Curcumin induces stress response, neurite outgrowth and prevent nf-κb activation by inhibiting the proteasome function

Curcumin, a natural polyphenolic compound, has long been known as an anti-tumour and anti-inflammatory compound; although, the common mechanism through which it exhibits such prop-erties are remains unclear. Recently, we reported that the curcumin-induced apoptosis is mediated through the impairment of ubiquitin proteasome system (UPS). Here, we show that curcumin dis-rupts UPS function by directly inhibiting the enzyme activity of the proteasome's 20S core cata-lytic component. Like other proteasome inhibitors, curcumin exposure induces neurite outgrowth and the stress response, as evident from the induction of various cytosolic and endoplasmic reticulum chap-erones as well as induction of transcription factor CHOP/GADD153. The direct inhibition of protea-some activity also causes an increase in half-life of IκB-α that ultimately leads to the down-regulation of NF-κB activation. These results suggest that cur-cumin-induced proteasomal malfunction might be linked with both anti-proliferative and anti-inflam-matory activities.

Keywords: Curcumin; Proteasome; IκB; NF-κB; Stress

INTRODUCTION

The ubiquitin-proteasome system (UPS) is the cell's principal mechanism for controlled protein degrada-tion. The pathway has been shown to be involved in the regulation of critical cellular processes such as transcription, cell cycle progression, oncogenesis, growth and development, selective elimination of abnormal proteins and antigen processing (Glickman and Ciechanover, 2001). Degradation of a protein by UPS involves two distinct and successive steps: (a) covalent attachment of multiple ubiquitin molecules to

the target protein and (b) degradation of the targeted protein by proteasome. The 20S core catalytic com-plex of the proteasome is a cylindrical stack of four seven-membered rings and is flanked on both sides by 19S regulatory complexes. Its proteolytic site faces an interior chamber that can be entered only through pores at either end of the cylinder. Because folded proteins can not enter this chamber, the isolated 20S complex hydrolyses only small peptides and denatured proteins. Three distinct types of proteolytic activities have been defined for 20S proteasome: chymotrypsin-like (Tyr or Phe at P1), trypsin-like (Arg or Lys at P1) and post-glu-tamyl peptidyl hydrolytic-like (Glu at P1). Since UPS plays a crucial role in the degradation of many regula-tory proteins that are necessary for cell growth, it is expected that the altered function of this pathway will affect cell survival. In fact, it has long been known that the inhibition of proteasome function induces stress response and programmed cell death, depending on the cell types and conditions (Sadoul et al., 1996; Bush et al., 1997; Drexler, 1997; Lopes et al., 1997; Meriin et al., 1998; Drexler et al., 2000; Jana et al., 2001). Curcumin is the major active and yellow compo-nent of turmeric, a commonly used spice derived from the rhizome of plant Curcuma longa. It has been demonstrated to have anti-inflammatory and anti-pro-liferative activities (Aggarwal et al., 2003; Ammon and Wahl, 2003). Ample evidence exists to support its use in cancer prevention for its anti-proliferative and anti-carcinogenic properties (Huang MT et al., 1994; Mehta et al., 1997; Khar et al., 1999; Chuang et al., 2000; Pal et al., 2001; Bharti et al., 2003; Woo et al., 2003). Although its precise mode of action remains unclear, studies have shown that the chemo-preventive action of curcumin might be due to its ability to induce apoptosis (Khar et al., 1999; Pal et. al., 2001; Bharti et al., 2003; Woo et al., 2003). However, the precise

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Curcumin Induces Stress Response, Neurite Outgrowth and Prevent NF-κB Activation by Inhibiting the Proteasome Function

PRIYANKA DIKSHIT, ANAND GOSWAMI, AMIT MISHRA, MOU CHATTERJEE and NIHAR RANJAN JANA*

Cellular and Molecular Neuroscience Laboratory, National Brain Research Centre, Manesar, Gurgaon - 122 050, India. [email protected]

(Received 15 April 2005; Revised 19 July 2005; In final form 25 August 2005)

*Corresponding author. Tel. +91-124-2338922; FAX: +91-124-2338910; E-mail: [email protected] 1029 8428 print/ ISSN 1476-3524 online. © 2006 FP Graham Publishing Co., www.NeurotoxicityResearch.com

Neurotoxicity Research, 2006, VOL. 9(1). pp. 29-37

mechanism through which curcumin induces apopto-sis remains unknown. Curcumin has been shown to inhibit the activation of transcription factors NF-κB and AP-1 (Huang TS et al., 1991; Singh and Aggarwal, 1995), inhibit the activity of c-Jun N-terminal kinase (Chen and Tan, 1998), and protein tyrosine kinase (Aggarwal et al., 2003). Modulation of the activities of these factors might be linked with the initiation of the apoptotic signal. Earlier, we reported that curcumin-induced pro-grammed cell death is mediated by the altered function of UPS (Jana et al., 2004). However, the mechanism through which curcumin induces UPS malfunction is not clear. Here, we show for the first time that the cur-cumin-induced UPS malfunction is mediated through the direct inhibition of proteasome activity. Finally, we show that this impaired proteasomal function is responsible for induction of the stress response and down-regulation of NF-κB activation.

MATERIALS AND METHODS

MaterialsCurcumin, lactacystin, MG132, TNF-α, purified 20S proteasome, proteasome substrates and all cell culture reagents were obtained from Sigma. LipofactAMINE 2000 was purchased from Invitrogen. Rabbit poly-clonal anti-IκB-α, anti-p-IκB-α, anti-NF-κB p65 subunit, anti-ubiquitin and mouse monoclonal anti-GADD153 and anti-β-tubulin were from Santa Cruz Biotechnology. Mouse monoclonal anti-GFP was from Roche diagnostics and mouse monoclonal anti-Hsp70 and rabbit polyclonal anti-Hsp40 and anti-Grp78 were from Stressgen. Goat anti-rabbit IgG-rhodamine was purchased from Vector laboratories and HRP-con-jugated anti-mouse and anti-rabbit IgG were from Amersham Life Science. The plasmids IκB-α-EGFP and NF-κB-luciferase (contains multiple copies of NF-κB response elements) were obtained from Clontech. The plasmid PRL-SV40 and the dual luciferase report-er assay system were obtained from Promega. Mouse Neuro 2a was obtained from National Centre for Cell Science, India.

Cell Culture, Transfection, Treatments and Reporter Gene AssayNeuro 2a cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inac-tivated fetal bovine serum and antibiotics penicil-lin/streptomycin. For transfection experiments, cells were plated into 6-well tissue cultured plates at a sub confluent density. Twenty-four hours later, cells were transiently transfected with various plasmids

using LipofectAMINE 2000 reagent according to the manufacturer's instruction. Transfection efficiency was about 80-90%. For reporter gene assay, cells were tran-siently transfected with NF-κB-luciferase and PRL-SV40 plasmids together, 24 h of post-transfection, cells were treated with different doses of curcumin and then processed for luciferase assay. Luciferase activity was measured using a dual luciferase reporter assay sys-tem as per the manufacturer instructions. PRL-SV40 was used for co-transfection to normalize the data and transfected at very low concentration (150-fold lower than NF-κB luciferase plasmid). Data were represented as relative luciferase activity (the ratio of Firefly to Renilla values).

Immunofluorescence StainingCells grown on two-well chamber slides were treated with curcumin and lactacystin. Eight hours after treat-ment, cells were washed twice with PBS, fixed with 4% paraformaldehyde in PBS for 20 min, permeabi-lized with 0.5% Triton X-100 in PBS for 5 min, washed extensively, then blocked with 5% nonfat dried milk in TBST for 1 h. Primary antibody (anti-GADD153, anti-IκB-α and anti-NF-κB) incubation was carried out overnight at 4°C. After several washings with TBST, cells were incubated with rhodamine-conjugated sec-ondary antibody for 2 h, washed several times, and visualized in the fluorescence microscope. The digital images were assembled using Adobe Photoshop®.

Assay of Proteasome ActivityNeuro 2a cells were plated in a 6-well tissue culture plate and on the following day, cells were treated with varying doses of curcumin for 8 h. Cells were then isolated and suspended in 100 µl of proteasome assay buffer (10 mM Tris pH 7.4, 1 mM EDTA, 5 mM ATP, 5 mM DTT and 20% (v/v) glycerol), lysed by sonication and then centrifuged at 15,000g for 15 min at 4°C. The supernatant (25 µg) was incubated in the proteasome activity assay buffer (50 mM Tris pH 7.4, 0.5 mM EDTA and 50 µM of each proteasome substrate) for different time periods to obtain linearity of the reaction. The substrates Suc-Leu-Leu-val-Tyr-MCA and Z-Leu-Leu-Glu-MCA were used to determine chymotrypsin and post-glutamyl peptidyl hydrolytic-like activity respectively. To evaluate the direct effect of curcumin on proteasome's protease activity, pure 20S proteasome (250 ng/reaction) was used instead of cell supernatant in the protease activity assay buffer. Protease activities at a particular time point (30 min) within the linear range were used to calculate the data. The fluorescence intensity was measured at 355 nm excitation and 460 nm emissions using a Zemini fluorescence micro plate

P. DIKSHIT et al.30

reader. Statistical analysis was performed using the Student's t-test and P < 0.05 was considered to indicate statistical significance.

Cycloheximide-Chase ExperimentNeuro 2a cells were plated in a 6-well tissue culture plate and on the following day, cells were chased with 15 µg/ml of cycloheximide for different time periods in the presence or absence of curcumin. Cells collected at each time point were then processed for immunoblot-ting by anti-IκB-α and anti-NF-κB.

Co-Immunoprecipitation and Immunoblotting ExperimentTwenty-four hours after platting, cells were transiently transfected with IκB-α-EGFP plasmids, and then were treated with curcumin or lactacystin for 4 h. Cells were collected, lysed in RIPA buffer and processed for immunoprecipitation using anti-GFP as described earlier (Jana et al., 2004). In some experiments, cells were treated with different doses of curcumin and lac-tacystin for 8 h. Cells were then washed with cold PBS, scraped, pelleted by centrifugation, and lysed with SDS sample buffer. The samples were then separated through SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were successively incubated in blocking buffer (5% skim milk in TBST [50 mM Tris; pH 7.5, 0.15 M NaCl, 0.05% Tween]), with primary antibody in TBST, and then with secondary antibody conjugated with HRP in TBST. Detection was carried out with enhanced che-miluminescence reagent. All primary antibodies were used in 1:1000 dilutions for immunoblotting.

RESULTS

Curcumin Inhibits Proteasome ActivityInitially we investigated the effect of curcumin on proteasome activity in the in vitro cell culture model. Mouse neuro 2a cells were treated with different doses of curcumin for 8 h. Cells were then collected and subjected to the chymotrypsin-like and post glutamyl peptidyl-like protease assay of the 20S proteasome. As shown in figure 1A, curcumin treatment dose-depen-detly decreased the proteasome's protease activity. Curcumin at a dose of 5 and 10 µM decreased protea-some activity to approximately 25% and 45%, respec-tively. Since curcumin inhibits proteasome activity in cell culture, we were further interested in the possible mechanism of proteasome inhibition. Therefore, we tested the direct effect of curcumin on proteasome activity. The purified 20S proteasome was incubated with varying doses of curcumin in the presence of

chymotrypsin-like and post glutamyl peptidyl-like pro-tease substrate. As shown in figure 1B, curcumin dose-dependently decreased the purified 20S proteasome's protease activity. Subsequently, we compared the direct inhibitory effect of curcumin on 20S proteasome's pro-tease activity with the known proteasome substrates

CURCUMIN DIRECTLY INHIBITS PROTEASOME FUNCTION 31

FIGURE 1 Proteasome activity is inhibited by curcumin. A) Curcumin inhibits proteasome activity in cell culture. Neuro 2a cells were treated with different doses of curcumin for 8 h. Cells were then collected and processed for proteasome's protease activ-ity assay as described in Materials and Methods. Results are means ± SD of two independent experiments, each performed in triplicate. *p <0.01 vs control. B) Direct inhibition of proteasome activity by curcumin. Purified 20S proteasome was incubated with different doses of curcumin in the presence of different protease substrates. Results are means ± SD of three independent experiments each performed in triplicate. *p <0.01 vs control. C) Comparison of the proteasome inhibitory effect of curcumin with the known protea-some inhibitors lactacystin and MG132. Purified 20S proteasome was incubated in the presence of different protease substrates with 5 µM curcumin, lactacystin and MG132. Results are means ± SD of two independent experiments each performed in triplicate. Con, control; Lact, lactacystin; Cur, curcumin.

lactacystin and MG132. Curcumin was approximately 4-fold and 2.5-fold less potent, respectively, in com-parison with lactacystin and MG132 (FIG.1C).

Curcumin Exposure Induced Stress ResponseSince the inhibition of proteasome function induces the stress response by blocking rapid degradation of abnormal cytosolic and endoplasmic reticulum (ER) associated proteins, we therefore tested whether curcumin, by causing the accumulation of abnormal proteins, might stimulate the expression of various

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FIGURE 2 Curcumin treatment increases the expression of Hsp70, Hsp40 and Grp78. A) Neuro 2a cells were treated with curcumin or lactacystin (Lact) for 8 h. Cells were then collected and processed for immunoblotting using antibody against Hsp70, Hsp40 and Grp78 and β-tubulin. B) Cells were transiently transfected with empty pcDNA, Hsp70 and Hsp40 plasmids. Media was replaced 24 h later and the cells were treated with curcumin (20 µM) for 8 h. Cell viability was determined by trypan blue dye exclusion method under the microscope. Values are means ± SD of three independent experiments. *p <0.01 vs pcDNA transfected experiments. **p <0.01 vs pcDNA transfected and curcumin treated experiments.

FIGURE 3 Curcumin exposure increases the expression of tran-scription factor GADD153. A) Neuro 2a cells were treated with curcumin or lactacystin (Lact) for 8 h. Cells were collected and subjected to immunoblotting using antibody against GADD153 and β-tubulin. B) Cells were treated in a similar way as described above. The cells were then processed for immunofluorescence staining using anti-GADD153 as described in the Materials and Methods.

FIGURE 4 Curcumin treatment induces neurite outgrowth. Neuro 2a cells were plated into a 60 mm tissue cultured plate at sub conflu-ent density. On the following day, cells were treated with 5 µM cur-cumin for different time periods. The cells were then observed under the microscope and photos were taken using a digital camera. A, Control; B,C,D are 1, 2 or 4 h of curcumin exposure, respectively.

cytosolic and ER chaperones. For this purpose, cells were treated with different doses of curcumin or lacta-cystin for 8 h, and then the cell lysates were processed for immunoblotting using antibodies against various cytosolic and ER chaperones. As shown in figure 2A, curcumin or lactacystin exposure dose-dependently increased the expression of Hsp70, Hsp40 and Grp78. Curcumin at a dose of 10 µM increased the levels of Hsp70 and Hsp40 to about 8 and 3 fold, respectively. The levels of Grp78 also increased approximately 1.5 fold upon 10 µM of curcumin exposure. This increased expression of various chaperones indicates that the cell is under stress and attempts to protect itself from the stress. Next, we assessed the effect of over-expression of Hsp70 and Hsp40 chaperones on the protection of cell death induced by curcumin. Curcumin, at a dose 20 µM reduced cell viability to about 20%, and over-expression of both Hsp70 and Hsp40 significantly protected from curcumin-induced cell death (FIG. 2B). Because curcumin induced ER stress, as evident from the induction of Grp78, we further assessed the expression levels of GADD153. GADD153 is a C/EBP family transcription factor whose expression levels are increased in response to cellular stresses, especially by ER stress. As expected, curcumin or lactacystin exposure dramatically increased the expression of GADD153/CHOP (FIG. 3).

Treatment of Curcumin Induces Neurite OutgrowthThe impairment of proteasome function is also known to induce the neurite outgrowth. Since curcumin inhib-its proteasome function, we further tested its possible effect on neurite outgrowth. Neuro 2a cells were plated at sub confluent density and then treated with 5 µM curcumin for different time periods. As shown in fig-ure 4, curcumin exposure time-dependently induced neurite outgrowth. Approximately 80-90% of the cells showed long processes after 4 hours of curcumin expo-sure. In most cases, cells had more than two neurites and the neurites were branched. As a positive control, we treated neuro 2a cells with lactacystin. Lactacystin treatment also induced the neurite outgrowth, but in most cases, the cells appeared to be bipolar (having two neurites) and the neurites were not branched (data not shown). After longer exposure of either curcumin or lactacystin, the cells became round and then died through apoptosis.

Curcumin Exposure Prevents Degradation of IκB-αCurcumin is known to inhibit NF-κB-dependent tran-scriptional activation. But the mechanism through

which it does so was not known. Since curcumin inhibits proteasome function, and IκB-α is a substrate of proteasome, we hypothesized that probably slower degradation of IκB-α might be responsible for the down-regulation of NF-κB activation. Therefore, we tested the accumulation of IκB-α and its phosphory-lated form upon curcumin exposure. As expected, cur-cumin treatment increased the accumulation of IκB-α as well as its phosphorylated forms (FIG. 5). Curcumin had no effect on the levels of NF-κB p65 subunit.

CURCUMIN DIRECTLY INHIBITS PROTEASOME FUNCTION 33

FIGURE 5 Curcumin exposure causes increased accumulation of IκB-α and its phosphorylated forms. A) Neuro 2a cells were treated with different doses of curcumin for 8 h. Cells were then collected and processed for immunoblotting using antibody against IκB-α, p-IκB-α, NF-κB p65 subunit, ubiquitin and β-tubulin. B) Quantitation of the band intensities of the IκB-α, p-IκB-α and NF-κB p65 subunit blots (represented in A), collected from three independent experiments, was performed using NIH image analysis software. Values are means ± SD. Data were normalised against β-tubulin. C indicates control. p65 indicates NF-κB p65 subunit.

Curcumin exposure also increased the accumulation of ubiquitinated proteins (FIG. 5). We further confirm the extended half-life of IκB-α by the cycloheximide chase experiment. As shown in figure 6, curcumin treatment dramatically increased the half-life of IκB-α. The altered degradation of IκB-α upon curcumin expo-sure was also confirmed by transiently transfecting the IκB-α-EGFP plasmids into the mouse neuro2a cells. The cells were then exposed to curcumin and lactacys-tin for 4 hours and the collected cells were processed for either immunoblot analysis with GFP antibody or immunoprecipitation experiments using GFP antibody and sequential blot detection by ubiquitin and GFP antibody. As shown in figure 7, curcumin or lactacystin exposure increased the accumulation of IκB-α-EGFP and also their ubiquitinated derivatives. We also per-formed immunofluorescence staining of NF-κB p65 subunit before and after curcumin exposure (FIG. 8).

In normal cells, NF-κB p65 predominantly localised in the cytoplasm with diffuse staining in the nucleus. Exposure of curcumin prevents the nuclear transloca-tion and thereby increased the cytoplasmic levels of NF-κB p65.

Down-regulation of NF-κB-Dependent Transcriptional ActivitySince curcumin treatment increases the half-life and accumulation of IκB-α, and IκB-α prevents nuclear translocation of NF-κB by forming complex with it, it is expected that the treatment of curcumin will inhibit NF-κB activation. As shown in figure 9A and 9B, cur-cumin exposure dose-dependently inhibited the normal as well as TNF-α-induced NF-κB activation. Finally, we show that curcumin also prevents TNF-α-induced degradation of IκB-α (FIG. 9C)

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FIGURE 6 Curcumin treatment increases the half-life of IκB-α. Neuro 2a cells were plated into a 6-well tissue culture plate. Cells were treated 24 h later with cycloheximide (15 µg/ml) and chased in the presence or absence of curcumin for different time periods. Cells were then collected and processed for immunoblotting using anti-body against IκB-α, NF-κB p65 subunit. B) Quantitation of the band intensities of the blot shown in A using NIH image analysis software. Results are means ± SD of three independent experiments.

FIGURE 7 Curcumin treatment increases the accumulation of IκB-α-EGFP protein and their ubiquitinated derivatives. A) Neuro 2a cells were transiently transfected with IκB-α-EGFP plasmids. Cells were treated after 24 h of post-transfection with curcumin (Cur) or lactacystin (Lact) for 4 h and then processed for immu-noblotting with GFP and β-tubulin antibody. B) Cells were tran-siently transfected with IκB-α-EGFP plasmids and treated with 10 µM Cur or Lact, as above. The cell lysate were then processed for immunoprecipitation (IP) using GFP antibody. Blot was detected with ubiquitin (Ub) and GFP antibody.

DISCUSSION

Curcumin has long been used as herbal medicine and popular dietary spice in several Southeastern countries. Recent evidence suggests that curcumin has anti-inflammatory and anti-tumour activities because of its ability to down-regulate NF-κB activity and to induce apoptosis, respectively. However, the common mecha-nisms through which curcumin elicits such activities are not known. Here, we find that curcumin directly inhibits proteasome's protease activity and thereby inhibits the degradation of its substrate IκB-α. The inhibition of IκB-α degradation leads to down-regu-lation of NF-κB activation. We also demonstrate that inhibition of proteasome function by curcumin induces the stress response and neurite outgrowth. Earlier, we had reported that curcumin-induced apop-tosis is mediated through the impairment of UPS (Jana

CURCUMIN DIRECTLY INHIBITS PROTEASOME FUNCTION 35

FIGURE 8 Immunofluorescence staining of NF-κB p65 subunit before and after curcumin treatment. Neuro 2a cells were treated with 5 µM of curcumin for 8 h. Cells were then fixed and subjected to immunofluorescence staining using antibody against NF-κB p65 subunit, as described in the Materials and Methods.

FIGURE 9 Down-regulation of NF-κB activation by curcumin. A) Neuro 2a cells were transiently transfected with NF-κB luciferase and PRL-SV40 plasmids. Cells were treated 24 h after post-trans-fection with different doses of curcumin, and were then collected and processed for dual luciferase reporter gene assay. Results are means ± SD of three independent experiments each performed in triplicate. *p <0.01 vs control. B) Cells were transiently transfected and treated with curcumin as described in A. TNF-α was added 1 h before collecting the cells. Results are means ± SD of three independent experiments, each performed in triplicate. *p <0.01 vs control. **p <0.01 vs TNF-α treated experiment. C) Cells were treated as described in B, and then were collected and processed for immunoblotting using antibody against IκB-α and NF-κB p65 subunit and β-tubulin.

et al., 2004). However, we did not know how curcumin induced the malfunction of UPS. Our current findings lend support for a direct inhibitory role of curcumin on proteasome function. However, we cannot exclude the additional possibility of a role of oxidative stress or an inhibition of ubiquitin isopeptidase activity on curcumin-induced proteasomal malfunction. Because curcumin has been reported to induce oxidative stress (Bhaumik et al., 1999; Woo et al., 2003) and inhibits isopeptidase activity (Mullally and Fitzpatrick, 2002), both these phenomenon could conceivably disturb pro-teasome function (Okada et al., 1999; Ding and Keller, 2001). It has long been known that inhibition of proteasome function induces stress response, neurite outgrowth and apoptosis (Sadoul et al., 1996; Bush et al., 1997; Drexler, 1997; Lopes et al., 1997; Meriin et al., 1998; Drexler et al., 2000; Jana et al., 2001). Though the pre-cise link between proteasome inhibition and induction of apoptosis is unknown, it is believed that the altered degradation or the altered expression of proteins involved in these pathways will affect cell survival and promote apoptosis. In fact, curcumin, like other protea-some inhibitors, also has been shown to induce dual (caspase 8 and caspase 9) apoptotic signalling pathways (Mitsiades et al., 2002; Jana et al., 2004). Here, we observed that curcumin also induces the stress response and neurite outgrowth like other proteasome inhibitors. The induction of stress response could be due to the accumulation of misfolded proteins in cytosol and ER leading to the increased expression of cytosolic and ER-resident chaperones. We have also observed the increased expression of GADD153, a C/EBP family transcription factor that is induced in response to cel-lular stresses, especially by ER stress. GADD153 has been shown to be involved in the process of apoptosis associated with endoplasmic reticulum stress, although the mechanisms is still unclear (Wang et al., 1998). The curcumin-induced neurite outgrowth could be due to accumulation of ubiquitinated proteins or activation of stress kinases. Both processes are reportedly linked with proteasome inhibitor-induced neurite outgrowth (Giasson et al., 1999; Obin et al., 1999). Curcumin is known to inhibit NF-κB activation, although the underlying mechanism was not clear (Singh and Aggarwal, 1995). NF-κB is a ubiquitous transcription factor that regulates the transcription of many genes involved in immune and inflammatory responses as well as cell survival (Garg and Aggarwal, 2002). In normal cells, NF-κB is localized in the cyto-plasm, associated with the IκB family, rendering IκB inactive. In response to multiple activating signals, IκB

is phosphorylated and subsequently degraded by UPS. Here, we have shown clearly that curcumin-induced proteasomal malfunction inhibits degradation of IκB-α and thereby blocks the nuclear translocation and tran-scriptional activation of NF-κB. Altogether, our results indicate that the curcumin-induced stress response and down-regulation of NF-κB activation is mediated through the direct inhibition of proteasomal function. This provides a common link between curcumin-induced apoptosis and down-regu-lation of NF-κB activation. Acknowledgements

This work was supported by the Department of Biotechnology, Government of India. P.D. and A.M. were supported by a research fellowship from the Council of Scientific and Industrial Research, Government of India.

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