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Cancer Research 68, 8861, November 1, 2008. doi: 10.1158/0008-5472.CAN-08-1902
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Modification of Cysteine Residue in p65 Subunit of Nuclear Factor-{kappa}B (NF-{kappa}B) by Picroliv Suppresses NF-{kappa}B–Regulated Gene Products and Potentiates Apoptosis

Preetha Anand1, Ajaikumar B. Kunnumakkara1, Kuzhuvelil B. Harikumar1, Kwang Seok Ahn1, Vladimir Badmaev2 and Bharat B. Aggarwal1

1 Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas and 2 Sabinsa Corp., Piscataway, New Jersey

Requests for reprints: Bharat B. Aggarwal, Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Unit 143, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-1817; Fax: 713-745-6339; E-mail: aggarwal{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Picroliv, an iridoid glycoside derived from the plant Picrorhiza kurroa, is used traditionally to treat fever, asthma, hepatitis, and other inflammatory conditions. However, the exact mechanism of its therapeutic action is still unknown. Because nuclear factor-{kappa}B (NF-{kappa}B) activation plays a major role in inflammation and carcinogenesis, we postulated that picroliv must interfere with this pathway by inhibiting the activation of NF-{kappa}B–mediated signal cascade. Electrophoretic mobility shift assay showed that pretreatment with picroliv abrogated tumor necrosis factor (TNF)–induced activation of NF-{kappa}B. The glycoside also inhibited NF-{kappa}B activated by carcinogenic and inflammatory agents, such as cigarette smoke condensate, phorbol 12-myristate 13-acetate, okadaic acid, hydrogen peroxide, lipopolysaccharide, and epidermal growth factor. When examined for the mechanism of action, we found that picroliv inhibited activation of I{kappa}B{alpha} kinase, leading to inhibition of phosphorylation and degradation of I{kappa}B{alpha}. It also inhibited phosphorylation and nuclear translocation of p65. Further studies revealed that picroliv directly inhibits the binding of p65 to DNA, which was reversed by the treatment with reducing agents, suggesting a role for a cysteine residue in interaction with picroliv. Mutation of Cys38 in p65 to serine abolished this effect of picroliv. NF-{kappa}B inhibition by picroliv leads to suppression of NF-{kappa}B–regulated proteins, including those linked with cell survival (inhibitor of apoptosis protein 1, Bcl-2, Bcl-xL, survivin, and TNF receptor–associated factor 2), proliferation (cyclin D1 and cyclooxygenase-2), angiogenesis (vascular endothelial growth factor), and invasion (intercellular adhesion molecule-1 and matrix metalloproteinase-9). Suppression of these proteins enhanced apoptosis induced by TNF. Overall, our results show that picroliv inhibits the NF-{kappa}B activation pathway, which may explain its anti-inflammatory and anticarcinogenic effects. [Cancer Res 2008;68(21):8861–70]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Identification of the active components and molecular basis for the action of a traditional medicine is likely to make it more acceptable for use in humans, an approach sometimes referred to as "reverse pharmacology." The extracts from roots and rhizomes of the plant Picrorhiza kurroa (commonly called as katuka, kutki, or kutaki) are used to treat a variety of ailments, including fever, hepatitis, allergies, asthma, and other inflammatory diseases (1, 2). One of the major active constituents of P. kurroa is picroliv, which consists of the iridoid glycosides picroside-1 and kutkoside at a ratio 1.0:1.5 (w/w; Fig. 1A ).


Figure 1
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  		Figure 1. A, structure of picroside-I and kutkoside. B, picroliv suppresses TNF-induced NF-{kappa}B activation in a dose-dependent manner. KBM-5 cells (2 x 106) were incubated with picroliv at different concentrations for 12 h and then treated with TNF (0.1 nmol/L) for 30 min. Nuclear extracts of the cells were prepared and assayed for NF-{kappa}B activation using EMSA. Cell viability was measured by trypan blue assay. C, picroliv suppresses TNF-induced NF-{kappa}B activation in a time-dependent manner. KBM-5 cells (2 x 106) were incubated with 150 µg/mL picroliv for the indicated times and then treated with TNF (0.1 nmol/L) for 30 min. Nuclear extracts of the cells were then prepared and assayed for NF-{kappa}B activation using EMSA. D, picroliv blocks activation of NF-{kappa}B induced by TNF, OA, CSC, H2O2, PMA, LPS, and EGF. KBM-5 cells (2 x 106) were preincubated with 150 µg/mL picroliv at 37°C for 12 h and then treated with TNF (0.1 nmol/L, 30 min), OA (500 nmol/L, 4 h), CSC (10 µg/mL, 1 h), H2O2 (500 µmol/L, 2 h), PMA (25 ng/mL, 1 h), LPS (100 ng/mL, 2 h), and EGF (100 ng/mL, 2 h). Nuclear extracts were prepared and assayed for NF-{kappa}B activation using EMSA. M, medium.

 
Studies have shown that picroliv exhibits hepatoprotective effects against aflatoxin (3–5), oxytetracycline (6), carbon tetrachloride (7), and alcohol (8); protects against ischemia-reperfusion injury of the liver (9) and kidneys (10); and exhibits anti-inflammatory (11), immunomodulatory (12), and anticarcinogenic (13–16) effects. For instance, studies have shown that oral administration of picroliv suppresses 20-methylcholanthrene–induced sarcoma and 7,12-dimethylbenz(a)anthracene (DMBA)–initiated papilloma formation in mice (15) and 1,2-dimethylhydrazine hydrochloride–induced liver tumor formation (16) and N-nitrosodiethylamine–induced hepatocarcinogenesis (13, 14) in rats. How picroliv mediates these effects is not fully understood. Studies have also shown that picroliv changes the antioxidant status of cells (17), down-regulates the expression of c-Jun and c-fos (18), and inhibits hypoxia-induced down-regulation of insulin-like growth factor (IGF)-1 and IGF-2 in rats (19). Because pro-oxidant, proinflammatory, immunomodulatory, and carcinogenic effects have been linked with activation of nuclear factor-{kappa}B (NF-{kappa}B), picroliv may mediate these effects through modulation of the NF-{kappa}B activation pathway.

NF-{kappa}B is an inducible transcription factor that is activated by various carcinogens, inflammatory stimuli, and growth factors and controls the expression of genes linked with survival, proliferation, invasion, and metastasis of tumors (20). Whether picroliv modulates this pathway is not known. We performed the study described herein to evaluate the effect of picroliv on NF-{kappa}B pathway. The results showed that picroliv can inhibit the NF-{kappa}B activation pathway leading to suppression of the expression of NF-{kappa}B–regulated proteins and potentiation of apoptosis in tumor cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Reagents. A 50-mg/mL solution of picroliv (supplied by Sabinsa Corp.) was prepared in DMSO, stored as small aliquots at –20°C, and then diluted as needed in a cell culture medium. Bacteria-derived human recombinant tumor necrosis factor (TNF), purified to homogeneity with a specific activity of 5 x 107 units/mg, was provided by Genentech. Cigarette smoke condensate (CSC) was prepared as described previously (21). N-acetyl-leucyl-leucyl-norleucinal (ALLN) was purchased from Calbiochem. Penicillin, streptomycin, RPMI 1640, Iscove's modified Dulbecco's medium, DMEM, and fetal bovine serum (FBS) were obtained from Invitrogen. Phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA), lipopolysaccharide (LPS), epidermal growth factor (EGF), hydrogen peroxide (H2O2), and DTT were obtained from Sigma. Anti-p65, anti-p50, anti-I{kappa}B{alpha}, anti-cyclin D1, anti-matrix metalloproteinase-9 (MMP-9), β-actin, anti-poly(ADP-ribose) polymerase (PARP), anti-inhibitor of apoptosis protein (IAP) 1, anti-Bcl-2, anti-intercellular adhesion molecule-1 (ICAM-1), and anti-Bcl-xL antibodies were obtained from Santa Cruz Biotechnology. An anti-cyclooxygenase-2 (COX-2) antibody was obtained from BD Biosciences. An anti-vascular endothelial growth factor (VEGF) antibody was purchased from NeoMarkers. Phosphospecific anti-I{kappa}B (Ser32) and anti-p65 (Ser536) antibodies were purchased from Cell Signaling Technology. Anti-I{kappa}B kinase (IKK)-{alpha} and anti-IKK-β antibodies were provided by Imgenex. pcDNA3.1 and pcDNA expression vectors for murine p65 and murine p65C38S were provided by Dr. T.D. Gilmore (Boston University, Boston, MA).

Cell lines. The human cell lines KBM-5 (chronic myeloid leukemia), H1299 (lung adenocarcinoma), and A293 (embryonic kidney carcinoma) were obtained from the American Type Culture Collection. The cell lines were cultured as follows: KBM-5 in Iscove's modified Dulbecco's medium with 15% FBS, H1299 and U266 in RPMI 1640, and A293 in DMEM supplemented with 10% FBS. Culture media were supplemented with 100 units/mL penicillin and 100 µg/mL streptomycin.

Electrophoretic mobility shift assay. To determine activation of NF-{kappa}B, electrophoretic mobility shift assay (EMSA) was performed as described previously (22). In brief, nuclear extracts of TNF-treated cells were incubated with a 32P-end-labeled, 45-mer double-stranded NF-{kappa}B oligonucleotide (15 µg of protein with 16 fmol DNA) from the HIV long terminal repeat 5'-TGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' (NF-{kappa}B binding sites are in boldface) for 30 min at 37°C. The DNA-protein complex that formed was separated from the free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3', was used to evaluate the specificity of NF-{kappa}B binding to DNA. The specificity was also determined using competition with an unlabeled oligonucleotide. The gels were dried and visualized, and the radioactive bands were quantitated using a Storm 220 PhosphorImager (Amersham Biosciences) with the ImageQuant software program (Molecular Dynamics).

Transfection. A293 cells (5 x 105 per well) were plated in six-well plates and transiently transfected with FuGENE 6 (Roche Molecular Biochemicals) with the pcDNA3.1 or pcDNA expression vector for murine p65 or murine p65C38S for 48 h (23). Thereafter, nuclear extracts of transfected cells were prepared and incubated with picroliv for 30 min, and the DNA binding was measured using EMSA.

Western blot analysis. To determine the levels of protein expression in the cytoplasm and nucleus, extracts were fractionated using SDS-PAGE as described previously (22). The proteins were then electrotransferred to nitrocellulose membranes and blotted with each antibody, and the protein expression was detected using an enhanced chemiluminescence reagent (Amersham Biosciences).

IKK assay. To determine the effects of picroliv on TNF-induced activation of IKK, we performed the IKK assay as described previously (22). To determine the total amounts of IKK-{alpha} and IKK-β in each sample, 30 µg of whole-cell protein were resolved using 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and blotted with anti-IKK-{alpha} or anti-IKK-β antibodies.

NF-{kappa}B–dependent reporter gene expression assay. A NF-{kappa}B–dependent reporter gene expression assay was performed as described previously (22). The effects of picroliv on NF-{kappa}B–dependent reporter gene transcription activated by TNF, TNF receptor (TNFR), TNFR-associated death domain (TRADD), TNFR-associated factor 2 (TRAF2), NF-{kappa}B–inducing kinase (NIK), IKK-β, and TAK/TAB were analyzed using the secretary alkaline phosphatase (SEAP) assay.

Immunocytochemical analysis of NF-{kappa}B p65 localization. The effects of picroliv on nuclear p65 translocation in KBM-5 cells were evaluated using immunocytochemical analysis as described previously (22).

Invasion assay. Extracellular matrix invasion is a crucial step in tumor metastasis. Therefore, the effect of picroliv on this invasion was assessed using an invasion assay as described previously (22). A Matrigel basement membrane matrix extracted from a murine Engelbreth-Holm-Swarm tumor (BD Biosciences) was reconstituted and used for this assay.

Live/Dead assay. To assess the membrane permeability, we used the Live/Dead assay (Molecular Probes), which measures intracellular esterase activity and plasma membrane integrity, as described previously (22).

Annexin V assay. To identify phosphatidylserine externalization during apoptosis, cells were stained with an Annexin V antibody conjugated with the fluorescent dye FITC. In brief, 5 x 105 cells were preincubated with 150 µg/mL picroliv for 12 h and then treated with 1 nmol/L TNF for 24 h, stained with the Annexin V-FITC conjugate, and analyzed using a flow cytometer (FACSCalibur, BD Biosciences; ref. 22).

Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. To measure the DNA strand breaks during apoptosis, the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay, which uses the in situ cell death detection reagent (Roche Molecular Biochemicals), was performed. In brief, 5 x 105 cells were preincubated with 150 µg/mL picroliv for 12 h, treated with 1 nmol/L TNF for 24 h, and then incubated with a reaction mixture. Cells were analyzed using a flow cytometer (FACSCalibur; ref. 22).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
The goal of this study was to evaluate the effects of picroliv on the NF-{kappa}B signaling pathway, NF-{kappa}B–regulated proteins, and NF-{kappa}B–mediated cellular responses. We used TNF to examine the effect of picroliv on the NF-{kappa}B activation pathway because the pathway activated by this agent is well understood. We carried out most of these studies using KBM-5 cells because they express both types of TNFRs. Under the conditions that we used for examination of the NF-{kappa}B pathway and NF-{kappa}B–regulated gene expression, picroliv had a minimal effect on the cell viability.

Suppression of NF-{kappa}B activation by picroliv is dose and time dependent. We first examined the effect of picroliv on TNF-dependent activation of NF-{kappa}B. To do so, we pretreated KBM-5 cells with different concentrations of picroliv for 12 h and then exposed them to TNF for 30 min. We found that picroliv suppressed TNF-induced activation of NF-{kappa}B in a dose-dependent manner, with maximum suppression occurring at 150 µg/mL (Fig. 1B). The inhibition of TNF-induced NF-{kappa}B activation by picroliv was found to be not due to loss of cell viability (Fig. 1B). Whether this inhibition of TNF-induced NF-{kappa}B activation by picroliv is due to the down-regulation of TNFRs was examined. Our results showed that picroliv at these experimental conditions had no effect on TNFR expression (Supplementary Fig. S1A).

We next determined the minimum duration of exposure to picroliv required to inhibit TNF-mediated activation of NF-{kappa}B. We exposed KBM-5 cells to picroliv for 1, 2, 4, 8, or 12 h and then treated them with TNF for 30 min. The results showed that picroliv suppressed TNF-induced activation of NF-{kappa}B in a time-dependent manner, with maximum inhibition occurring at 12 h (Fig. 1C). Under these conditions, picroliv alone had no effect on activation of NF-{kappa}B.

Picroliv inhibits activation of NF-{kappa}B induced by carcinogens, inflammatory stimuli, and growth factors. Whether picroliv could inhibit NF-{kappa}B activation induced by agents other than TNF was examined. Numerous agents, including OA, LPS, H2O2, PMA, EGF, and CSC, are known for their activation of NF-{kappa}B, but the pathways by which these agents activate NF-{kappa}B differ (21, 24–26). We found that all of the agents activated NF-{kappa}B in KBM-5 cells and that pretreatment of the cells with picroliv blocked this activation (Fig. 1D). These results suggested that picroliv acts at a step in the NF-{kappa}B activation pathway that is common to all of these agents.

Picroliv-induced NF-{kappa}B suppression is not reversible. Whether picroliv-induced inhibition of NF-{kappa}B activation is reversible was examined. To determine this, U266 cells were treated with picroliv for 12 h, washed twice with PBS, resuspended in fresh medium, harvested after 0, 2, 4, 8, 12, and 24 h, prepared nuclear extracts, and analyzed for NF-{kappa}B activity by EMSA. Our results showed that picroliv-induced NF-{kappa}B inhibition is not reversible (Supplementary Fig. S1B).

NF-{kappa}B activation inhibited by picroliv is specific and consists of p50 and p65 subunits. To determine the composition of the NF-{kappa}B band inhibited by picroliv, we incubated nuclear extracts of TNF-treated KBM-5 cells with an anti-p50 antibody, an anti-p65 antibody, unlabeled oligonucleotides, or mutated oligonucleotides. Both antibodies shifted the band to high molecular weight, indicating that the band was composed of p50 and p65 (Supplementary Fig. S1B). Displacement of the band with wild-type oligonucleotides but not mutant oligonucleotides indicates that the band was specific (Supplementary Fig. S1C).

Picroliv inhibits TNF-dependent degradation of I{kappa}B{alpha}. We investigated how picroliv inhibits TNF-induced activation of NF-{kappa}B in detail. Because degradation of I{kappa}B{alpha} is critical for activation of NF-{kappa}B, we investigated picroliv to determine whether it affects TNF-induced degradation of I{kappa}B{alpha}. We exposed the KBM-5 cells to picroliv for 12 h and then treated them with TNF for different periods. We then prepared nuclear extracts and cytoplasmic extracts and analyzed them for NF-{kappa}B and for degradation of I{kappa}B{alpha}, respectively. The results showed that TNF induced activation of NF-{kappa}B in a time-dependent manner and that the earliest activation occurred within 5 min after TNF addition (Fig. 2A ). However, we did not observe activation of NF-{kappa}B in cells pretreated with picroliv. These results suggested that picroliv is a very effective inhibitor of TNF-induced activation of NF-{kappa}B.


Figure 2
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  		Figure 2. A, picroliv inhibits time-dependent TNF-induced activation of NF-{kappa}B. KBM-5 cells (2 x 106) were preincubated with 150 µg/mL picroliv for 12 h. They were then treated with 0.1 nmol/L TNF for the indicated times and analyzed for NF-{kappa}B activation using EMSA. B, picroliv suppresses TNF-induced degradation of I{kappa}B{alpha}. KBM-5 cells (2 x106) were preincubated with 150 µg/mL picroliv for 12 h and then treated with TNF (0.1 nmol/L) for the indicated times. Cytoplasmic extracts of the cells were prepared, fractionated using 10% SDS-PAGE, and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-I{kappa}B{alpha} antibodies. An anti-β-actin antibody was used as the loading control. C, effect of picroliv on the phosphorylation of I{kappa}B{alpha} by TNF. The same gel used in experiment B was reprobed with phosphorylated I{kappa}B{alpha} (phospho-I{kappa}B{alpha}) antibody. D, effect of picroliv on the phosphorylation of I{kappa}B{alpha} by TNF in the presence of ALLN. KBM-5 cells were preincubated with 150 µg/mL picroliv for 12 h, incubated with 50 µg/mL ALLN for 30 min, and then treated with 0.1 nmol/L TNF for 10 min. Cytoplasmic extracts of the cells were fractionated and then subjected to Western blot analysis using a phosphospecific anti-I{kappa}B{alpha} antibody.

 
When we examined cytoplasmic extracts, we found that TNF induced degradation of I{kappa}B{alpha} as quickly as 10 min, and resynthesis occurred at 60 min (Fig. 2B). Treatment of these cells with picroliv completely inhibited the degradation of I{kappa}B{alpha}. Thus, picroliv apparently suppresses TNF-induced activation of NF-{kappa}B through inhibition of degradation of I{kappa}B{alpha}.

Picroliv inhibits TNF-dependent phosphorylation of I{kappa}B{alpha}. Because phosphorylation of I{kappa}B{alpha} is required for degradation of it, picroliv may inhibit TNF-induced degradation of I{kappa}B{alpha} by inhibiting its phosphorylation. To determine whether inhibition of TNF-induced degradation of I{kappa}B{alpha} is caused by inhibition of phosphorylation of I{kappa}B{alpha}, we probed the samples used in experiment in Fig. 2B with antibody that detects I{kappa}B{alpha} phosphorylated at Ser32 residue. Results show that TNF induced phosphorylation of I{kappa}B{alpha} within 5 min (Fig. 2C), and picroliv treatment inhibited this phosphorylation. Because a rapid degradation of phosphorylated I{kappa}B{alpha} occurred beyond 5 min, we used the proteasome inhibitor ALLN to block degradation of I{kappa}B{alpha} (27). Specifically, we pretreated KBM-5 cells with picroliv and then treated them with ALLN for 30 min before exposing them to TNF. We then examined the cells to determine the I{kappa}B{alpha} phosphorylation status in them using Western blot analysis with an antibody that recognizes specifically the serine-phosphorylated form of I{kappa}B{alpha}. We observed that TNF induced phosphorylation of I{kappa}B{alpha} and that picroliv suppressed it (Fig. 2D). These results suggest that picroliv inhibits TNF-induced phosphorylation of I{kappa}B{alpha}, thereby preventing its degradation.

Picroliv inhibits TNF-induced activation of IKK. TNF-induced phosphorylation of I{kappa}B{alpha} is mediated through the activation of IKK (22). Because picroliv inhibits phosphorylation of I{kappa}B{alpha}, we studied its effects on TNF-induced activation of IKK by immune complex kinase assays. The results showed that TNF induced activation of IKK in a time-dependent manner, and treatment of the cells with picroliv suppressed this activation (Fig. 3A ). At these experimental conditions, neither TNF nor picroliv affected the expression of IKK proteins.


Figure 3
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  		Figure 3. A, picroliv suppresses the TNF-induced activation of IKK in vivo. KBM-5 cells were pretreated with 150 µg/mL picroliv for 12 h and then treated with TNF (1 nmol/L) for the indicated times. Whole-cell extracts of the cells were immunoprecipitated with an antibody against IKK-{alpha} and analyzed using an immune complex kinase assay as described in Materials and Methods. To determine the effect of picroliv on the IKK proteins, a part of the above whole-cell extracts was fractionated using SDS-PAGE and examined using Western blot analysis with anti-IKK-{alpha} and anti-IKK-β antibodies. B, picroliv suppresses the TNF-induced activation of IKK in vitro. KBM-5 cells were treated with TNF (1 nmol/L) for 10 min; whole-cell extracts were immunoprecipitated with an antibody against IKK-{alpha}; and indicated concentrations of picroliv were added to the immunoprecipitated IKK complex, incubated for 30 min, and analyzed using an immune complex kinase assay as described in Materials and Methods. C, immunocytochemical analysis of p65 localization. KBM-5 cells (2 x 106) were preincubated with 150 µg/mL picroliv for 12 h and TNF (1 nmol/L) for 15 min and subjected to immunocytochemical analysis as described in Materials and Methods. D, effect of picroliv on TNF-induced nuclear translocation of p65. KBM-5 cells (2 x 106) were preincubated with 150 µg/mL picroliv for 12 h and then treated with 0.1 nmol/L TNF for the indicated times. Nuclear extracts of the cells were prepared, fractionated using 10% SDS-PAGE, and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-p65 and phosphospecific anti-p65 antibodies. Blotting of the membrane with an anti-PARP antibody was performed as a loading control for nuclear protein.

 
Whether picroliv directly inhibits IKK induced by TNF was examined. We immunoprecipitated the IKK with an anti-IKK-{alpha} antibody and then incubated with picroliv at different concentrations and examined them for activation of IKK. The results showed that picroliv had no effects on the activity of IKK (Fig. 3B).

Picroliv inhibits TNF-induced nuclear translocation of NF-{kappa}B. Whether picroliv could inhibit TNF-induced nuclear translocation of NF-{kappa}B using immunocytochemistry was examined. As shown in Fig. 3C, TNF induced the nuclear translocation of NF-{kappa}B, and picroliv blocked this translocation. The suppression of TNF-induced nuclear translocation of p65 was independently confirmed by Western blot analysis. The results showed a time-dependent nuclear translocation of p65 induced by TNF but picroliv abrogated the translocation (Fig. 3D).

Picroliv inhibits TNF-induced phosphorylation of p65. Studies have shown that the p65 subunit of NF-{kappa}B undergoes phosphorylation, which is required for the transactivation of NF-{kappa}B. We examined whether picroliv can inhibit the phosphorylation of p65 in KBM-5 cells using a phosphospecific anti-p65 (Ser536) antibody. As shown in Fig. 3D (middle), TNF induced phosphorylation of p65 in a time-dependent manner, and picroliv inhibited this phosphorylation.

Picroliv directly interferes with the binding of p65 to DNA. Certain NF-{kappa}B inhibitors can suppress NF-{kappa}B activation by directly blocking the binding of p65 to DNA (28–30). We determined whether picroliv mediates suppression of NF-{kappa}B activation using a similar mechanism. We incubated nuclear extracts of TNF-treated cells with picroliv at different concentrations and then examined DNA binding using EMSA. The results showed that picroliv inhibited the binding of p65 to DNA in a dose-dependent manner, with maximum inhibition occurring at a concentration of ~25 µg/mL (Fig. 4A ). These results suggest that picroliv may inhibit the binding of NF-{kappa}B to DNA through modification of p65.


Figure 4
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  		Figure 4. A, picroliv inhibits direct binding of NF-{kappa}B to DNA. Nuclear extracts (NE) of untreated KBM-5 cells or KBM-5 cells treated with 0.1 nmol/L TNF for 30 min were prepared, incubated for 30 min with picroliv at the indicated concentrations, and then assayed for NF-{kappa}B binding to DNA using EMSA. B, DTT inhibits picroliv-mediated suppression of NF-{kappa}B in vitro. Left, nuclear extracts of untreated KBM-5 cells or KBM-5 cells treated with 0.1 nmol/L TNF for 30 min were prepared, incubated for 30 min with 25 µg/mL picroliv in the presence or absence of 100 µmol/L DTT, and then assayed for NF-{kappa}B binding to DNA using EMSA. DTT inhibits picroliv-mediated suppression of NF-{kappa}B activation induced by TNF. KBM-5 cells were pretreated with picroliv (150 µg/mL, 12 h) with or without 100 µmol/L DTT for 4 h and then incubated with 0.1 nmol/L TNF for 30 min. Right, nuclear extracts of the cells were then prepared and assayed for NF-{kappa}B activation using EMSA. C, DTT inhibits picroliv-mediated suppression of overexpressed p65 in A293 cells in vitro. Nuclear extracts of cells overexpressing p65 were incubated with 25 µg/mL picroliv with or without 100 µmol/L DTT for 30 min and then assayed for NF-{kappa}B binding to DNA using EMSA. D, picroliv suppresses overexpression of wild-type p65 but not mutated p65C38S in A293 cells in vitro. Cells were transfected with an expression vector for murine p65 or mutant p65C38S, and nuclear extracts of the cells were prepared, treated with 25 µg/mL picroliv for 30 min, and subjected to EMSA.

 
Reducing agents can reverse picroliv-induced inhibition of p65 binding to DNA. Our laboratory has reported that suppression of p65 binding by caffeic acid phenylethyl ester can be reversed by reducing agents (28). Whether reducing agents can also reverse picroliv-mediated binding of p65 to DNA was investigated. We coincubated nuclear extracts with picroliv (25 µg/mL) in the presence or absence of DTT and then examined DNA binding. We found that DTT completely reversed the effect of picroliv (Fig. 4B, left).

Whether DTT can reverse the NF-{kappa}B–suppressing effect of picroliv in intact cells was examined. We treated KBM-5 cells with picroliv (150 µg/mL) in the presence of DTT and then activated NF-{kappa}B in the cells by treating them with TNF. The results showed that TNF activated NF-{kappa}B, picroliv inhibited this activation, and DTT completely reversed this inhibition (Fig. 4C, right).

Picroliv inhibits the binding of recombinant p65 to DNA, and DTT reverses this inhibition. To determine whether picroliv inhibits binding of the recombinant p65 subunit of NF-{kappa}B to DNA, we induced overexpression of p65 in A293 cells by transfecting a p65-containing plasmid into them. We then prepared nuclear extracts and treated them with picroliv (25 µg/mL) in the presence or absence of DTT. The recombinant p65 subunit bound to DNA, and treatment with picroliv suppressed this binding. DTT reversed the inhibitory effect of picroliv (Fig. 4C).

Mutation of Cys38 to serine in p65 abolishes the inhibitory effect of picroliv. Reversal of picroliv-induced inhibition of DNA binding by reducing agents suggests a role for cysteine residues in p65. Previous studies showed that Cys38 in p65, in particular, is highly susceptible to various agents (31, 32). Therefore, we explored whether picroliv interacts with Cys38 in p65 and thus inhibiting DNA binding. Specifically, we used a p65 plasmid with Cys38 mutated to a serine residue. We transiently transfected A293 cells with a pcDNA3.1 or pcDNA expression vector wild-type for p65 or p65C38S for 48 h, prepared nuclear extracts treated with picroliv (25 µg/mL) for 30 min, and measured the DNA binding using EMSA. The results showed that picroliv inhibited the binding of wild-type p65 but not mutated p65 to DNA, indicating that Cys38 in p65 is a target of picroliv (Fig. 4D).

Picroliv suppresses TNF-induced NF-{kappa}B–dependent reporter gene expression. Because DNA binding does not always correlate with NF-{kappa}B–dependent gene transcription, we determined the effects of picroliv on TNF-induced NF-{kappa}B reporter activity. We transiently transfected A293 cells with the NF-{kappa}B–regulated SEAP reporter construct, incubated them with picroliv at different concentrations, and then stimulated with TNF. The results indicated that TNF induced NF-{kappa}B reporter activity and that this activity was substantially diminished by picroliv in a dose-dependent manner (Fig. 5A ). In addition, dominant-negative I{kappa}B{alpha} plasmid suppressed TNF-induced reporter activity, indicating the specificity. These results suggested that picroliv inhibits TNF-induced reporter gene expression.


Figure 5
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  		Figure 5. A, picroliv inhibits NF-{kappa}B–dependent reporter gene expression induced by TNF. A293 cells were transiently transfected with a NF-{kappa}B–containing plasmid for 24 h. After transfection, the cells were coincubated with picroliv at the indicated concentrations and 1 nmol/L TNF for 24 h. The supernatants of the culture medium were assayed for SEAP activity. B, picroliv inhibits the NF-{kappa}B–dependent reporter gene expression induced by TNF, TNFR1, TRAF2, TAK/TAB, NIK, and IKK. A293 cells were transiently transfected with a NF-{kappa}B–containing plasmid alone or with the indicated plasmids. After 24 h, the cells were coincubated with 150 µg/mL picroliv and 1 nmol/L TNF for 24 h. The supernatants of the culture medium were assayed for SEAP activity. C, picroliv inhibits the expression of NF-{kappa}B–regulated proteins involved in survival (survivin, IAP1, Bcl-xL, Bcl-2, and TRAF2), proliferation (cyclin D1 and COX-2), invasion and metastasis (MMP-9 and ICAM-1), and angiogenesis (VEGF). KBM-5 cells were coincubated with 150 µg/mL picroliv and TNF (1 nmol/L) for the indicated times. Whole-cell extracts were prepared and examined using Western blot analysis with the indicated antibodies. The viability of cells under these conditions was >90% as examined using trypan blue exclusion.

 
Picroliv inhibits activation of NF-{kappa}B induced by TNFR1, TRADD, TRAF2, NIK, and IKK. TNF-induced activation of NF-{kappa}B is mediated through sequential interaction of TNFR with TRADD, TRAF2, TAK1, and IKK, resulting in phosphorylation of I{kappa}B{alpha} (33). We transiently transfected A293 cells with the NF-{kappa}B–regulated SEAP reporter construct together with plasmids for TNFR1, TRADD, TAK1/TAB1, TRAF2, NIK, or IKK-β and then treated them with picroliv and examined them for NF-{kappa}B–dependent SEAP expression. Picroliv suppressed activation of NF-{kappa}B induced by TNFR1, TRADD, TAK1/TAB1, TRAF2, NIK, and IKK-β (Fig. 5B).

Picroliv inhibits TNF-induced NF-{kappa}B–regulated expression of cell survival proteins. Studies have linked activation of NF-{kappa}B with tumor cell survival. This activation is mediated through NF-{kappa}B–regulated expression of cell survival proteins. Because picroliv inhibits TNF-induced activation of NF-{kappa}B, we hypothesized that it would also inhibit TNF-induced expression of cell survival proteins, such as survivin, Bcl-2, Bcl-xL, IAP1, and TRAF2, all of which are regulated by NF-{kappa}B (20). We performed Western blot analysis and found that picroliv inhibited the expression of all these proteins (Fig. 5C).

Picroliv inhibits TNF-induced expression of proliferative proteins. The activation of NF-{kappa}B regulates the expression of cyclin D1 (20) linked with the proliferation of various tumor cells. We examined whether the expression of this protein is modulated by picroliv in KBM-5 cells. We observed that TNF induced the expression of cyclin D1 and that picroliv significantly suppressed its expression (Fig. 5C).

Picroliv inhibits TNF-induced expression of COX-2 proteins. The activation of NF-{kappa}B regulates the expression of COX-2 linked to inflammation. We examined whether the expression of this protein is modulated by picroliv in KBM-5 cells. We observed that TNF induced the expression of COX-2 and that picroliv significantly suppressed its expression (Fig. 5C).

Picroliv suppresses TNF-induced expression of MMP-9, ICAM-1, and VEGF. The expression of MMP-9 and ICAM-1, which are involved in tumor cell invasion and metastasis, are regulated by NF-{kappa}B (34). VEGF is the most potent angiogenic factor and its expression is also regulated by NF-{kappa}B (35). We investigated whether picroliv affects the expression of these proteins; we found that TNF induced the expression of MMP-9, ICAM-1, and VEGF and that treatment with picroliv suppressed this expression (Fig. 5C).

Picroliv potentiates apoptosis induced by TNF. Because TNF-induced expression of cell survival proteins is down-regulated by picroliv, we examined whether picroliv enhances apoptosis induced by TNF. A DNA strand break assay using TUNEL revealed that picroliv increased the TNF-induced apoptosis rate in these cells from 2% to 60% (Fig. 6A, left ). We also examined whether picroliv potentiates TNF-induced apoptosis in KBM-5 cells as assessed according to phosphatidylserine externalization using the Annexin V assay. The results shown in Fig. 6A (right) indicated that picroliv increased the TNF-induced apoptosis rate from 8% to 33%. Using the Live/Dead assay, which measures cell membrane permeability, we found that picroliv increased the rate of TNF-induced cytotoxicity in KBM-5 cells from 8% to 60% (Fig. 6B). In addition, studies have shown that TNF activates caspase-3, which leads to cleavage of PARP. Thus, we examined whether picroliv enhances TNF-induced caspase-3–mediated cleavage of PARP. The results shown in Fig. 6C indicated that picroliv enhanced TNF-induced cleavage of PARP. Taken together, the results of these assays suggested that picroliv enhances the apoptotic effects of TNF.


Figure 6
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  		Figure 6. A, picroliv potentiates TNF-induced apoptosis in KBM-5 cells as determined by TUNEL and Annexin V assay. Cells (1 x 106/mL) were pretreated with 150 µg/mL picroliv for 12 h and then incubated with 1 nmol/L TNF for 24 h. The cells were then stained for TUNEL positivity or incubated with a FITC-conjugated Annexin V antibody and analyzed using flow cytometry. B, Live/Dead assay results indicating that picroliv up-regulates TNF-induced cytotoxicity. KBM-5 cells were preincubated with 150 µg/mL picroliv for 12 h and then treated with 1 nmol/L TNF for 24 h. Cells were stained with Live/Dead assay reagent for 30 min and then analyzed under a fluorescence microscope. C, picroliv potentiates TNF-induced apoptosis as determined by PARP assay. KBM-5 cells were preincubated with 150 µg/mL picroliv for 12 h and then treated with 1 nmol/L TNF for the indicated times. Whole-cell extracts of the cells were prepared, subjected to SDS-PAGE, and blotted with an anti-PARP antibody. D, picroliv suppresses TNF-induced invasion. H1299 cells were seeded in a Matrigel invasion chamber overnight in the absence of serum, coincubated with 150 µg/mL picroliv and 1 nmol/L TNF for 24 h in the presence of 1% serum, and then subjected to an invasion assay as described in Materials and Methods.

 
Picroliv suppresses TNF-induced invasion of tumor cells. The expression of COX-2 and MMP-9 has been linked with tumor cell invasion (36). Because picroliv suppressed the TNF-induced expression of COX-2 and MMP-9, we determined its effects on TNF-induced invasion of tumor cells. Using a Matrigel invasion assay, we showed that picroliv suppressed the TNF-induced invasive activity of the tumor cells (Fig. 6D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Previously, it has been reported that picroliv, a component of the traditional medicine, exhibits anti-inflammatory, hepatoprotective, and anticarcinogenic effects through an undefined mechanism. In the current report, we show that this iridoid can inhibit the activation of NF-{kappa}B and NF-{kappa}B–regulated proteins. We observed that picroliv inhibited NF-{kappa}B activation induced by different carcinogens and proinflammatory agents, thus suggesting that it must act at a step in the NF-{kappa}B activation pathway common to all of the NF-{kappa}B inducers tested in this study. Our results also revealed that picroliv acts at two different steps in the NF-{kappa}B signaling pathway: first, via its direct interaction with the p65 subunit of NF-{kappa}B, and second, through its effect on TNF-induced activation of IKK.

We found that picroliv inhibits NF-{kappa}B activation by modifying Cys38 of p65, which is crucial for DNA binding (32). How picroliv targets the critical cysteine in p65 is not clear. Most polyphenols mediate their cellular effects through two different mechanisms: redox recycling and reaction with reduced glutathione. Redox cycling results in the generation of the semiquinone radicals followed by formation of superoxide radical and H2O2. Because picroliv directly modified p65 not only in vivo but also in vitro (see Fig. 4A), it is unlikely that the effect of picroliv is mediated through generation of reactive oxygen species (ROS). Moreover, this iridoid is a scavenger of free radicals (17). It is also less likely that ROS is being produced by the nuclear extracts in in vitro conditions used for the modification of p65 by picroliv. All these results suggest that picroliv is interacting with cysteine residue of p65 directly. This is consistent with reports on polyphenols, such as sesquiterpene lactones, epoxyquinone A, and plumbagin, shown to directly alkylate Cys38 of p65 (23, 31, 32). In addition to its effects on p65, we found that picroliv inhibits TNF-induced activation of IKK, which leads to inhibition of phosphorylation and degradation of I{kappa}B{alpha}.

We also found for the first time that picroliv inhibits the TNF-induced expression of cell survival proteins, such as IAP1, Bcl-2, Bcl-xL, and survivin, all known to be regulated by NF-{kappa}B. Because these proteins play a major role in suppression of apoptosis, picroliv is expected to enhance apoptosis induced by cytokines and chemotherapeutic agents. Indeed, we did find that TNF-induced apoptosis was potentiated by the polyphenol as indicated by the DNA strand breaks, phosphatidylserine staining, esterase staining, and caspase-mediated PARP cleavage. Similarly, picroliv was also found to potentiate the effects of chemotherapeutic agents (data not shown). Besides cell survival proteins, we found that picroliv down-regulated the expression of cyclin D1 required for G1 to S transition of the cell cycle. Because more than 30% of the tumors are known to overexpress cyclin D1 (37), this polyphenol is likely to inhibit the proliferation of these tumor cells as well.

Our results also indicate that picroliv can down-regulate the expression of COX-2 protein, one of the major mediators of inflammation (38) and carcinogenesis (39). Because picroliv is used for the treatment of various inflammatory diseases (1, 2), it is possible that these effects are mediated through inhibition of expression of COX-2 as shown here. Picroliv has been shown to inhibit carcinogenesis, such as 20-methylcholanthrene–induced sarcoma, DMBA-initiated papilloma formation (15), 1,2-dimethylhydrazine hydrochloride–induced liver tumor formation (16), and N-nitrosodiethylamine–induced hepatocarcinogenesis (13, 14). These anticarcinogenic effects of picroliv are likely to be mediated through suppression of COX-2 expression. Additionally, several carcinogens, such as PMA (40), OA (23), benzopyrene (41), DMBA (42), and urethane (43), are known to activate NF-{kappa}B. The suppression of NF-{kappa}B activated by these agents by picroliv could explain its anticarcinogenic activity.

The enzyme MMP-9 is a major mediator of tumor cell invasion (44). We found that the expression of this enzyme was down-regulated by picroliv. The down-regulation of this protein was accompanied with suppression of tumor cell invasion as indicated by the Boyden chamber assay. Cell surface adhesion molecules, such as ICAM-1, which also plays a role in tumor cell invasion, were also found to be suppressed by the polyphenol.

Our results indicate that the expression of VEGF, one of the major mediators of angiogenesis, is suppressed by picroliv. The latter has been reported to modulate angiogenesis (19, 45). Gaddipati and colleagues (19) found that the expression of VEGF was enhanced by treatment with picroliv during normoxia and hypoxia in HUVEC and Hep 3B cells; on reoxygenation, the expression of VEGF was significantly reduced, whereas simultaneous treatment with picroliv during hypoxia inhibited VEGF expression in glioma cells. Because invasion and angiogenesis are known to mediate tumor metastasis, it is likely that this polyphenol can suppress not only survival and proliferation of cancer cells but also metastasis of cancer.

Because NF-{kappa}B activation is cytoprotective, it is possible that the hepatoprotective effects of picroliv against aflatoxin (5), oxytetracycline (6), carbon tetrachloride (7), and alcohol (8) may be mediated through suppression of NF-{kappa}B. The protection from ischemia-reperfusion injury of the liver (9) and kidney (10) by picroliv may also involve suppression of NF-{kappa}B activation.

In summary, suppression of the NF-{kappa}B pathway by picroliv may explain its anti-inflammatory, hepatoprotective, and anticarcinogenic effects. Human clinical trials have shown that picroliv is well tolerated and is hepatoprotective (46, 47). Our results will pave the way for further studies to validate these findings.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Clayton Foundation and NIH Cancer Center Support grant 5P30 CA016672-32. B.B. Aggarwal is the Ransom Horne, Jr., Professor of Cancer Research.

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.

We thank Donald Norwood for carefully editing the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

B.B. Aggarwal is the Ransom Horne, Jr., Professor of Cancer Research.

Received 5/20/08. Revised 7/24/08. Accepted 8/20/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Mehrotra R, Rawat S, Kulshreshtha DK, Patnaik GK, Dhawan BN. In vitro studies on the effect of certain natural products against hepatitis B virus. Indian J Med Res 1990;92:133–8.[Medline]
  2. Baruah CC, Gupta PP, Nath A, Patnaik LG, Dhawan BN. Anti-allergic and anti-anaphylactic activity of picroliv—a standardised iridoid glycoside fraction of Picrorhiza kurroa. Pharmacol Res 1998;38:487–92.[CrossRef][Medline]
  3. Rastogi R, Srivastava AK, Srivastava M, Rastogi AK. Hepatocurative effect of picroliv and silymarin against aflatoxin B1 induced hepatotoxicity in rats. Planta Med 2000;66:709–13.[CrossRef][Medline]
  4. Rastogi R, Srivastava AK, Rastogi AK. Long term effect of aflatoxin B(1) on lipid peroxidation in rat liver and kidney: effect of picroliv and silymarin. Phytother Res 2001;15:307–10.[CrossRef][Medline]
  5. Rastogi R, Srivastava AK, Rastogi AK. Biochemical changes induced in liver and serum of aflatoxin B1-treated male wistar rats: preventive effect of picroliv. Pharmacol Toxicol 2001;88:53–8.[Medline]
  6. Saraswat B, Visen PK, Patnaik GK, Dhawan BN. Protective effect of picroliv, active constituent of Picrorhiza kurrooa, against oxytetracycline induced hepatic damage. Indian J Exp Biol 1997;35:1302–5.[Medline]
  7. Santra A, Das S, Maity A, Rao SB, Mazumder DN. Prevention of carbon tetrachloride-induced hepatic injury in mice by Picrorhiza kurrooa. Indian J Gastroenterol 1998;17:6–9.[Medline]
  8. Rastogi R, Saksena S, Garg NK, Kapoor NK, Agarwal DP, Dhawan BN. Picroliv protects against alcohol-induced chronic hepatotoxicity in rats. Planta Med 1996;62:283–5.[CrossRef][Medline]
  9. Singh AK, Mani H, Seth P, et al. Picroliv preconditioning protects the rat liver against ischemia-reperfusion injury. Eur J Pharmacol 2000;395:229–39.[CrossRef][Medline]
  10. Seth P, Kumari R, Madhavan S, et al. Prevention of renal ischemia-reperfusion-induced injury in rats by picroliv. Biochem Pharmacol 2000;59:1315–22.[CrossRef][Medline]
  11. Jia Q, Hong MF, Minter D. Pikuroside: a novel iridoid from Picrorhiza kurroa. J Nat Prod 1999;62:901–3.[CrossRef][Medline]
  12. Sinha S, Mehrotra J, Bala L, Jaiswal AK, Dhawan BN. Picroliv, the iridoid glycoside fraction of Picrorhiza kurroa, selectively augments human T cell response to mycobacterial protein antigens. Immunopharm Immun 1998;20:579–88.[CrossRef]
  13. Jeena KJ, Joy KL, Kuttan R. Effect of Emblica officinalis, Phyllanthus amarus and Picrorrhiza kurroa on N-nitrosodiethylamine induced hepatocarcinogenesis. Cancer Lett 1999;136:11–6.[CrossRef][Medline]
  14. Rajeshkumar NV, Kuttan R. Inhibition of N-nitrosodiethylamine-induced hepatocarcinogenesis by picroliv. J Exp Clin Cancer Res 2000;19:459–65.[Medline]
  15. Rajeshkumar NV, Kuttan R. Protective effect of picroliv, the active constituent of Picrorhiza kurroa, against chemical carcinogenesis in mice. Teratog Carcinog Mutagen 2001;21:303–13.[CrossRef][Medline]
  16. Rajeshkumar NV, Kuttan R. Modulation of carcinogenic response and antioxidant enzymes of rats administered with 1,2-dimethylhydrazine by picroliv. Cancer Lett 2003;191:137–43.[CrossRef][Medline]
  17. Chander R, Kapoor NK, Dhawan BN. Picroliv, picroside-I and kutkoside from Picrorhiza kurrooa are scavengers of superoxide anions. Biochem Pharmacol 1992;44:180–3.[CrossRef][Medline]
  18. Seth P, Sundar SV, Seth RK, et al. Picroliv modulates antioxidant status and down-regulates AP1 transcription factor after hemorrhage and resuscitation. Shock 2003;19:169–75.[CrossRef][Medline]
  19. Gaddipati JP, Madhavan S, Sidhu GS, Singh AK, Seth P, Maheshwari RK. Picroliv—a natural product protects cells and regulates the gene expression during hypoxia/reoxygenation. Mol Cell Biochem 1999;194:271–81.[CrossRef][Medline]
  20. Aggarwal BB. Nuclear factor-{kappa}B: the enemy within. Cancer Cell 2004;6:203–8.[CrossRef][Medline]
  21. Anto RJ, Mukhopadhyay A, Shishodia S, Gairola CG, Aggarwal BB. Cigarette smoke condensate activates nuclear transcription factor-{kappa}B through phosphorylation and degradation of I{kappa}B({alpha}): correlation with induction of cyclooxygenase-2. Carcinogenesis 2002;23:1511–8.[Abstract/Free Full Text]
  22. Kunnumakkara AB, Nair AS, Ahn KS, et al. Gossypin, a pentahydroxy glucosyl flavone, inhibits the transforming growth factor β-activated kinase-1-mediated NF-{kappa}B activation pathway, leading to potentiation of apoptosis, suppression of invasion, and abrogation of osteoclastogenesis. Blood 2007;109:5112–21.[Abstract/Free Full Text]
  23. Sandur SK, Ichikawa H, Sethi G, Ahn KS, Aggarwal BB. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) suppresses NF-{kappa}B activation and NF-{kappa}B-regulated gene products through modulation of p65 and I{kappa}B{alpha} kinase activation, leading to potentiation of apoptosis induced by cytokine and chemotherapeutic agents. J Biol Chem 2006;281:17023–33.[Abstract/Free Full Text]
  24. Garg A, Aggarwal BB. Nuclear transcription factor-{kappa}B as a target for cancer drug development. Leukemia 2002;16:1053–68.[CrossRef][Medline]
  25. Takada Y, Mukhopadhyay A, Kundu GC, Mahabeleshwar GH, Singh S, Aggarwal BB. Hydrogen peroxide activates NF-{kappa}B through tyrosine phosphorylation of I{kappa}B{alpha} and serine phosphorylation of p65: evidence for the involvement of I{kappa}B{alpha} kinase and Syk protein-tyrosine kinase. J Biol Chem 2003;278:24233–41.[Abstract/Free Full Text]
  26. Sethi G, Ahn KS, Chaturvedi MM, Aggarwal BB. Epidermal growth factor (EGF) activates nuclear factor-{kappa}B through I{kappa}B{alpha} kinase-independent but EGF receptor-kinase dependent tyrosine 42 phosphorylation of I{kappa}B{alpha}. Oncogene 2007;26:7324–32.[CrossRef][Medline]
  27. Ichikawa H, Takada Y, Murakami A, Aggarwal BB. Identification of a novel blocker of I{kappa}B{alpha} kinase that enhances cellular apoptosis and inhibits cellular invasion through suppression of NF-{kappa}B-regulated gene products. J Immunol 2005;174:7383–92.[Abstract/Free Full Text]
  28. 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-{kappa}B. Proc Natl Acad Sci U S A 1996;93:9090–5.[Abstract/Free Full Text]
  29. Finco TS, Beg AA, Baldwin AS, Jr. Inducible phosphorylation of I{kappa}B{alpha} is not sufficient for its dissociation from NF-{kappa}B and is inhibited by protease inhibitors. Proc Natl Acad Sci U S A 1994;91:11884–8.[Abstract/Free Full Text]
  30. Mahon TM, O'Neill LA. Studies into the effect of the tyrosine kinase inhibitor herbimycin A on NF-{kappa}B activation in T lymphocytes. Evidence for covalent modification of the p50 subunit. J Biol Chem 1995;270:28557–64.[Abstract/Free Full Text]
  31. Liang MC, Bardhan S, Pace EA, et al. Inhibition of transcription factor NF-{kappa}B 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.[CrossRef][Medline]
  32. Garcia-Pineres AJ, Castro V, Mora G, et al. Cysteine 38 in p65/NF-{kappa}B plays a crucial role in DNA binding inhibition by sesquiterpene lactones. J Biol Chem 2001;276:39713–20.[Abstract/Free Full Text]
  33. Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 2003;3:745–56.[CrossRef][Medline]
  34. Esteve PO, Chicoine E, Robledo O, et al. Protein kinase C-{zeta} regulates transcription of the matrix metalloproteinase-9 gene induced by IL-1 and TNF-{alpha} in glioma cells via NF-{kappa}B. J Biol Chem 2002;277:35150–5.[Abstract/Free Full Text]
  35. Kim I, Moon SO, Kim SH, Kim HJ, Koh YS, Koh GY. Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-{kappa}B activation in endothelial cells. J Biol Chem 2001;276:7614–20.[Abstract/Free Full Text]
  36. Liotta LA, Thorgeirsson UP, Garbisa S. Role of collagenases in tumor cell invasion. Cancer Metastasis Rev 1982;1:277–88.[CrossRef][Medline]
  37. Fu M, Wang C, Li Z, Sakamaki T, Pestell RG. Minireview: Cyclin D1: normal and abnormal functions. Endocrinology 2004;145:5439–47.[Abstract/Free Full Text]
  38. Kuwano T, Nakao S, Yamamoto H, et al. Cyclooxygenase 2 is a key enzyme for inflammatory cytokine-induced angiogenesis. FASEB J 2004;18:300–10.[Abstract/Free Full Text]
  39. Dannenberg AJ, Lippman SM, Mann JR, Subbaramaiah K, DuBois RN. Cyclooxygenase-2 and epidermal growth factor receptor: pharmacologic targets for chemoprevention. J Clin Oncol 2005;23:254–66.[Abstract/Free Full Text]
  40. Zheng X, Chang RL, Cui XX, et al. Inhibition of NF-{kappa}B by (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY11-7082; BAY) is associated with enhanced 12-O-tetradecanoylphorbol-13-acetate-induced growth suppression and apoptosis in human prostate cancer PC-3 cells. Int J Oncol 2008;32:257–64.[Medline]
  41. Ding J, Wu K, Zhang D, et al. Activation of both nuclear factor of activated T cells and inhibitor of nuclear factor-{kappa}B kinase β-subunit-/nuclear factor-{kappa}B is critical for cyclooxygenase-2 induction by benzo[a]pyrene in human bronchial epithelial cells. Cancer Sci 2007;98:1323–9.[CrossRef][Medline]
  42. Shin SR, Sanchez-Velar N, Sherr DH, Sonenshein GE. 7,12-Dimethylbenz(a)anthracene treatment of a c-rel mouse mammary tumor cell line induces epithelial to mesenchymal transition via activation of nuclear factor-{kappa}B. Cancer Res 2006;66:2570–5.[Abstract/Free Full Text]
  43. Stathopoulos GT, Sherrill TP, Cheng DS, et al. Epithelial NF-{kappa}B activation promotes urethane-induced lung carcinogenesis. Proc Natl Acad Sci U S A 2007;104:18514–9.[Abstract/Free Full Text]
  44. Watanabe H, Nakanishi I, Yamashita K, Hayakawa T, Okada Y. Matrix metalloproteinase-9 (92 kDa gelatinase/type IV collagenase) from U937 monoblastoid cells: correlation with cellular invasion. Journal of cell science 1993;104:991–9.[Abstract]
  45. Singh AK, Sharma A, Warren J, et al. Picroliv accelerates epithelialization and angiogenesis in rat wounds. Planta Med 2007;73:251–6.[CrossRef][Medline]
  46. Antarkar DS, Vaidya AB, Doshi JC, et al. A double-blind clinical trial of Arogya-wardhani—an ayurvedic drug–in acute viral hepatitis. Indian J Med Res 1980;72:588–93.[Medline]
  47. Vaidya AB, Antarkar DS, Doshi JC, et al. Picrorhiza kurroa (Kutaki) Royle ex Benth as a hepatoprotective agent—experimental & clinical studies. J Postgrad Med 1996;42:105–8.[Medline]



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