Thymoquinone (TQ), derived from the medicinal plant Nigella sativa, exhibits antiinflammatory and anticancer activities through mechanism(s) that is not fully understood. Because numerous effects modulated by TQ can be linked to interference with the nuclear factor-κB (NF-κB) signaling, we investigated in detail the effect of this quinone on NF-κB pathway. As examined by DNA binding, we found that TQ suppressed tumor necrosis factor–induced NF-κB activation in a dose- and time-dependent manner and inhibited NF-κB activation induced by various carcinogens and inflammatory stimuli. The suppression of NF-κB activation correlated with sequential inhibition of the activation of IκBα kinase, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-κB–dependent reporter gene expression. TQ specifically suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by DTT. However, TQ did not inhibit p65 binding to DNA when cells were transfected with the p65 plasmid containing cysteine residue 38 mutated to serine. TQ also down-regulated the expression of NF-κB–regulated antiapoptotic (IAP1, IAP2, XIAP Bcl-2, Bcl-xL, and survivin), proliferative (cyclin D1, cyclooxygenase-2, and c-Myc), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products. This led to potentiation of apoptosis induced by tumor necrosis factor and chemotherapeutic agents. Overall, our results indicate that the anticancer and antiinflammatory activities previously assigned to TQ may be mediated in part through the suppression of the NF-κB activation pathway, as shown here, and thus may have potential in treatment of myeloid leukemia and other cancers. (Mol Cancer Res 2008;6(6):1059–70)

Although folk medicine is generally regarded as safe, neither the active component nor their mechanism of action is clearly understood. The black cumin (Nigella sativa) is one such spice that has been used in the treatment of a variety of illnesses, including bronchial asthma, headache, dysentery, infections, obesity, back pain, hypertension, gastrointestinal problems, and eczema (1). Several components of black cumin have been identified, including thymoquione, thymol, thymohydroquinone, and dithymquinone (2, 3). Thymoquinone (TQ), the most abundant component of black seed oil, has been reported to exhibit antioxidant (4-6), antiinflammatory, and chemopreventive (7-9) effects. For instance, TQ has been shown to suppress the proliferation of various tumor cells, including colorectal carcinoma, breast adenocarcinoma, osteosarcoma, ovarian carcinoma, myeloblastic leukemia, and pancreatic carcinoma (7, 10-14), although it is minimally toxic to normal cells (15).

In animal models, TQ has been shown to suppress acetic acid–induced colitis in rats (16), inhibit tumor necrosis factor-α (TNF-α) production in murine septic peritonitis (17), and reduce carrageenan-induced paw edema in rats (18). TQ has also been reported to enhance antitumor activity of ifosfamide in Ehrlich ascites carcinoma–bearing mice (8), prevent cisplatin-induced nephrotoxicity in mice and rats (19), ameliorate benzo(a)pyrene-induced forestomach carcinogenesis (20), inhibit cyclooxygenase 2 (COX-2) expression and prostaglandin production in a mouse model of allergic airway inflammation (21), and protect against doxorubicin-induced cardiotoxicity in mice (22). How TQ manifests these activities is not fully understood, but it has been shown to down-regulate the expression of Bcl-xL (11), COX-2 (21), iNOS (23), 5-lipooxygenase (24), TNF (25), and cyclin D1 (26), all known to be regulated by nuclear factor-κB (NF-κB).

NF-κB is an ubiquitous transcription factor, consisting of p50, p65, and IκBα, that resides in the cytoplasm and is activated in response to various inflammatory stimuli, environmental pollutants, prooxidants, carcinogens, stress, and growth factors (27). On activation, NF-κB translocates from the cytoplasm to the nucleus, binds DNA, and causes gene transcription. Numerous kinases have been linked with activation of NF-κB, including IκBα kinase (IKK). Its activation has been shown to cause the expression of various gene products that regulate apoptosis, proliferation, chemoresistance, radioresistance, invasion, angiogenesis, metastasis, and inflammation (28, 29).

Because TQ has been shown to modulate cellular proliferation and inflammation, we postulated that it must mediate its effect through regulation of NF-κB pathway. Furthermore, various gene products known to be modulated by TQ are also regulated by NF-κB. Therefore, we investigated the effect of TQ in detail on NF-κB activation pathway and NF-κB–regulated cellular responses. We found that TQ suppressed the NF-κB activation pathway through modulation of the p65 subunit of NF-κB and inhibition of IKK; down-regulated the NF-κB–regulated gene products involved in cell survival, proliferation, and invasion; and potentiated apoptosis induced by TNF and chemotherapeutic agents.

We investigated the effect of TQ on NF-κB activation pathway induced by various carcinogens and inflammatory stimuli, NF-κB–regulated gene expression, and apoptosis induced by cytokines and chemotherapeutic agents. Most of our studies were done using human chronic myeloid leukemia cells (KBM-5) because these cells express both types of TNF receptors. Under the conditions that we used to examine the NF-κB pathway and NF-κB–regulated gene products, TQ had no effect on the viability of these cells (data not shown). The chemical structure of TQ is shown in (Fig. 1A).

TQ Suppresses NF-κB Activation in a Dose-Dependent and Time-Dependent Manner

We first determined the optimum dose and time of exposure to TQ required to suppress TNF-induced NF-κB activation. To determine the dose response, cells were pretreated with different doses of TQ for 4 hours and then stimulated with 0.1 nmol/L TNF for 30 minutes. As indicated by electrophoretic mobility shift assay (EMSA), TQ suppressed TNF-induced NF-κB activation in a dose-dependent manner (Fig. 1B).

We next investigated whether suppression of TNF-induced NF-κB by TQ is time dependent. For this, we incubated the cells with 25 μmol/L TQ for different times and then exposed the cells to 0.1 nmol/L TNF for 30 minutes. The EMSA results showed that TQ alone even for 6 hours did not activate NF-κB, but it abolished TNF-induced NF-κB activation almost maximally at 4 hours (Fig. 1C).

To determine the specificity of the NF-κB band, the nuclear extracts from TNF-activated cells were incubated with antibodies to the p50 (NF-κB) and the p65 (RelA) subunit of NF-κB; the resulting bands were shifted to higher molecular masses (Fig. 1D), 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 complete disappearance of the band, whereas mutated oligonucleotide had no effect on the DNA binding.

TQ Inhibits NF-κB Activation Induced by Carcinogens and Inflammatory Stimuli

Phorbol myristate acetate, okadaic acid, lipopolysaccharide, and cigarette smoke condensate are potent activators of NF-κB, but the pathways by which these agents activate NF-κB, however, differs (30-33). The effect of TQ on the activation of NF-κB by these agents was examined by EMSA. Pretreatment of cells with TQ completely suppressed the activation of NF-κB induced by all agents (Fig. 1E). These results clearly suggest that TQ acts at a step in the NF-κB activation pathway that must be common to all the agents.

TQ Inhibits TNF-Induced IκBα Degradation

The translocation of NF-κB to the nucleus is preceded by the proteolytic degradation of IκBα (27). In our study, TNF induced IκBα degradation in control cells within 5 minutes and reached a maximum in 15 minutes, but in TQ-pretreated cells, TNF had no effect on IκBα (Fig. 2A). These results indicate that TQ prevents IκBα degradation by acting at a step upstream to IκBα degradation.

TQ Inhibits TNF-Dependent IκBα Phosphorylation

To determine whether the inhibition of TNF-induced IκBα degradation was due to inhibition of IκBα phosphorylation, we used the proteasome inhibitor N-acetyl-leucylleucyl-norleucinal to block degradation of IκBα (34). Cells were pretreated with TQ, treated with N-acetyl-leucylleucyl-norleucinal for 30 minutes, exposed to TNF, and then examined for IκBα phosphorylation status by Western blot analysis. TQ completely suppressed IκBα phosphorylation induced by TNF in the presence of the proteasome inhibitor (Fig. 2B).

TQ Inhibits TNF-Induced IKK Activation

Because TQ inhibits the phosphorylation of IκBα, we tested its effect on TNF-induced IKK activation, which is required for TNF-induced phosphorylation of IκBα (34). Results from the immune complex kinase assay showed that TNF activated IKK as early as 5 minutes after treatment and that TQ strongly suppressed this activation (Fig. 2C). Neither TNF nor TQ had any direct effect on the expression of either IKK-α or IKK-β proteins.

TQ Inhibits TNF-Induced Nuclear Translocation of p65

Degradation of IκBα is known to cause the nuclear translocation of the p65 subunit of NF-κB (27). We examined whether TQ modulates TNF-induced nuclear translocation of p65. Western blot analysis showed that TNF induced nuclear translocation of p65 in a time-dependent manner in KBM-5 cells and that pretreatment with TQ inhibited it (Fig. 2D, middle).

TNF induces the phosphorylation of p65, which is required for its transcriptional activity (35). We examined whether TQ modulates TNF-induced phosphorylation of p65. Western blot analysis showed that TNF induced the phosphorylation of p65 within 15 min and that TQ strongly suppressed this phosphorylation (Fig. 2D, top).

TQ Directly Interferes with the Binding of NF-κB to the DNA

Results from our laboratory and others have shown that certain NF-κB inhibitors suppress NF-κB activation by directly interacting with NF-κB proteins and thus suppressing the binding to the DNA (36-38). We determined whether TQ mediates suppression of NF-κB activation through a similar mechanism. To determine this, we incubated nuclear extracts from TNF-treated cells with TQ. The EMSA results showed that TQ significantly inhibited NF-κB binding to the DNA (Fig. 3A). Thus, it is possible that TQ inhibits the binding of NF-κB to the DNA through modification of NF-κB proteins. We found that indeed coincubation of nuclear extracts with TQ in the presence of the DTT reversed the effect of TQ completely (Fig. 3B).

We also investigated whether DTT could reverse the NF-κB–suppressing effect of TQ in intact cells. The results in Fig. 3C showed that TQ induced the inhibition of TNF-induced NF-κB activation and that DTT completely reversed the effect of TQ. To determine whether TQ targets the p65 subunit of NF-κB, we overexpressed p65 by transfecting p65-containing plasmid into A293 cells. We then prepared nuclear extracts and treated them with TQ in the presence or absence of DTT. The recombinant p65 subunit bound to the DNA and TQ treatment resulted in suppression of binding. Like to endogenous p65, DTT significantly reversed the effect of TQ on ectopically expressed p65 (Fig. 3D).

It has been reported that the cysteine residue located at position 38 in p65 is highly susceptible to various agents (38-40). Whether Cys38 is a target for TQ was investigated. Therefore, we used p65 plasmid with Cys38 mutated to serine residue. A293 cells were transiently transfected with pcDNA3.1 or pcDNA expression vectors for wild-type p65 or p65C38S for 48 hours, prepared the nuclear extracts, treated with TQ for 30 minutes, and measured the DNA binding by EMSA. These results show that TQ modifies the DNA binding of wild-type p65 but not the mutated p65 (Fig. 3E). Thus, these results show that Cys38 in p65 is one of the targets of TQ.

TQ Represses TNF-Induced NF-κB–Dependent Reporter Gene Expression

Although we determined by EMSA that TQ inhibits NF-κB activation, DNA binding alone is not always associated with NF-κB–dependent gene transcription, suggesting that additional regulatory steps are involved (41). We examined whether TQ modulates TNF-induced transcription of the NF-κB reporter activity. The results showed that TNF induced the NF-κB reporter activity and that TQ inhibited TNF-induced NF-κB reporter activity in a dose-dependent manner (Fig. 4A).

We next investigated where TQ acts in the sequence of TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-β recruitment that characterizes TNF-induced NF-κB activation (42). In cells transfected with TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, IKK-β, and p65 plasmids, NF-κB–dependent secretory alkaline phosphatase (SEAP) expression was induced; TQ substantially suppressed NF-κB–dependent SEAP expression in all cases (Fig. 4B).

TQ Represses the Expression of TNF-Induced NF-κB–Dependent Antiapoptotic Gene Products

It is possible that potentiation of TNF-induced apoptosis is through suppression of NF-κB–regulated antiapoptotic gene products. NF-κB regulates the expression of the antiapoptotic proteins IAP1/IAP2 (43, 44), XIAP (45), Bcl-2 (46), Bcl-xL (47), and survivin (48). Whether TQ could modulate TNF-induced expression of these antiapoptotic gene products was investigated. The results of Western blot analysis showed that TNF induced expression of these antiapoptotic proteins in a time-dependent manner and that TQ suppressed it (Fig. 5A).

TQ Suppresses TNF-Induced NF-κB–Dependent Gene Products Involved in Cell Proliferation

Numerous gene products that mediate cellular proliferation, such as cyclin D1, COX-2, and c-Myc, have NF-κB–binding sites in their promoters (49-51). We investigated whether the expression of these gene products is modulated by TQ. Cells were pretreated with TQ for 4 hours and then treated with TNF for the indicated times. Western blot analysis indicated that TQ suppressed the expression of these proteins (Fig. 5B). The results provide further evidence of the role of TQ in blocking cellular proliferation through suppression of NF-κB–regulated gene products.

TQ Suppresses TNF-Induced NF-κB–Dependent Gene Products Involved in Invasion and Angiogenesis

The gene products that have been linked with invasion and angiogenesis [matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF)] are regulated by NF-κB (52, 53). We determined whether the expression of these gene products is modulated by TQ. Western blot analysis indicated that TNF induced these gene products and that TQ suppressed the expression (Fig. 5C). The results provide evidence for the role of TQ in blocking invasion and angiogenesis.

TQ Potentiates Apoptosis Induced by TNF and Chemotherapeutic Agents

NF-κB activation has been shown to suppress apoptosis induced by TNF and chemotherapeutic agents through the expression of gene products regulated by NF-κB (54). We investigated whether TQ modulates the cytotoxic effects of TNF, paclitaxel, and doxorubicin. The effect of TQ on TNF-induced and chemotherapeutic agent–induced apoptosis was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We found that TQ significantly enhanced the cytotoxic effects of TNF, paclitaxel, and doxorubicin (Fig. 6A). The Live/Dead assay, which measures intracellular esterase activity and plasma membrane integrity, further indicated that TQ up-regulates TNF-induced apoptosis from 8% to 68% (Fig. 6B1) and STI571-induced apoptosis from 18% to 76% (Fig. 6B2). The results of Annexin V staining, which examines early apoptosis, also showed that TNF-induced apoptosis was enhanced by incubation with TQ (Fig. 6C). When examined for caspase-mediated poly(ADP-ribose) polymerase (PARP) cleavage, we found that the TQ enhanced apoptosis induced by TNF (Fig. 6D). Together, these results support the conclusion that TQ potentiates the apoptotic effect of TNF and chemotherapeutic agents.

We investigated the molecular mechanism by which TQ mediates its antiproliferative, antiinflammatory, and chemopreventive effects. We found that TQ suppressed the NF-κB activation induced by various carcinogens and inflammatory agents and that the inhibition of NF-κB was due to the inhibition of IKK activation, leading to the suppression of IκBα phosphorylation and degradation. Suppression of NF-κB activation by TQ is due to inhibition of TNF-induced IKK activation, which led to inhibition of IκBα phosphorylation and degradation, suppression of p65 phosphorylation, and translocation to the nucleus. Apart from this, TQ also inhibited the binding of the p65 subunit of NF-κB to the DNA and reporter gene expression. TQ also down-regulated NF-κB–dependent gene products involved in antiapoptosis, proliferation, invasion, and angiogenesis. This down-regulation led to the potentiation of apoptosis induced by cytokines and chemotherapeutic agents (Fig. 7).

This is the first report to suggest that TQ could suppress NF-κB activation induced by a variety of inflammatory stimuli and carcinogens. Because NF-κB activation induced by diverse stimuli was inhibited, this suggests that TQ must act at a step in the NF-κB activation pathway common to all NF-κB inducers.

TQ has been shown to inhibit NF-κB activation in a rat model of brain and spinal cord experimental autoimmune encephalomyelitis (55) and induced by lipopolysaccharide in mast cells (56) and advanced glycation end products in proximal tubular epithelial cells (57); however, none of these reports described the mechanism by which TQ suppresses NF-κB activation. We investigated in detail how TQ inhibits the NF-κB activation pathway. We found that TQ acts at two different steps in the NF-κB signaling pathway: firstly, through its direct interaction with the p65 subunit of NF-κB and, secondly, through its ability to suppress TNF-induced IKK activation. The observation that TQ could inhibit the binding of NF-κB to the DNA suggest that it is modifying the NF-κB protein, because the binding of recombinant p65 subunit of NF-κB, which has the transactivation domain, was suppressed, suggesting that TQ directly targets the p65 subunit. These results are consistent with our findings that TQ also suppressed the p65-induced NF-κB reporter activity. Because the effects of TQ could be reversed by reducing agent, it is possible that this agent modifies a cysteine residue in p65. These results are consistent with those previously reported from our laboratory and others with caffeic acid phenethyl ester (37), plumbagin (38), and sesquiterpene lactone parthenolide (40). A Cys38 residue has been identified in p65 subunits of NF-κB that is crucial for DNA binding (40). Our results indicate that when this Cys38 was replaced by serine in p65, TQ failed to inhibit the DNA binding ability of p65. Thus, our results indicate that TQ exerts its inhibitory effect by modifying this specific cysteine residue.

We found that, in addition to its effects on p65, TQ also inhibited TNF-induced IKK activation, which leads to inhibition of IκBα phosphorylation and degradation. How TQ suppresses IKK activation was also investigated. We found that TQ is not a direct inhibitor of IKK (data not shown). Several kinases, such as MAPK kinase kinase 1 (58), MAPK kinase kinase 3 (59), PKC (60); glycogen synthase kinase-3β (61), TAK1 (62), PDK1 (63), and Akt (64), have all been reported to function upstream of IKK. Recent studies, however, indicate that TAK1 plays a major role in TNF-induced NF-κB activation through its interaction with TAB1 and TAB2. For example, TAK1 can bind and activate IKK-β, leading to NF-κB activation (65). Indeed, our study showed for the first time that TAK1-induced NF-κB activation is inhibited by TQ.

TQ has been shown to suppress carcinogenesis (15, 66). Our results show that TQ inhibits the expression of COX-2, MMP-9, cyclin D1, VEGF, and c-Myc, all regulated by NF-κB. These results are in agreement with previous reports that TQ can suppress COX-2 expression both in vitro and in a mouse model of allergic airway inflammation (21, 67). NF-κB activation has been shown to mediate the suppression of apoptosis through the expression of several antiapoptotic gene products. We found that TQ down-regulated the expression of cIAP1/2, XIAP, Bcl-2, Bcl-xL, and survivin. Gali-Muhtasib et al. (7) indeed showed that TQ treatment resulted in a marked inhibition of the antiapoptotic Bcl-2 protein in HCT-116 cells.

We also found that TQ potentiated the apoptotic effects of cytokines and chemotherapeutic agents by down-regulating the NF-κB–dependent apoptosis gene products, including cIAP1/cIAP2, XIAP, Bcl-2, Bcl-xL, and survivin. It is possible that suppression of various other tumor cell lines previously reported (7, 10-14) is also through antiapoptotic and proliferative gene products, as described here. In animal models, TQ has been shown to suppress acetic acid–induced colitis in rats (16), inhibit TNF production in murine septic peritonitis (17), and reduce carrageenan-induced paw edema in rats (18). It is quite likely that several of these effects of TQ are mediated through the suppression of NF-κB activation.

Overall, our results show that TQ clearly inhibits NF-κB activation, which makes it a potentially effective suppressor of inflammation, tumor cell survival, proliferation, invasion, and angiogenesis. Further studies on TQ may provide important leads for potential treatment applications in cancer and other diseases.

Reagents

A 50 mmol/L solution each of TQ (molecular weight, 164.2; Sigma-Aldrich) was prepared in 100% DMSO, stored as small aliquots at −20°C, and then diluted as needed in cell culture medium. Bacteria-derived recombinant human TNF, purified to homogeneity with a specific activity of 5 × 107 units/mg, was kindly provided by Genentech. Cigarette smoke condensate, prepared as previously described (30), was kindly supplied by Dr. C. Gary Gairola (University of Kentucky). Paclitaxel, doxorubicin, phorbol 12-myristate 13-acetate, okadaic acid, lipopolysaacharide, DTT, and β-actin antibody were obtained from Sigma-Aldrich. N-acetyl-leucylleucyl-norleucinal was purchased from EMD Biosciences, Inc. Antibodies against p65, p50, IκBα, cyclin D1, c-Myc, MMP-9, PARP, IAP1, IAP2, Bcl-2, and Bcl-xL and the Annexin V staining kit were obtained from Santa Cruz Biotechnology. Anti–COX-2 and anti-XIAP antibodies were obtained from BD Biosciences. Phosphorylated-specific anti-IκBα (Ser32/36) and phosphorylated-specific anti-p65 (Ser536) antibodies were purchased from Cell Signaling. Anti–IKK-α and anti–IKK-β antibodies were kindly provided by Imgenex. Anti-VEGF was purchased from NeoMarkers. Survivin antibody was purchased from R&D System. STI571 (an active chemical component of imatinib mesylate) was kindly provided by Dr. Nicholas J. Donato, Department of Experimental Therapeutics, University of Texas M. D. Anderson Cancer Center.

Cell Lines

Human myeloid KBM-5 cells and human embryonic kidney A293 cells were obtained from American Type Culture Collection. KBM-5 cells were cultured in Iscove's modified Dulbecco's medium supplemented with 15% fetal bovine serum, and A293 cells were cultured in DMEM supplemented with 10% fetal bovine serum. All media were also supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin.

EMSA

To determine NF-κB activation by TNF, we did EMSA essentially as described previously (68). Briefly, nuclear extracts prepared from TNF-treated cells (2 × 106/mL) were incubated with 32P-end–labeled 45-mer double-stranded oligonucleotide (15 μg of protein with 16 fmol of DNA) from the HIV long terminal repeat, 5′-TTGTTACAA GGGACTTTC CGCTG GGGACTTTC CAGGGAGGCGTGG-3′ (boldface indicates NF-κB binding sites), for 30 min at 37°C. The DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5′-TTGTTACAA CTCACTTTC CGCTG CTCACTTTC CAGGGAGGCGTGG-3′, was used to examine binding specificity of NF-κB to the DNA. The binding specificity was also examined by competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with antibodies against either the p50 or the p65 subunit of NF-κB for 30 min at 37°C before the complex was analyzed by EMSA. Preimmune serum was included as a negative control. The dried gels were visualized with a Storm820, and radioactive bands were quantified using Imagequant software (Amersham Pharmacia Biotechnology).

Western Blot Analysis

To determine the effect of TQ on TNF-dependent IκBα phosphorylation and IκBα degradation, p65 phosphorylation, and nuclear translocation, we prepared cytoplasmic and nuclear extracts as previously described (69) and probed them with specific antibodies against IκBα, phosphorylated IκBα, phosphorylated p65, and p65. To determine the expression of cyclin D1, COX-2, MMP-9, IAP1, IAP2, Bcl-2, Bcl-XL, VEGF, and survivin whole-cell extracts from TNF-treated and TQ plus TNF–treated cells were prepared, and 30 μg of protein were resolved on SDS-PAGE and probed with specific antibodies according to the manufacturer's recommended protocol. The blots were washed, exposed to horseradish peroxidase–conjugated secondary antibodies for 1 h, and finally detected by enhanced chemiluminescence reagent (Amersham Pharmacia Biotechnology). The bands were quantified with a Personal Densitometer Scan v1.30 using Imagequant software version 3.3 (Molecular Dynamics).

IKK Assay

To determine the effect of TQ on TNF-induced IKK activation, we analyzed IKK essentially, as described previously (70). Briefly, the IKK complex from whole-cell extracts was precipitated with IKK-α and IKK-β and treated with protein A/G-agarose beads (Pierce Chemical). After 2 h, the beads were washed with lysis buffer and resuspended in a kinase assay mixture containing 50 mmol/L HEPES (pH 7.4), 20 mmol/L MgCl2, 2 mmol/L DTT, 20 μCi [γ-32P]ATP, 10 μmol/L unlabeled ATP, and 2 μg of substrate glutathione S-transferase–IκBα (amino acids 1-54) and incubated 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 a Storm 820. 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 antibodies.

NF-κB–Dependent Reporter Gene Transcription

To determine the effect of TQ on TNF-induced, TNFR-induced, TRADD-induced, TRAF2-induced, NIK-induced, TAK1/TAB1-induced, IKK-β–induced, and p65-induced NF-κB–dependent reporter gene transcription, we did the SEAP assay as previously described (71). Briefly, A293 cells (5 × 105 per well) were plated in six-well plates and transiently transfected by the calcium phosphate method with pNF-B-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 TQ and stimulated them with 1 nmol/L 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 μg of the control plasmid pCMV-FLAG1 for 24 h, treated with TQ, 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).

Plasmids

pcDNA3.1 and pcDNA expression vectors for mouse p65 and mouse p65C38S were kindly provided by Dr. T.D. Gilmore from Boston University.

Cytotoxicity Assay

Cytotoxicity was assayed by the modified tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, as described previously (72).

Live/Dead Assay

To measure apoptosis, we used the Live/Dead cell viability assay (Molecular Probes), which determines intracellular esterase activity and plasma membrane integrity (73). 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 5 μmol/L TQ for 4 h and treated with 1 nmol/L TNF/5 μmol/L STI-571 up to 16 h at 37°C. Cells were stained with the Live/Dead reagent (5 μmol/L ethidium homodimer and 5 μmol/L calcein-AM) and incubated at 37 °C for 30 min. Cells were analyzed under a fluorescence microscope (Labophot-2; Nikon).

PARP Cleavage Assay

To detect cleavage of PARP, we prepared whole-cell extracts by subjecting TQ-treated cells to lysis in lysis buffer [20 mmol/L Tris (pH 7.4), 250 mmol/L NaCl, 2 mmol/L EDTA (pH 8.0), 0.1% NP40, 0.01 μg/mL aprotinin, 0.005 μg/mL leupeptin, 0.4 mmol/L phenylmethylsulfonylfluoride, and 4 mmol/L sodium orthovanadate]. Lysates were spun at 14,000 rpm for 10 min to remove insoluble material, resolved by 10% SDS-PAGE, and probed with PARP antibody.

Annexin V Assay

Annexin V assay was done as described previously (72). An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phospholipid phosphatidylserine from the cytoplasmic interface of membrane to the extracellular surface. This loss of membrane asymmetry can be detected by using the binding properties of Annexin V. To identify apoptosis, we used an Annexin V antibody, which was conjugated with the FITC fluorescence dye. Briefly, 5 × 105 cells were pretreated with TQ, treated with TNF for 16 h at 37°C, and subjected to Annexin V staining. The cells were washed in PBS, resuspended in 100 μL of binding buffer containing a FITC-conjugated anti–Annexin V antibody, and then analyzed with a flow cytometer (FACSCalibur, BD).

No potential conflicts of interest were disclosed.

Grant support: Clayton Foundation for Research (B.B. Aggarwal) and Core grant 5P30CA016672-32 for flow cytometric analysis.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Dr. Aggarwal is a Ransom Horne, Jr., Professor of Cancer Research.

We thank Michael Worley for carefully editing the manuscript and providing valuable comments.

1
Salem ML. Immunomodulatory and therapeutic properties of the Nigella sativa L. seed.
Int Immunopharmacol
2005
;
5
:
1749
–70.
2
Morikawa T, Xu F, Kashima Y, Matsuda H, Ninomiya K, Yoshikawa M. Novel dolabellane-type diterpene alkaloids with lipid metabolism promoting activities from the seeds of Nigella sativa.
Org Lett
2004
;
6
:
869
–72.
3
Morikawa T, Xu F, Ninomiya K, Matsuda H, Yoshikawa M. Nigellamines A3, A4, A5, and C, new dolabellane-type diterpene alkaloids, with lipid metabolism-promoting activities from the Egyptian medicinal food black cumin.
Chem Pharm Bull Tokyo
2004
;
52
:
494
–7.
4
Badary OA, Taha RA, Gamal el-Din AM, Abdel-Wahab MH. Thymoquinone is a potent superoxide anion scavenger.
Drug Chem Toxicol
2003
;
26
:
87
–98.
5
Badary OA, Abd-Ellah MF, El-Mahdy MA, Salama SA, Hamada FM. Anticlastogenic activity of thymoquinone against benzo(a)pyrene in mice.
Food Chem Toxicol
2007
;
45
:
88
–92.
6
Mansour MA, Nagi MN, El-Khatib AS, Al-Bekairi AM. Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action.
Cell Biochem Funct
2002
;
20
:
143
–51.
7
Gali-Muhtasib H, Diab-Assaf M, Boltze C, et al. Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism.
Int J Oncol
2004
;
25
:
857
–66.
8
Badary OA. Thymoquinone attenuates ifosfamide-induced Fanconi syndrome in rats and enhances its antitumor activity in mice.
J Ethnopharmacol
1999
;
67
:
135
–42.
9
Badary OA, Gamal El-Din AM. Inhibitory effects of thymoquinone against 20-methylcholanthrene-induced fibrosarcoma tumorigenesis.
Cancer Detect Prev
2001
;
25
:
362
–8.
10
Gali-Muhtasib H, Roessner A, Schneider-Stock R. Thymoquinone: a promising anti-cancer drug from natural sources.
Int J Biochem Cell Biol
2006
;
38
:
1249
–53.
11
Roepke M, Diestel A, Bajbouj K, et al. Lack of p53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells. Cancer Biol Ther 2007;
6
:
160
–9.
12
Shoieb AM, Elgayyar M, Dudrick PS, Bell JL, Tithof PK. In vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone.
Int J Oncol
2003
;
22
:
107
–13.
13
El-Mahdy MA, Zhu Q, Wang QE, Wani G, Wani AA. Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells.
Int J Cancer
2005
;
117
:
409
–17.
14
Tan M, Norwood A, May M, Tucci M, Benghuzzi H. Effects of (-)epigallocatechin gallate and thymoquinone on proliferation of a PANC-1 cell line in culture.
Biomed Sci Instrum
2006
;
42
:
363
–71.
15
Worthen DR, Ghosheh OA, Crooks PA. The in vitro anti-tumor activity of some crude and purified components of blackseed, Nigella sativa L.
Anticancer Res
1998
;
18
:
1527
–32.
16
Mahgoub AA. Thymoquinone protects against experimental colitis in rats.
Toxicol Lett
2003
;
143
:
133
–43.
17
Haq A, Lobo PI, Al-Tufail M, Rama NR, Al-Sedairy ST. Immunomodulatory effect of Nigella sativa proteins fractionated by ion exchange chromatography.
Int J Immunopharmacol
1999
;
21
:
283
–95.
18
Hajhashemi V, Ghannadi A, Jafarabadi H. Black cumin seed essential oil, as a potent analgesic and antiinflammatory drug.
Phytother Res
2004
;
18
:
195
–9.
19
Badary OA, Nagi MN, al-Shabanah OA, al-Sawaf HA, al-Sohaibani MO, al-Bekairi AM. Thymoquinone ameliorates the nephrotoxicity induced by cisplatin in rodents and potentiates its antitumor activity.
Can J Physiol Pharmacol
1997
;
75
:
1356
–61.
20
Badary OA, Al-Shabanah OA, Nagi MN, Al-Rikabi AC, Elmazar MM. Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by thymoquinone.
Eur J Cancer Prev
1999
;
8
:
435
–40.
21
El Mezayen R, El Gazzar M, Nicolls MR, Marecki JC, Dreskin SC, Nomiyama H. Effect of thymoquinone on cyclooxygenase expression and prostaglandin production in a mouse model of allergic airway inflammation.
Immunol Lett
2006
;
106
:
72
–81.
22
Nagi MN, Mansour MA. Protective effect of thymoquinone against doxorubicin-induced cardiotoxicity in rats: a possible mechanism of protection.
Pharmacol Res
2000
;
41
:
283
–9.
23
El-Mahmoudy A, Matsuyama H, Borgan MA, et al. Thymoquinone suppresses expression of inducible nitric oxide synthase in rat macrophages.
Int Immunopharmacol
2002
;
2
:
1603
–11.
24
El-Dakhakhny M, Madi NJ, Lembert N, Ammon HP. Nigella sativa oil, nigellone and derived thymoquinone inhibit synthesis of 5-lipoxygenase products in polymorphonuclear leukocytes from rats.
J Ethnopharmacol
2002
;
81
:
161
–4.
25
El-Mahmoudy A, Shimizu Y, Shiina T, Matsuyama H, Nikami H, Takewaki T. Macrophage-derived cytokine and nitric oxide profiles in type I, type II. diabetes mellitus: effect of thymoquinone.
Acta Diabetol
2005
;
42
:
23
–30.
26
Gali-Muhtasib HU, Abou Kheir WG, Kheir LA, Darwiche N, Crooks PA. Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes.
Anticancer Drugs
2004
;
15
:
389
–99.
27
Aggarwal BB. Nuclear factor-κB: the enemy within.
Cancer Cell
2004
;
6
:
203
–8.
28
Shishodia S, Aggarwal BB. Nuclear factor-κB activation mediates cellular transformation, proliferation, invasion angiogenesis and metastasis of cancer.
Cancer Treat Res
2004
;
119
:
139
–73.
29
Ahn KS, Aggarwal BB. Transcription factor NF-{κ}B: a sensor for smoke and stress signals.
Ann N Y Acad Sci
2005
;
1056
:
218
–33.
30
Anto RJ, Mukhopadhyay A, Shishodia S, Gairola CG, Aggarwal BB. Cigarette smoke condensate activates nuclear transcription factor-κB through phosphorylation and degradation of IκB(α): correlation with induction of cyclooxygenase-2.
Carcinogenesis
2002
;
23
:
1511
–8.
31
Nelsen B, Hellman L, Sen R. The NF-κB-binding site mediates phorbol ester-inducible transcription in nonlymphoid cells.
Mol Cell Biol
1988
;
8
:
3526
–31.
32
Thevenin C, Kim SJ, Rieckmann P, et al. Induction of nuclear factor-κB and the human immunodeficiency virus long terminal repeat by okadaic acid, a specific inhibitor of phosphatases 1 and 2A.
New Biol
1990
;
2
:
793
–800.
33
Sen R, Baltimore D. Inducibility of κ immunoglobulin enhancer-binding protein Nf-κB by a posttranslational mechanism.
Cell
1986
;
47
:
921
–8.
34
Ghosh S, May MJ, Kopp EB. NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses.
Annu Rev Immunol
1998
;
16
:
225
–60.
35
Zhong H, Voll RE, Ghosh S. Phosphorylation of NF-κB p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300.
Mol Cell
1998
;
1
:
661
–71.
36
Mahon TM, O'Neill LA. Studies into the effect of the tyrosine kinase inhibitor herbimycin A on NF-κB activation in T lymphocytes. Evidence for covalent modification of the p50 subunit.
J Biol Chem
1995
;
270
:
28557
–64.
37
Natarajan K, Singh S, Burke TR, Jr., Grunberger D, Aggarwal BB. Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-κB.
Proc Natl Acad Sci U S A
1996
;
93
:
9090
–5.
38
Sandur SK, Ichikawa H, Sethi G, Ahn KS, Aggarwal BB. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) suppresses NF-κB activation and NF-κB-regulated gene products through modulation of p65 and IκBα kinase activation, leading to potentiation of apoptosis induced by cytokine and chemotherapeutic agents.
J Biol Chem
2006
;
281
:
17023
–33.
39
Liang MC, Bardhan S, Pace EA, et al. Inhibition of transcription factor NF-κB signaling proteins IKKβ and p65 through specific cysteine residues by epoxyquinone A monomer: correlation with its anti-cancer cell growth activity.
Biochem Pharmacol
2006
;
71
:
634
–45.
40
Garcia-Pineres AJ, Castro V, Mora G, et al. Cysteine 38 in p65/NF-κB plays a crucial role in DNA binding inhibition by sesquiterpene lactones.
J Biol Chem
2001
;
276
:
39713
–20.
41
Campbell KJ, Rocha S, Perkins ND. Active repression of antiapoptotic gene expression by RelA(p65) NF-κB.
Mol Cell
2004
;
13
:
853
–65.
42
Simeonidis S, Stauber D, Chen G, Hendrickson WA, Thanos D. Mechanisms by which IκB proteins control NF-κB activity.
Proc Natl Acad Sci U S A
1999
;
96
:
49
–54.
43
You M, Ku PT, Hrdlickova R, Bose HR, Jr. ch-IAP1, a member of the inhibitor-of-apoptosis protein family, is a mediator of the antiapoptotic activity of the v-Rel oncoprotein.
Mol Cell Biol
1997
;
17
:
7328
–41.
44
Chu ZL, McKinsey TA, Liu L, Gentry JJ, Malim MH, Ballard DW. Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-κB control.
Proc Natl Acad Sci U S A
1997
;
94
:
10057
–62.
45
Stehlik C, de Martin R, Kumabashiri I, Schmid JA, Binder BR, Lipp J. Nuclear factor (NF)-κB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor α-induced apoptosis.
J Exp Med
1998
;
188
:
211
–6.
46
Catz SD, Johnson JL. Transcriptional regulation of bcl-2 by nuclear factor κB and its significance in prostate cancer.
Oncogene
2001
;
20
:
7342
–51.
47
Tamatani M, Che YH, Matsuzaki H, et al. Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NFκB activation in primary hippocampal neurons.
J Biol Chem
1999
;
274
:
8531
–8.
48
Zhu L, Fukuda S, Cordis G, Das DK, Maulik N. Anti-apoptotic protein survivin plays a significant role in tubular morphogenesis of human coronary arteriolar endothelial cells by hypoxic preconditioning.
FEBS Lett
2001
;
508
:
369
–74.
49
Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin AS, Jr. NF-κB controls cell growth and differentiation through transcriptional regulation of cyclin D1.
Mol Cell Biol
1999
;
19
:
5785
–99.
50
Yamamoto K, Arakawa T, Ueda N, Yamamoto S. Transcriptional roles of nuclear factor κB and nuclear factor-interleukin-6 in the tumor necrosis factor α-dependent induction of cyclooxygenase-2 in MC3T3–1 cells.
J Biol Chem
1995
;
270
:
31315
–20.
51
Duyao MP, Kessler DJ, Spicer DB, et al. Transactivation of the c-myc promoter by human T cell leukemia virus type 1 tax is mediated by NF κB.
J Biol Chem
1992
;
267
:
16288
–91.
52
Esteve PO, Chicoine E, Robledo O, et al. Protein kinase C-ζ regulates transcription of the matrix metalloproteinase-9 gene induced by IL-1 and TNF-α in glioma cells via NF-κB.
J Biol Chem
2002
;
277
:
35150
–5.
53
Shibata A, Nagaya T, Imai T, Funahashi H, Nakao A, Seo H. Inhibition of NF-κB activity decreases the VEGF mRNA expression in MDA-MB-231 breast cancer cells.
Breast Cancer Res Treat
2002
;
73
:
237
–43.
54
Giri DK, Aggarwal BB. Constitutive activation of NF-κB causes resistance to apoptosis in human cutaneous T cell lymphoma HuT-78 cells. Autocrine role of tumor necrosis factor and reactive oxygen intermediates.
J Biol Chem
1998
;
273
:
14008
–14.
55
Mohamed A, Afridi DM, Garani O, Tucci M. Thymoquinone inhibits the activation of NF-κB in the brain and spinal cord of experimental autoimmune encephalomyelitis.
Biomed Sci Instrum
2005
;
41
:
388
–93.
56
El Gazzar MA, El Mezayen R, Nicolls MR, Dreskin SC. Thymoquinone attenuates proinflammatory responses in lipopolysaccharide-activated mast cells by modulating NF-κB nuclear transactivation.
Biochim Biophys Acta
2007
;
1770
:
556
–64.
57
Sayed AA, Morcos M. Thymoquinone decreases AGE-induced NF-κB activation in proximal tubular epithelial cells.
Phytother Res
2007
;
21
:
898
–9.
58
Lee FS, Peters RT, Dang LC, Maniatis T. MEKK1 activates both IκB kinase α and IκB kinase β.
Proc Natl Acad Sci U S A
1998
;
95
:
9319
–24.
59
Yang J, Lin Y, Guo Z, et al. The essential role of MEKK3 in TNF-induced NF-κB activation.
Nat Immunol
2001
;
2
:
620
–4.
60
Lallena MJ, Diaz-Meco MT, Bren G, Paya CV, Moscat J. Activation of IκB kinase β by protein kinase C isoforms.
Mol Cell Biol
1999
;
19
:
2180
–8.
61
Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, Woodgett JR. Requirement for glycogen synthase kinase-3β in cell survival and NF-κB activation.
Nature
2000
;
406
:
86
–90.
62
Takaesu G, Surabhi RM, Park KJ, Ninomiya-Tsuji J, Matsumoto K, Gaynor RB. TAK1 is critical for IκB kinase-mediated activation of the NF-κB pathway.
J Mol Biol
2003
;
326
:
105
–15.
63
Tanaka H, Fujita N, Tsuruo T. 3-Phosphoinositide-dependent protein kinase-1-mediated IκB kinase β (IkkB) phosphorylation activates NF-κB signaling.
J Biol Chem
2005
;
280
:
40965
–73.
64
Gustin JA, Maehama T, Dixon JE, Donner DB. The PTEN tumor suppressor protein inhibits tumor necrosis factor-induced nuclear factor κB activity.
J Biol Chem
2001
;
276
:
27740
–4.
65
Sakurai H, Miyoshi H, Toriumi W, Sugita T. Functional interactions of transforming growth factor β-activated kinase 1 with IκB kinases to stimulate NF-κB activation.
J Biol Chem
1999
;
274
:
10641
–8.
66
Ivankovic S, Stojkovic R, Jukic M, Milos M, Milos M, Jurin M. The antitumor activity of thymoquinone and thymohydroquinone in vitro and in vivo.
Exp Oncol
2006
;
28
:
220
–4.
67
Marsik P, Kokoska L, Landa P, Nepovim A, Soudek P, Vanek T. In vitro inhibitory effects of thymol and quinones of Nigella sativa seeds on cyclooxygenase-1- and -2-catalyzed prostaglandin E2 biosyntheses.
Planta Med
2005
;
71
:
739
–42.
68
Chaturvedi MM, Mukhopadhyay A, Aggarwal BB. Assay for redox-sensitive transcription factor.
Methods Enzymol
2000
;
319
:
585
–602.
69
Takada Y, Khuri FR, Aggarwal BB. Protein farnesyltransferase inhibitor (SCH 66336) abolishes NF-κB activation induced by various carcinogens and inflammatory stimuli leading to suppression of NF-κB-regulated gene expression and up-regulation of apoptosis.
J Biol Chem
2004
;
279
:
26287
–99.
70
Takada Y, Aggarwal BB. Betulinic acid suppresses carcinogen-induced NF-κB activation through inhibition of I κB α kinase and p65 phosphorylation: abrogation of cyclooxygenase-2 and matrix metalloprotease-9.
J Immunol
2003
;
171
:
3278
–86.
71
Ahn KS, Sethi G, Aggarwal BB. Simvastatin potentiates TNF-α-induced apoptosis through the down-regulation of NF-κB-dependent antiapoptotic gene products: role of IκBα kinase and TGF-β-activated kinase-1.
J Immunol
2007
;
178
:
2507
–16.
72
Sethi G, Ahn KS, Sandur SK, Lin X, Chaturvedi MM, Aggarwal BB. Indirubin enhances tumor necrosis factor-induced apoptosis through modulation of nuclear factor-κB signaling pathway.
J Biol Chem
2006
;
281
:
23425
–35.
73
Sethi G, Ahn KS, Pandey MK, Aggarwal BB. Celastrol, a novel triterpene, potentiates TNF-induced apoptosis and suppresses invasion of tumor cells by inhibiting NF-{κ}B-regulated gene products and TAK1-mediated NF-{κ}B activation. Blood 2006;
109
:
2727
–35.