transduction cascade of NF-kB activation that occurs tion factors are dimeric complexes composed of different mem- before IkBa phosphorylation but after the point at which var- bers of the Rel/NF-kB family of polypeptides. This family is ious signals transduced by different stimuli converge. This distinguished by the presence of a Rel homology domain of study shows that curcumin is a potential candidate for modu- about 300 amino acids that displays a 35 to 61% identity lation of NF-kB-dependent pathological conditions. between various family members (for review, see Ref. 1). Al- EXPERIMENTAL PROCEDURES though NF-kB is a ubiquitous transcription factor, it plays a critical role in the cells of the immune system, where it controls Materials—Penicillin, streptomycin, RPMI 1640 medium, and fetal calf serum were obtained from Life Technologies, Inc. Curcumin, gly- the expression of various cytokines and the major histocompat- cine, NaCl, and bovine serum albumin were obtained from Sigma, and ibility complex genes. The inappropriate regulation of NF-kB phenylarsine oxide from Aldrich. Bacteria-derived recombinant human and its dependent genes have been associated with various TNF, purified to homogeneity with a specific activity of 5 3 10 units/ pathological conditions including toxic/septic shock, graft ver- mg, was kindly provided by Genentech, Inc. (South San Francisco, CA). sus host reaction, acute inflammatory conditions, acute-phase Antibody against IkBa, cyclin D1, and NF-kB subunits p50 and p65 response, viral replication, radiation damage, atherosclerosis, and double-stranded oligonucleotides having AP-1 and Sp1 consensus sequences were obtained from Santa Cruz Biotechnology (Santa and cancer (1, 2). No wonder NF-kB is an important target for Cruz, CA). therapeutic intervention. Cell Lines—The cell line employed in this study was ML-1a, a human Unlike other transcription factors, the NF-kB proteins and myelomonoblastic leukemia cell line kindly provided by Dr. Ken Takeda * This research was supported by a grant from The Foundation for Research. The costs of publication of this article were defrayed in part The abbreviations used are: TNF, tumor necrosis factor; DTT, di- by the payment of page charges. This article must therefore be hereby thiothreitol; DMP, 2,3-dimercaptopropanol; HIV-LTR, human immuno- marked “advertisement” in accordance with 18 U.S.C. Section 1734 deficiency virus-1 long terminal repeat; PMA, phorbol 12-myristate solely to indicate this fact. 13-acetate; EMSA, electrophoretic mobility shift assay; TPCK, L-1-to- ‡ To whom correspondence should be addressed. Tel.: 713-792-3503/ sylamido-2-phenylethyl chloromethyl ketone; ROI, reactive oxygen 6459; Fax: 713-794-1613. intermediates. This is an Open Access article under the CC BY license. 24996 Inhibition of NF-kB Activation by Curcumin of Showa University, Japan. Cells were routinely grown in RPMI 1640 medium supplemented with glutamine (2 mM), gentamicin (50 mg/ml), and fetal bovine serum (10%). The cells were seeded at a density of 1 3 10 cells/ml in T25 flasks (Falcon 3013, Becton Dickinson Labware, Lincoln Park, NJ) containing 10 ml of medium and grown at 37 °C in an atmosphere of 95% air and 5% CO . Cell cultures were split every 3 or 4 days. Occasionally, cells were tested for mycoplasma contamination using the DNA-based assay kit purchased from Gen-Probe (San Diego, CA). Electrophoretic Mobility Shift Assays—ML-1a cells (2 3 10 cells/ml) were treated separately with different concentrations of an activator at 37 °C. Nuclear extracts were then prepared according to Schreiber et al. (15). Briefly, 2 3 10 cells were washed with cold phosphate-buffered saline and suspended in 0.4 ml of lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmeth- ylsulfonyl fluoride, 2.0 mg/ml leupeptin, 2.0 mg/ml aprotinin, and 0.5 mg/ml benzamidine). The cells were allowed to swell on ice for 15 min, after which 12.5 ml of 10% Nonidet P-40 was added. The tube was then vigorously mixed on a vortex machine for 10 s, and the homogenate was centrifuged for 30 s in a Microfuge E. The nuclear pellet was resus- pended in 25 ml of ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM phenyl- methylsulfonyl fluoride, 2.0 mg/ml leupeptin, 2.0 mg/ml aprotinin, and 0.5 mg/ml benzamidine), and the tube was incubated on ice for 30 min with intermittent mixing. The tube was then centrifuged for 5 min in a Microfuge E at 4 °C, and the supernatant (nuclear extract) was either used immediately or stored at 270 °C for later use. The protein content was measured by the method of Bradford (16). Electrophoretic mobility shift assays (EMSA) were performed by incubating 4 mg of nuclear extract (NE), with 16 fmol of P-end-labeled 45-mer double-stranded NF-kB oligonucleotide from the HIV-LTR, 59-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCG- TGG-39 (17), for 15 min at 37 °C. The incubation mixture included 2–3 mg of poly(dI-dC) in a binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM DTT, 1% Nonidet P-40, 5% glycerol, and 50 mM NaCl) (18, 19). The DNA-protein complex formed was separated from free oligonucleotide on 4.5% native polyacrylamide gel using buffer contain- ing 50 mM Tris, 200 mM glycine, pH 8.5, and 1 mM EDTA (20), and then the gel was dried. A double-stranded mutated oligonucleotide, 59-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCG- TGG-39, was used to examine the specificity of binding of NF-kBtothe DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with the antibodies against either p50 or p65 subunits of NF-kB for 30 min at room temperature before the complex was analyzed by EMSA (21). Antibodies against cyclin D1 and preim- mune serum were included as negative controls. The EMSAs for AP-1 and Sp1 were performed as described for NF-kB FIG.1. Dose response and kinetics of inhibition of TNF- using P-end-labeled double-stranded oligonucleotides. Specificity of dependent NF-kB activation by curcumin. a, ML-1a cells (2 3 binding was determined routinely by using an excess of unlabeled 10 /ml) were preincubated at 37 °C for 60 min with different concen- oligonucleotide for competition as described earlier (21). trations (2–60 mM) of curcumin followed by 30 min incubation with 0.1 Visualization and quantitation of radioactive bands was carried out nM TNF. b, ML-1a cells (2 3 10 /ml) were preincubated at 37 °C with 20 by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using mM curcumin for different times and then tested for NF-kB activation at “Image-quant” software. 37 °C for 30 min either with or without 0.1 nM TNF. 2 indicates time Western Blotting for IkBa and p65—After the NF-kB activation re- curcumin was present before the addition of TNF; 0 indicates co-incu- action described above, postnuclear extracts were resolved on 10% bation with TNF; and 1 indicates time curcumin was added after TNF. SDS-polyacrylamide gels for IkBa. To determine p65 levels, nuclear and 6 For panel c, ML-1a cells (2 3 10 /ml) were incubated at 37 °C with 50 postnuclear (cytoplasmic) extracts were resolved on 8% SDS-polyacryl- mM curcumin for 60 min followed by treatment with 10 nM TNF for amide gels. After the gels, the proteins were electrotransferred to ni- different times. After these treatments nuclear extracts were prepared trocellulose filters, probed with a rabbit polyclonal antibody against and then assayed for NF-kB as described under “Experimental Proce- IkBa or against p65, and detected by chemiluminescence (ECL, Amer- dures.” The arbitrary units represent the relative amounts of the ra- sham) (22). The bands obtained were quantitated using Personal Den- dioactivity present in respective bands. sitometer Scan v1.30 using Image Quanta software version 3.3 (Molec- ular Dynamics, Sunnyvale, CA). RESULTS trations of curcumin followed by treatment with TNF (0.1 nM) In this report we examined the effect of curcumin on the for 30 min at 37 °C. They were then examined for NF-kB activation of transcription factor NF-kB. We used human activation by electrophoretic mobility shift assay. The results ML-1a cells for these studies because their response to NF-kB in Fig. 1a indicate that 40–60 mM curcumin inhibited most of activation by various stimuli has been well characterized (21– the TNF response. Curcumin by itself did not activate NF-kB. 23). The time of incubation and the concentration of the drugs We next tested the kinetics of inhibition, incubating the cells used in our studies had no effect on the cell viability (data not with curcumin for 60, 30, and 10 min prior to the addition of shown). TNF, at the same time as the addition of TNF, or 10 min after Curcumin Inhibits TNF-dependent NF-kB Activation— the addition of TNF. The cells were treated with TNF for 30 ML-1a cells were preincubated for 1 h with different concen- min. TNF response was inhibited only when cells were pre- Inhibition of NF-kB Activation by Curcumin 24997 FIG.3. Effect of curcumin on PMA- and H O -mediated activa- 2 2 tion of NF-kB. ML-1a cells (2 3 10 /ml) were preincubated for 60 min at 37 °C with curcumin (50 mM) followed by PMA (25 ng/ml) or H O (0.5 2 2 mM) or the indicated combinations for 30 min and then tested for NF-kB activation as described under “Experimental Procedures.” Curcumin Also Blocks Phorbol Ester- and Hydrogen Perox- ide-mediated Activation of NF-kB—Besides TNF, NF-kB acti- vation is also induced by phorbol ester (PMA), and hydrogen peroxide (49). However, the initial signal transduction pathway induced by these agents that leads to the NF-kB activation differs. Therefore we examined the effect of curcumin on acti- vation of the transcription factor by these various agents. The results shown in Fig. 3 indicate that curcumin completely FIG.2. Supershift analysis and specificity of the effect of cur- blocked PMA and hydrogen peroxide-induced activation of NF- cumin on the NF-kB activation. For panel a, nuclear extracts were kB. Thus these results suggest that curcumin is a general prepared from untreated or TNF (0.1 nM)-treated ML-1a cells (2 3 10 /ml), incubated for 30 min with antibodies and then assayed for suppressor of NF-kB activation. NF-kB as described under “Experimental Procedures.” For panel b, Curcumin Down-modulates AP-1 but Not Sp1 Transcription nuclear extract prepared from TNF pretreated cells were incubated Factors—Whether curcumin specifically blocks the activation with different concentrations of curcumin for 15 min and then ana- of NF-kB or also affects other transcription factors was inves- lyzed for NF-kB by EMSA. DMSO, dimethyl sulfoxide; PIS, preimmune serum. tigated. Curcumin had no effect on the Sp1 transcription factor (Fig. 4); however, DNA binding of AP-1 transcription factors treated with curcumin (Fig. 1b). Cotreatment of cells with TNF was found to be down-modulated. This result is in agreement and curcumin was not effective. with an earlier report which showed that curcumin not only Previous studies from our laboratory have shown that a high inhibits the DNA binding activity of c-Jun/AP-1 binding factors concentration of TNF (10 nM) can activate NF-kB within 5 min but also down-modulates the level of these factors (24). and this induction is higher in its intensity than that obtained Reducing Agents Do Not Reverse the Effect of Curcumin—It with cells using 100-fold lower concentration of TNF for longer has been shown that agents like TPCK that modify the sulfhy- time (23). To determine the effect of curcumin on NF-kB acti- dryl group in NF-kB inhibit its activation but this inhibition is vation at higher TNF concentration and its effect on kinetics of prevented in the presence of DTT and DMP (25, 31). DTT and TNF-mediated activation of NF-kB, curcumin-pretreated cells DMP can also reverse the inhibitory effect of phenylarsine were exposed to 10 nM TNF for various times (Fig. 1c). In oxide (a potent protein tyrosine phosphatase inhibitor) on agreement with our previous results, the induction of NF-kBby NF-kB activation (21). To determine if the inhibitory effect of 10 nM TNF was very high and occurred within 5 min. Curcumin curcumin on NF-kB was reversed by these reducing agents, could completely inhibit the activation of NF-kB induced by 10 ML-1a cells were treated with curcumin in the presence and nM as efficiently as it did with 0.1 nM TNF. This suggests that absence of either DTT or DMP and then examined for the curcumin is a very potent inhibitor of NF-kB activation. activation of NF-kB by TNF. As shown in Fig. 5, DTT and DMP To show that the retarded band observed by EMSA in TNF- did not reverse the inhibition caused by curcumin but com- treated cells was indeed NF-kB we incubated the nuclear ex- pletely reversed the phenylarsine oxide-mediated inhibition. tracts with antibody to either p50 (NF-kB1) or p65 (Rel A) These results thus suggest that the mechanism of action of subunits and then carried out EMSA. The results from this curcumin is different from that of protein tyrosine phosphatase experiment (Fig. 2a) show that antibodies to either subunit of inhibitors. NF-kB shifted the band to higher molecular weight, thus sug- Curcumin Inhibits TNF-dependent Phosphorylation and gesting that the TNF-activated complex consisted of p50 and Degradation of IkBa and Hence Translocation of p65 Subunit of p65 subunits. Neither preimmune serum nor irrelevant anti- NF-kB to the Nucleus—The translocation of NF-kB to the nu- body against cyclin Di had any affect on the mobility of NF-kB. cleus is preceded by the phosphorylation and proteolytic deg- Both TPCK and herbimycin A have been shown to interfere radation of IkBa (for review, see Ref. 26). To determine with the binding of NF-kB to the DNA (25, 52). To determine whether the inhibitory action of curcumin was due to its effect the effect of curcumin on the binding of NF-kB to the DNA, the on IkBa degradation, the cytoplasmic levels of IkBa protein nuclear extracts from TNF-preactivated cells were incubated were examined by Western blot analysis. IkBa was phospho- with curcumin and then EMSA was performed. The results of rylated within 5 min of TNF treatment of ML-1a cells and then this experiment (Fig. 2b) show that curcumin did not modify disappeared within 15 min. However, curcumin abolished both the ability of NF-kB to bind to the DNA. the phosphorylation (as indicated by absence of the slow mi- 24998 Inhibition of NF-kB Activation by Curcumin FIG.4. Effect of curcumin on AP-1 and Sp1 transcription fac- tors. Cells were treated with different concentrations of curcumin for 60 min at 37 °C, and nuclear extract were then prepared and used for EMSA of AP-1 and Sp1 transcription factors as described. FIG.5. Effect of DTT and DMP on the curcumin and phenyl- arsine oxide (PAO)-induced inhibition of NF-kB activation. FIG.6. Effect of curcumin on TNF-induced phosphorylation ML-1a (2 3 10 /ml) were incubated at 37 °C for 60 min with DTT (100 and degradation of IkBa and on level of p65 in cytoplasm and mM) or DMP (100 mM) in the presence of curcumin (50 mM) or phenyl- 6 nucleus. ML-1a (2 3 10 /ml) pretreated (for 60 min at 37 °C) with or arsine oxide (2.4 mM) or the indicated combinations followed by 30 min without curcumin (50 mM) were incubated for different times with TNF incubation with 0.1 nM TNF and then assayed for NF-kB activation as (0.1 nM), and then assayed for IkBa (panel A) and for p65 (panel B)in described under “Experimental Procedures.” cytosolic fractions by Western blot analysis as described under “Exper- imental Procedures.” For panel C, ML-1a (2 3 10 /ml) pretreated (for 60 min at 37 °C) with curcumin were incubated with TNF (0.1 nM) for 30 min. Nuclear and cytoplasmic extracts were assayed by Western blot grating band) and degradation of IkBa induced by TNF analysis for p65. The arbitrary units represent the relative amounts of (Fig. 6A). the respective proteins as described under “Experimental Procedures.” We also measured the level of p65 in the cytoplasm and nucleus. As expected upon TNF treatment, the level of p65 declined in the cytoplasm with a concurrent increase in the ious other agents including phorbol ester and H O was also 2 2 nucleus (Fig. 6, B and C). The treatment of cells with curcumin inhibited by curcumin. As has been shown with other inhibi- abolished the TNF-dependent change in the nuclear and cyto- tors, the effect of curcumin was not due to the chemical modi- plasmic p65 levels. These results show that curcumin inhibits fication of NF-kB proteins (25, 31, 52). The inhibition of NF-kB the TNF-induced translocation of p65 to the nucleus and this is activation was accompanied by the inhibition of p65 transloca- consistent with the inhibition of TNF-dependent degrdation of tion to the nucleus and of IkBa degradation. IkBa by curcumin. Identifying how curcumin blocks the activation of NF-kB DISCUSSION requires an understanding of the mechanism by which various Curcumin is a pharmacologically safe compound with known inducers activate this important transcription factor. The role anti-inflammatory, anticarcinogenic, and free radical scav- of different TNF-activated signals including acidic and neutral enger properties (6, 10, 27–30). However, how curcumin carries sphingomyelinase-generated ceramides, proteases, serine/thre- out these functions is not very clear. We investigated cur- onine protein kinase, protein tyrosine kinase, protein tyrosine cumin’s effect on NF-kB activation because NF-kB is involved phosphatase, and superoxide radicals in the activation of in so many of the activities that curcumin is known to block. NF-kB have been implicated (1, 21, 22, 32–35). Whether these NF-kB plays a pivotal role in cells of the immune system signals are generated by TNF sequentially or independently of because it is rapidly activated by a wide variety of pathogenic each other, however, is not understood. signals and functions as a potent and pleiotropic transcrip- All three inducers of NF-kB used in our studies are known to tional activator. Intervention in NF-kB activation may be ben- produce reactive oxygen intermediates (ROI). Therefore, it is eficial in suppressing toxic/septic shock, graft versus host reac- possible that the effect of curcumin is through quenching of tions, acute inflammatory reactions, HIV replication, acute- ROI production. The inhibitors of mitochondrial electron trans- phase response, and radiation damage. port have been shown to impair the TNF-induced activation of Our results show that curcumin completely blocked the TNF- NF-kB (36), thus also suggesting the role of ROI. Several ad- dependent activation of NF-kB. The activation induced by var- ditional, indirect lines of evidence suggest a role for ROI as a Inhibition of NF-kB Activation by Curcumin 24999 shown that TPCK chemically modifies NF-kB, thus altering its release from IkBa (25). Curcumin, however, does not chemi- cally modify the DNA binding properties of NF-kB. Another level of modification that could prevent formation of p50/p65 heterodimer is down-modulation of the cytoplasmic pool of p65 subunit of NF-kB. Our results, however, show that p65 was not down-modulated by curcumin but its translocation to the nucleus was inhibited, most likely through inhibition of degradation of IkBa. The observation that TNF-induced phosphorylation and deg- radation of IkBa is abolished by curcumin indicate that the step in the signal transduction pathway of NF-kB activation inhibited by this agent is at or before the phosphorylation step of NF-kB (Fig. 7). That it can inhibit NF-kB activation by diverse agents indicate that this step is after or at the step where the diverse signals converge. Overall we conclude that because of its very low pharmacological toxicity and its ability FIG.7. Possible site of action of curcumin on TNF- PMA- and to modulate activation of NF-kB by various agents, curcumin H O -induced NF-kB activation. 2 2 has a high potential for use in modulating expression of genes regulated by NF-kB. Acknowledgment—We are thankful to Dr. B. G. Darnay for his common and critical denominator (40, 41), including evidence thoughtful criticism of the manuscript. that cellular levels of ROI increase in response to TNF, inter- leukin-1, PMA, lipopolysaccharide, UV light, and g-irradiation REFERENCES (for review, see Ref. 1). But among the various ROI adminis- 1. Siebenlist, U., Franzo, G., and Brown, K. (1994) Annu. Rev. Cell Biol. 10, tered to cells in culture, only hydrogen peroxide was found to be 405–455 2. Baeuerle, P. 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