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Up-stream events in the nuclear factor κB activation cascade in response to sparsely ionizing radiation
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Advances in Space Research 44 (2009) 907–916 www.elsevier.com/locate/asr
Up-stream events in the nuclear factor jB activation cascade in response to sparsely ionizing radiation Christine E. Hellweg *, Britta Langen, Galina Klimow, Roland Ruscher, Claudia Schmitz, Christa Baumstark-Khan, Gu¨nther Reitz Division of Radiation Biology, Institute of Aerospace Medicine, German Aerospace Center (DLR), Linder Hoehe, 51147 Koeln, Germany Received 10 October 2008; received in revised form 13 July 2009; accepted 13 July 2009
Abstract Radiation is a potentially limiting factor for manned long-term space missions. Prolonged exposure to galactic cosmic rays may shorten the healthy life-span after return to Earth due to cancer induction. During the mission, a solar flare can be life threatening. For better risk estimation and development of appropriate countermeasures, the study of the cellular radiation response is necessary. Since apoptosis may be a mechanism the body uses to eliminate damaged cells, the induction by cosmic radiation of the nuclear antiapoptotic transcription factor nuclear factor jB (NF-jB) could influence the cancer risk of astronauts exposed to cosmic radiation by improving the survival of radiation-damaged cells. In previous studies using a screening assay for the detection of NF-jB-dependent gene induction (HEK-pNF-jB-d2EGFP/Neo cells), the activation of this transcription factor by heavy ions was shown [BaumstarkKhan, C., Hellweg, C.E., Arenz, A., Meier, M.M. Cellular monitoring of the nuclear factor kappa B pathway for assessment of space environmental radiation. Radiat. Res. 164, 527–530, 2005]. Studies with NF-jB inhibitors can map functional details of the NF-jB pathway and the influence of radiation-induced NF-jB activation on various cellular outcomes such as survival or cell cycle arrest. In this work, the efficacy and cytotoxicity of four different NF-jB inhibitors, caffeic acid phenethyl ester (CAPE), capsaicin, the proteasome inhibitor MG-132, and the cell permeable peptide NF-jB SN50 were analyzed using HEK-pNF-jB-d2EGFP/Neo cells. In the recommended concentration range, only CAPE displayed considerable cytotoxicity. CAPE and capsaicin partially inhibited NF-jB activation by the cytokine tumor necrosis factor a. MG-132 completely abolished the activation and was therefore used for experiments with X-rays. NF-jB SN-50 could not reduce NF-jB dependent expression of the reporter destabilized Enhanced Green Fluorescent Protein (d2EGFP). MG-132 entirely suppressed the X-ray induced NF-jB activation in HEK-pNF-jB-d2EGFP/Neo cells. In conclusion, the degradation of the inhibitor of NF-jB (IjB) in the proteasome is essential for X-ray induced NF-jB activation, and MG-132 will be useful in studies of the NF-jB pathway involvement in the cellular response to heavy ion exposure and other space-relevant radiation qualities. Ó 2009 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: Nuclear factor jB inhibitors; Ionizing radiation; Human cells; Proteasome; Capsaicin; Caffeic acid phenethyl ester
1. Introduction During space missions, astronauts are exposed to not only greater amounts of natural radiation than they receive
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Corresponding author. Tel.: +49 2203 601 3243; fax: +49 2203 61970. E-mail addresses: [email protected] (C.E. Hellweg), claudia. [email protected] (C. Schmitz), [email protected] (C. BaumstarkKhan), [email protected] (G. Reitz).
on earth but also to a differing radiation quality, which can result in immediate and long-term risks. The cellular radiation response is essential to the development of acute and chronic radiation exposure sequels. A possible modulator of the cellular radiation response might be the ubiquitously expressed transcription factor nuclear factor jB (NF-jB), which has been shown to play a role in apoptosis regulation, mostly by inducing the expression of anti-apoptotic proteins in many cell types (Baichwal and Baeuerle, 1997; Chen and Greene, 2003; Kucharczak et al., 2003).
0273-1177/$36.00 Ó 2009 COSPAR. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.asr.2009.07.009
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In order to examine the role of NF-jB in the cellular response to space-relevant radiation qualities, a stably transfected human embryonic kidney cell line (HEKpNF-jB-d2EGFP/Neo cells) indicating NF-jB activation by increased expression of the reporter destabilized Enhanced Green Fluorescent Protein (d2EGFP, Chalfie et al., 1994; Li et al., 1998) was constructed and evaluated (Hellweg et al., 2003). HEK cells are a useful tool to study many different processes in human cells, as they can easily be transiently and stably transfected. This allows reporter gene studies or functional studies with overexpression of the proteins in question (examples: Sung et al., 2009; Lodeiro et al., 2009). Many studies, including own investigations, have shown that the NF-jB pathway is functional in HEK cells (examples: Muroi and Tanamoto, 2008; Rajagopal et al., 2008; Ait-Ghezala et al., 2007; Muscat et al., 2007; Simon and Samuel, 2007; Bladh et al., 2005; Matsuda et al., 2003; Hellweg et al., 2006). The HEK-pNF-jB-d2EGFP/Neo cell line demonstrates the translocation of NF-jB containing p65 to the cell nucleus, its binding to NF-jB-dependent promoters and subsequent increased mRNA transcription by an increase in production of the reporter protein d2EGFP. The dose effect relationship for NF-jB activation in HEK cells in response to several known NF-jB activators such as tumor necrosis factor a (TNF-a), interleukin 1b (IL-1b), phorbol ester and lipopolysaccharide was previously examined (Hellweg et al., 2006), revealing the functionality of the NF-jB sub-pathways which start at membrane receptors (IL-1 and TNF-a receptors). NF-jB activation by DNA damaging agents was also detected using this stably transfected cell line, showing the functionality of the NF-jB subpathway which starts in the cell nucleus from DNA double strand breaks induced by the topoisomerase I inhibitor camptothecin. Camptothecin induces the NF-jB dependent d2EGFP expression in this cell line with slower kinetics compared to cytokines (Hellweg et al., 2006). Different radiation qualities such as heavy ions with a medium linear energy transfer (LET 270 keV/lm), X-rays and Pm-147, a b-emitter, were shown to activate the NF-jB pathway in this cell line (Baumstark-Khan et al., 2005; Hellweg and BaumstarkKhan, 2007; Hellweg et al., 2007). Activation of the NFjB pathway is supposed to play a role in the negative regulation of apoptosis in many cell types, which in turn would favor survival of cells with residual DNA damage. This again would result in survival of mutation-prone cells. Furthermore, the production of NF-jB-regulated inflammatory cytokines could initiate a tissue reaction; production of NF-jB dependent degrading enzymes could facilitate tumor progression and metastasis of cancer cells. Therefore, further investigation of the NF-jB pathway activated in response exposure to ionizing radiation might reveal possible pharmacological targets for modulation of the cellular radiation response. As the NF-jB pathway was identified to be a central element in the pathogenesis of inflammatory diseases and also in therapy resistance of
tumors (Chastel et al., 2004), many different NF-jB inhibitors with varying specificity and efficiency are described. A detailed overview of the involvement of NF-jB in various diseases and a list of agents that inhibit NF-jB activation is given by Aggarwal et al. (2006). Bhattacharjee (2008) describes the influence of NF-jB modulators on radiation resistance. Caffeic acid phenethyl ester (CAPE), a component of propolis from honeybee hives, belongs to the group of natural chemopreventive agents which are also potent NF-jB inhibitors, such as resveratrol and curcumin (Aggarwal et al., 2006). It has been used in traditional medicine due to its anti-inflammatory and anti-oxidative potential. The efficacy of CAPE in inhibiting NF-jB activation and proinflammatory production has been demonstrated in various animal models of inflammation (Fitzpatrick et al., 2001; Orban et al., 2000). CAPE was shown to suppress the binding of the p50–p65 complex to the DNA (Natarajan et al., 1996). The NF-jB suppressive effect of CAPE was also present in vivo in irradiated rats and resulted in a reduced inflammatory response (Linard et al., 2004). Capsaicin, a homovanillic acid derivative, is an active component of the red pepper of the genus Capsicum and has been found to protect against experimentally induced mutagenesis and tumorigenesis (Surh et al., 1998; Watson et al., 1988). In vitro, capsaicin has been found to inhibit the growth of various immortalized and malignant cells (Morre et al., 1995; Takahata et al., 1999; Zhang et al., 2003) and induce apoptosis in transformed cells (Macho et al., 1999; Wolvetang et al., 1996). In addition, capsaicin is also found to be a potent inhibitor of TNF-mediated NF-jB activation in human myeloid ML-1a cells (Singh et al., 1996). Many inflammatory mediators (e.g. TNF) trigger NFjB activation by causing the phosphorylation of the inhibitor of NF-jB (IjB), which leads to its rapid ubiquitination and degradation. The proteasomal degradation of IjB proteins is an essential step in the activation of NF-jB not only in the canonical NF-jB pathway, but also in the atypical pathway which is activated in response to DNA double strand breaks (Habraken and Piette, 2006). Pharmacological inhibitors of the proteasome, which is responsible for degradation of cytosolic proteins, are available since 1994 (Lee and Goldberg, 1998). The activity of the 26S proteasome can be suppressed by the peptide aldehydes, such as Cbz-leu-leu-leucinal (MG-132) (Lee et al., 1998; Schenkein, 2002), and therefore, degradation of IjB proteins is blocked, leading to the accumulation of IjB in a phosphorylated form. Degradation of IjB allows the liberated p50– p65 complex to translocate into the nucleus and activate gene transcription. Other inhibitors are synthetic peptides interacting with the agonist induced nuclear translocation of NF-jB (Lin et al., 1995). The cell permeable peptide NF-jB SN50 carries the p50 nuclear localization sequence (NLS) bound to the hydrophobic region of the signal peptide of Kaposi fibroblast growth factor which interacts with lipid bilayers.
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Thus, the recognition mechanism for the p50 NLS is blocked and translocation of p50 containing NF-jB dimers is inhibited (Lin et al., 1995). These inhibitors can suppress radiation-induced NF-jB activation, especially the strong activation which was observed after exposure of HEK cells to high-LET radiation (Baumstark-Khan et al., 2005). Studies with NF-jB inhibitors targeting different components of the pathway can reveal the architecture of the radiation-induced NFjB pathway. Furthermore, the influence of radiationinduced NF-jB activation on various cellular outcomes such as survival or cell cycle arrest can be assessed by NF-jB inhibition. The cytotoxicity and potency of these inhibitors in suppressing heavy-ion-radiation induced NF-jB activation is unknown. As TNF-a is a strong NFjB activator, and the down-stream pathway of the TNFa receptor and the DNA double strand induced NF-jB pathway share many components – IjBa, IjB kinase (IKK) consisting of NEMO, IKK-a, IKK-b and ELKS, the 26S proteasome and the NF-jB subunits p50 and p65 (Habraken and Piette, 2006) – the potency of the inhibitors can be monitored in TNF-a treated HEK cells. In this study, the potency and cytotoxicity of several chemical inhibitors (Table 1) of different components of the NF-jB pathway was determined in the stably transfected NF-jB reporter cell line. Reduction in vitality after incubation with the inhibitors was measured using the MTT assay. Concerning the inhibitory effects, first their interference with TNF-a activated NF-jB was determined. Inhibitors which effectively blocked this pathway were applied in experiments with ionizing radiation. X-rays (150 kV) were used as model radiation.
2. Materials and methods 2.1. Cell line and growth conditions Human Embryonic Kidney (293/HEK) cells (CRL1573) were obtained from the American Type Culture Collection, Manassas, VA, USA. Construction of the plasmid
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pNF-jB-d2EGFP/Neo and stable transfection of HEK cells was already delineated in detail (Hellweg et al., 2003). The vector contains a synthetic promoter consisting of four j enhancer elements (jB4 = 4 NF-jB response elements) and the thymidine kinase minimal promoter for controlling expression of d2EGFP in response to NF-jB activation after challenging the cells with chemicals or radiation. Cells were grown under standard conditions in 80 cm2 flasks (Nunc, Wiesbaden, Germany) in a-MEMmedium (modified MEM, Biochrom KG, Berlin, Germany), with 10% fetal bovine serum (FBS) at 37 °C, saturated humidity and in a 5% CO2/95% air atmosphere. Cultures were split (1:20) every 7 days using standard detachment procedures (0.05% trypsin containing 0.02% EDTA solution, Biochrom KG, Berlin, Germany). Medium was changed after 4 days. Culture vessels were coated with poly-D-lysine for 5 min at room temperature and washed with PBS three times prior to seeding of HEK cells.
2.2. Inhibition of the NF-jB pathway NF-jB signaling pathways can be inhibited at different points by various agents. An overview of the inhibitors used and their modes of action are given in Table 1. Capsaicin was prepared freshly before use and dissolved in ethanol, then mixed into complete media. Ethanol concentration in the medium was maintained below 0.1 vol.% and this concentration was not found to influence cell growth. CAPE and MG-132 were dissolved in DMSO, aliquoted and stored at 20 °C until use. SN50 was dissolved in sterile deionised water and stored at 20 °C. The IC50 of MG-132 is a few micromolar for the inhibition of proteolysis in cultured cells (Lee et al., 1998). Toxicity of inhibitors was tested using the MTT test (Section 2.3) and the efficacy of NF-jB inhibition was analyzed in HEK-pNF-jB-d2EGFP/Neo L2 cells treated with the respective inhibitor and the NF-jB activator TNF-a. Therefore, HEK cells were seeded in a density of 3 104 cells per cm2 with 1 ml complete a-medium in poly-D-lysine coated 24-well plates. After 2 days, cells reached 30–40%
Table 1 Inhibitors of the NF-jB signaling pathway. Inhibitor
Mode of action
CAPE (caffeic acid phenylester)a
Specific p65 inhibitor, decreases NF-jB DNA binding and promoter activity (Joo et al., 2003; Natarajan et al., 1996) Decreases phosphorylated IjBa, which is marked for degradation, and inhibition of p65 translocation and its subsequent binding to jB sites (Zhang et al., 2003) Proteasome inhibitor (Munshi et al., 2004) which prevents the degradation of IjB proteins by the proteasome
Capsaicin (trans-8-methyl-N-vanillyl-6nonenamide)a MG-132 (carbobenzoxy-L-leucyl-Lleucyl-L-leucinal)b NF-jB SN50c
a b c
Cell permeable peptide carrying the p50 nuclear localization sequence (NLS) bound to the hydrophobic region of the signal peptide of Kaposi fibroblast growth factor which interacts with lipid bilayers – the recognition mechanism for the p50 NLS is blocked and translocation p50 containing NF-jB dimers is inhibited (Lin et al., 1995)
Sigma–Aldrich, Taufkirchen, Germany. Calbiochem, San Diego, CA, USA. Biomol GmbH, Hamburg, Germany.
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confluence. The NF-jB activation potential was shown to be maximal in this confluence range (Hellweg et al., 2006). The inhibitors were added in concentration ranges recommended by the literature and as determined by the cytotoxicity test. The solvents (ethanol, DMSO, sterile deionised water) were added to controls. After pre-incubation with the inhibitor for 1 h, TNF-a stock solution (10 ng/ll in PBS) was added to a final concentration of 0.6 nmol/l. Cells were incubated for another 20 h and prepared for FACS analysis (Section 2.5). 2.3. MTT test HEK-pNF-jB-d2EGFP/Neo L2 cells were seeded into polylysin-coated 96-well plates at a density of 3 104 cells per cm2 in 200 ll a-medium per well. 48 h later, inhibitors in increasing concentration were added. After 20 h, cells in four wells were killed by adding pure DMSO to generate the blank, medium was exchanged in all wells by serum free medium containing 0.5 mg/ml MTT reagent (3-(4,5dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma M5655, Taufkirchen, Germany) and cells were incubated at 37 °C for one hour. Medium was removed and the formazan crystals were dissolved in DMSO containing 0.6% v/v acetic acid and 10% w/v sodium dodecyl sulphate (SDS) by mixing for 20 min. Absorption was measured at 562 nm in a microplate reader (Lambda Fluoro 320 plus, MWG Biotech, Ebersberg, Germany). Relative absorption was calculated according to the following formula: relative absorption ¼
ODtreated ODblank ODuntreated ODblank
ð1Þ
OD = optical density at 562 nm. 2.4. Irradiation of human cells For radiation challenging, cells were seeded into poly-Dlysine-coated (Section 2.1) petri dishes (diameter 3 cm) in a density of 3 104 cells per cm2 and were incubated for 2 days resulting in 30–40% confluence. Cells were preincubated with the selected inhibitors (2 lmol/l MG-132) as described in Section 2.2. Cells were irradiated at 37 °C on a warming device coupled to a thermostat (mgw Lauda RDS RC20, Lauda-Ko¨nigshofen, Germany) with X-rays generated by an X-ray unit (Mu¨ller Typ MG 150, MCN 165, Philips, Hamburg, Germany) operated at 150 kV and 19 mA yielding a dose rate of 2 Gy/min (focus-object distance: 312 mm). TNF-a treated cells served as positive control of NF-jB activation. After incubation for 20 h, cells were prepared for FACS analysis (Section 2.5). 2.5. Flow cytometry Distribution patterns of fluorescent protein expressing cells were analyzed using a fluorescence activated cell scanner (FACS, Becton–Dickinson; San Jose, CA, USA) equipped with an argon laser (488 nm) as excitation source
for d2EGFP. The samples’ forward and sideward scatter and d2EGFP fluorescence in fluorescence channel FL1 were determined and analyzed using the CellQuest software (version 1.2, Becton–Dickinson). Samples of cells treated with different agents were prepared as follows: cells were detached from the growth surface using trypsine and fixed with cold 3.5% formaldehyde in PBS for 30 min at 4 °C. The fixative was diluted with PBS (1:3) and cells were stored at 4 °C. Before FACS analysis, cells were centrifuged and resuspended in PBS. Cells (2 104) were analyzed at a rate of 200–600 cells per second. The markers M1 (EGFP()) and M2 (EGFP(+)) were set by means of untreated and TNF-a treated HEK-pNF-jB-d2EGFP/ Neo cells. The population fractions displaying fluorescence intensities in the zone of marker M2 (EGFP(+)-cells) were used as a measure of induction. 2.6. Measurement of NF-jB activation Cells were seeded into petri dishes (diameter 6 cm), incubated for 2 days, and treated with 0.6 nmol/l TNF-a or exposed to X-rays (Section 2.4). After incubation for up 4 h, petri dishes were washed with ice-cold PBS, and cells were scraped in 3 ml ice-cold PBS, centrifuged, lysed using the lysis buffer provided by the TransAMTM supplier and stored at 80 °C until analysis. Protein concentration was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Mu¨nchen, Germany), based on the Bradford dye-binding procedure. Activated NF-jB in the cell lysates (adjusted for 7.5 lg total protein) was measured using the NF-jB p65 Transcription Factor Assay Kit (TransAMTM, Active Motif Europe, Rixensart, Belgium), containing an olignucleotide with a NF-jB consensus binding site for p65 dimers (50 -GGGACTTTCC-30 ). Results were expressed as optical density measured at 450 nm per lg protein. As internal controls, lysates were incubated with mutated and wildtype oligonucleotides. The experiments were repeated three times. 2.7. Statistics Each experiment was repeated up to five times with one to three replicates each. Means, standard errors and significance levels in the t test were calculated with MicrosoftÒ Office Excel 2003. 3. Results As activation of the NF-jB pathway represents one important cellular stress response with anti-apoptotic functions which can be involved in the cellular radiation response, we evaluated several inhibitors of different steps in the NF-jB pathway. For that purpose, cytotoxicity and potential of suppression of NF-jB activation were tested using a fluorescent screening assay for NF-jB activation dependent gene expression, based on recombinant
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human cell lines. Using this HEK-pNF-jB-d2EGFP/Neo clone, NF-jB activation in presence of the inhibitors was measured by flow cytometric analysis of d2EGFP in cells treated with TNF-a, or exposed to X-rays. 3.1. Up-stream events in the NF-jB pathway To investigate a crucial step in the pathway leading finally to the increased d2EGFP expression, the activation of NF-jB by X-rays was verified by means of an oligonucleotide based NF-jB-ELISA (Fig. 1). Incubation with TNF-a resulted in an increased NF-jB binding to the oligonucleotide (jB binding sequence) 1–4 h after addition of the cytokine. Controls incubated with a mutated jB site oligonucleotide showed no competition in p65 binding in treated and untreated cells, while addition of an excess amount of wildtype oligonucleotide nearly completely abolished p65 binding. Exposure of HEK cells to 150 kV X-rays resulted in a dose-dependent p65 translocation 2 and 4 h after irradiation. Even after exposure to a high radiation dose of 16 Gy, activation was less pronounced compared to TNF-a-treated cells.
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3.2. Chemical inhibition of the NF-jB pathway at different levels 3.2.1. Cytotoxicity of the inhibitors and of the cytokine TNF-a In order define the concentration range in which the inhibitors under investigation suppress NF-jB-dependent gene expression at an acceptable cell viability level (>80%), the cytotoxicity of the inhibitors was assessed by means of the MTT test. For functional testing, combined treatment of cells with the NF-jB activator TNF-a and the inhibitor was necessary. Therefore, cytotoxicity of both was assessed using the MTT test 20 h after addition of the agents (Fig. 2). This test measures the activity of mitochondrial dehydrogenases and is used as marker of cell viability. In a wide concentration range, TNF-a elicits no cytotoxicity in HEK cells (Fig. 2A). The inhibitor of NF-jB translocation SN50 even enhances cellular viability, only at the highest tested concentration, viability is slightly reduced to 90%. E-capsaicin has no significant effect on cell viability even at the highest concentrations tested. Incubation of HEK cells with MG-132 with concentrations up to 64 lmol/l affected the viability moderately, but significantly. CAPE displays the strongest cytotoxic effect in the examined concentration range, with a reduction to 20% at the highest dose (Fig. 2B). 3.2.2. Inhibition of TNF-a induced NF-jB activation CAPE is a partial inhibitor of TNF-a induced NF-jB activation in the examined dose range, with concentrations above 12.5 lmol/l, the inhibition ranges around 50%. Further dose increase up to 300 lmol/l does not enhance the inhibitory effect (Fig. 3 CAPE). The inhibitory effect of capsaicin is also only partial (Fig. 3 Capsaicin), already with the lowest tested concentration (1 lmol/l) the percentage of d2EGFP-expressing cells drops from 60% to 30%, with no further enhancement with dose increase. There is a slight increase of d2EGFP expression in control cells with increasing CAPE and capsaicin concentrations. With doses of 2 lmol/l and higher, MG-132 is a potent inhibitor of TNF-a induced NF-jB activation, resulting in nearly total abolishment of d2EGFP expression to the control level (Fig. 3 MG-132). Incubation with SN50 increases d2EGFP to a higher level, while control cells remain unaffected (Fig. 3 SN50).
Fig. 1. Translocation of the NF-jB subunit p65 in the nucleus after exposure to X-rays (TransAMTM p65 Oligo ELISA of nuclear extracts). HEK-pNF-jB-d2EGFP/Neo clone L2 cells were seeded into petri dishes in medium containing 10% FBS, grown for 2 days, and irradiated with 150 kV X-rays at 37 °C. TNF-a (10 ng/ml) was used as positive control for NF-jB activation. For control of binding specificity, nuclear extracts of solvent and of TNF-a-treated cells were incubated with excess wild type oligonucleotide with p65 binding specificity (wt Oligo), or with a mutated oligonucleotide without p65 binding capacity (mut Oligo). Bars show the standard deviation of three independent experiments (2 and 4 h).
3.2.3. Inhibition of X-ray induced NF-jB activation As MG-132 was shown to be the most potent NF-jB inhibitor in HEK cells, its effectiveness in suppression of radiation-induced NF-jB activation was examined. After exposure to X-rays, an increase in NF-jB activation was seen with high doses (P8 Gy) and comprises a small part of the cell population (Fig. 4). During proteasome inhibition with 2 lmol/l MG-132, this response is suppressed. MG-132 incubation results in a slight, but not significant increase of d2EGFP expression in mock irradiated cells.
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pathway in the cellular radiation response. Initially, it was shown that exposure to X-rays results in nuclear translocation of the NF-jB subunit p65. 4.1. Translocation after X-ray exposure In the canonical pathway, here initiated by TNF-a, through a series of intracellular steps, the activation of the receptor promotes the phosphorylation and subsequent dissociation of the inhibitor protein IjB from the inactive NF-jB complex, allowing liberated NF-jB to translocate to the nucleus. Once active and inside the nucleus, NFjB binds to the j enhancer element on the DNA and activates transcription of several apoptosis-related, cell growth-dependent, and B-cell-proliferation genes. Activation of NF-jB by ionizing radiation is thought to occur via ATM, which is activated in response to DNA double strand breaks. The pathway involves shuttling of NEMO between the cytoplasm and the nucleus and occurs with lower intensity and slower kinetics compared to the canonical pathway (Habraken et al., 2006). 4.2. Chemical inhibition of the NF-jB activation by TNF-a
Fig. 2. Effect of TNF-a (A) and inhibitors (B) on HEK cell viability as determined by the MTT test. TNF-a was used as positive control for NFjB activation in HEK-pNF-jB-d2EGFP/Neo cells in further experiments. The cells were seeded into microtiter plates in medium with 10% FBS, treated with TNF-a or inhibitors 2 days later, incubated for 20 h after addition, and treated with MTT reagent for one hour. Bars show standard errors of three independent experiments. t Tests were performed with TNF-a or inhibitor treated versus solvent treated cells. significant (p < 0.05), highly significant (p < 0.01), no symbol – not significant.
4. Discussion Low cytotoxicity and high inhibitory efficiency of the proteasome inhibitor MG-132 suggest this peptide aldehyde as a useful tool in studying the role of the NF-jB
In mammalian systems, the NF-jB signal transduction pathway is induced by stimulation of the TNF or IL-1 (or other) lymphokine receptors by their respective ligands. In the cellular monitoring system used here, the HEKpNF-jB-d2EGFP/Neo cells show increased NF-jB-dependent d2EGFP expression in response to the known NF-jB activator TNF-a. The inhibitors differ strongly in the specificity for NF-jB pathway inhibition, which might partially explain the differences in their cytotoxicity. The proteasomal inhibitor MG132 disturbs the metabolism of all proteins and therefore has a potentially higher toxicity than the highly specific peptide SN50, which was even shown to have a paradoxical effect in HEK cells. Capsaicin shows no cytotoxic effect in the examined dose range. CAPE was shown to strongly induce apoptosis in cells of the immune system (Orban et al., 2000), which might explain its cytotoxic effect in HEK cells. In this work, CAPE was shown to be a partial inhibitor of TNF-a induced NF-jB activation with considerable cytotoxicity. Linard et al. (2004) show that CAPE treatment after irradiation did not completely inhibit the NFjB p65 DNA-binding activity, although it had some effects on proinflammatory cytokine production. The selective blockade of the NF-jB pathway did not inhibit the radiation-induced increase in TNF-a expression, and it reduced IL-1b expression only slightly. Similar observations have been already reported in alveolar epithelial cells, where CAPE did not totally abrogate lipopolysaccharide-mediated TNF-a biosynthesis (Haddad and Land, 2001). A total blockade of TNF-induced NF-jB activation was achieved in the human monocyte cell line U937, with a maximal effect at 90 lmol/l (Natarajan et al., 1998;
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Fig. 3. Inhibition of NF-jB activation by TNF-a in HEK-pNF-jB-d2EGFP/Neo cells. The cells were seeded into 24-well plates in medium with 10% FBS. Two days later, they were preincubated with increasing inhibitor concentrations (CAPE, Capsaicin, MG-132, NF-jB SN-50) for 1 h, treated with TNF-a, and collected for FACS analysis of d2EGFP expression 20 h after treatment. Bars show the standard deviation of up to five independent experiments. t Tests were performed with TNF-a treated versus untreated cells, both without inhibitor (dose 0 lmol/l), to show the effect of TNF-a alone. t Tests demonstrating the inhibitor effectiveness were operated with inhibitor treated cells versus cells without inhibitor, for each TNF-a treated (black ) and control cells (gray ). significant (p < 0.05), highly significant (p < 0.01), no symbol – not significant.
Natarajan et al., 1996). As human embryonic kidney cells are epithelial cells and not closely related to cells of the immune system, cell-type specific differences in the TNF response might be responsible for the differences in NFjB inhibition by CAPE. In HEK cells, capsaicin was shown to be a partial inhibitor of TNF-induced NF-jB activation. A possible mechanism is the decrease of phosphorylated IjBa, which is
marked for degradation, and inhibition of p65 translocation and subsequent binding to jB sites (Zhang et al., 2003). MG-132 was an effective inhibitor of TNF-a and X-ray induced NF-jB activation in HEK cells. In interpreting these results, it should be considered that this proteasome inhibitor is not a specific NF-jB inhibitor. As the degradation of many cellular proteins occurs at the proteasome, its inhibition by MG-132 influences many pathways in
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Fig. 4. NF-jB-dependent expression of the reporter protein d2EGFP after X-ray exposure in presence of the proteasome inhibitor MG-132 (FACS analysis of d2EGFP expression). The HEK-pNF-jB-d2EGFP/Neo clone L2 cells were seeded into petri dishes in medium containing 10% FBS, grown for 2 days, and irradiated with 150 kV X-rays at 37 °C. TNF-a (10 ng/ml) was used as positive control for NF-jB activation. For proteasome inhibition during and after irradiation, 2 lmol/l MG-132 was added one hour before irradiation. Bars show the standard deviation of four independent experiments. t Tests were performed with irradiated treated versus mock irradiated cells and with TNF-a treated versus mock treated cells, to show the effect of X-ray exposure or TNF-a treatment on NF-jB activation in absence of MG-132 (black ). T tests demonstrating the inhibitor effectiveness (no significant NF-jB activation in presence of the inhibitor) were operated with inhibitor treated and irradiated cells versus in absence of MG-132 mock irradiated/TNF-a treated cells (gray ). significant (p < 0.05), highly significant (p < 0.01), no symbol – not significant.
mammalian cells by altering the level of signaling molecules, transcription factors and splicing regulatory proteins (Katzenberger et al., 2009). For example, the stability of complexes formed in response to double strand breaks consisting of ATM, CHK2 and p53 or of ATR and CHK1 is controlled by the 26S proteasome. Therefore, treatment of tumor cells with MG-132 prior to irradiation radiosensitizes them (Choudhury et al., 2006). This effect might be explained also by the NF-jB inhibition elicited by MG132. High MG-132 concentrations (100 lmol/l) are not only toxic to tumor cells, but also to normal peripheral neutrophil granulocytes, resulting in 100% apoptosis after 20 h incubation (Ward et al., 1999). Short-term viability of HEK cells was not strongly decreased in this study. The long-term viability of cells treated with MG-132 may be reduced as other transcription factors than NF-jB, cyclins and enzymes are degraded by the proteasome (Lee et al., 1998). In other cases, proteasome inhibition induces the heat-shock response in mammalian cells (Lee et al., 1998) and may therefore result in an adaptive response to various stressors including ionizing radiation.
The paradoxical NF-jB activation by MG-132 in control cells after 20 h was also found by other researchers in some cell lines after prolonged incubation: The authors demonstrate an NF-jB dependent luciferase expression 14–16 h after treatment of a human endometrial adenocarcinoma cell line (Ishikawa cells) carrying a NF-jB-luciferase reporter construct with 0.5 lmol/l MG-132 (Dolcet et al., 2006). Eighteen hours after treatment of human colon cancer HT-29 cells with 0.3–10 lmol/l MG-132, interleukin-8 (IL-8) and growth-related oncogene protein a (Gro-a) expression increased NF-jB activation (Nemeth et al., 2004). Although the mechanism leading to NF-jB activation after treatment of the cells with proteasome inhibitors is unclear, Nemeth et al. (2004) proposed the following explanations: Proteasome inhibitors can activate stress kinases, including c-Jun terminal kinase, p42/p44 mitogen-activated protein kinase or p38. These pathways are known to mediate NF-jB signaling. Also, the FasL– Fas pathway could be involved, since it can be activated by proteasome inhibitors and FasL–Fas interaction can activate the IKK complex and hence NF-jB. The NF-jB inhibitor SN50 should prevent the translocation of NF-jB dimers containing p50 by binding to its NLS (Lin et al., 1995). In myeloma cell lines, 20 lmol/l SN50 slightly reduced cell viability, as determined by the MTT assay (Goel et al., 2005). In rat endothelial cells, 18 lmol/l SN-50 effectively suppressed TNF-a induced (via NF-jB) expression of bone morphogenetic protein 2 (BMP-2) (Csiszar et al., 2005). In HEK cells, SN50 was ineffective; it even enhanced NF-jB activation. One possible explanation for the ineffectiveness of SN50 is that redundant processes could allow HEK cells to escape from NF-jB inhibition. 4.3. Chemical inhibition of the NF-jB Activation by sparsely ionizing radiation Activation of the transcription factor NF-jB has been shown to play a key role in the anti-apoptotic pathway of apoptosis in X-irradiated cells (Piette et al., 1997). High doses are required to induce nuclear translocation of p65 and subsequent increased reporter gene expression in HEK cells. This activation can be suppressed using the proteasome inhibitor MG-132. As NF-jB activates prosurvival pathways in many cell types, this suppression is not only relevant for long-term manned space missions, resulting in exposure to radiation qualities much more effective in NF-jB activation (Baumstark-Khan et al., 2005), but also for radiotherapy. Proteasome inhibitors can induce apoptosis in tumor cells or sensitize them to radiation or chemotherapy (Schenkein, 2002). 4.4. Outlook The study presented here revealed the potency of several chemical NF-jB inhibitors in human embryonic kidney cells. CAPE and capsaicin, which interfere with p65
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DNA binding and IjBa phosphorylation, respectively, partially inhibited the pathway, or complete inhibition was reached only at highly cytotoxic concentrations. The proteasome inhibitor MG-132 most effectively suppressed NF-jB activation, having the drawback of inhibiting other processes in the cell. Therefore, this work is a considerable contribution to the understanding of the pharmacological targets for modulation of the NF-jB pathway activated in response to exposure to ionizing radiation. In future experiments, the proteasome inhibitor MG-132 will be applied in experiments with heavy ions to analyze the involvement of the NF-jB pathway in the cellular response to densely ionizing radiation. Acknowledgement The authors thank Susanne Bogner for technical support. References Aggarwal, B.B., Sethi, G., Nair, A., Ichikawa, H. Nuclear factor-kappaB: A holy grail in cancer prevention and therapy. Current Signal Transduction Therapy 1, 25–52, 2006. Ait-Ghezala, G., Volmar, C.H., Frieling, J., Paris, D., Tweed, M., Bakshi, P., Mullan, M. CD40 promotion of amyloid beta production occurs via the NF-kappaB pathway. Eur. J. Neurosci. 25, 1685–1695, 2007. Baichwal, V.R., Baeuerle, P.A. Activate NF-kappa B or die? Curr. Biol. 7, R94–R96, 1997. Baumstark-Khan, C., Hellweg, C.E., Arenz, A., Meier, M.M. Cellular monitoring of the nuclear factor kappa B pathway for assessment of space environmental radiation. Radiat. Res. 164, 527–530, 2005. Bhattacharjee, Y. Medicine. Drug bestows radiation resistance on mice and monkeys. Science 320, 163, 2008. Bladh, L.G., Liden, J., Pazirandeh, A., Rafter, I., Dahlman-Wright, K., Nilsson, S., Okret, S. Identification of target genes involved in the antiproliferative effect of glucocorticoids reveals a role for nuclear factor-(kappa)B repression. Mol. Endocrinol. 19, 632–643, 2005. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C. Green fluorescent protein as a marker for gene expression. Science 263, 802– 805, 1994. Chastel, C., Jiricny, J., Jaussi, R. Activation of stress-responsive promoters by ionizing radiation for deployment in targeted gene therapy. DNA Repair (Amst.) 3, 201–215, 2004. Chen, L.F., Greene, W.C. Regulation of distinct biological activities of the NF-kappaB transcription factor complex by acetylation. J. Mol. Med. 81, 549–557, 2003. Choudhury, A., Cuddihy, A., Bristow, R.G. Radiation and new molecular agents part I: targeting ATM-ATR checkpoints, DNA repair, and the proteasome. Semin. Radiat. Oncol. 16, 51–58, 2006. Csiszar, A., Smith, K.E., Koller, A., Kaley, G., Edwards, J.G., Ungvari, Z. Regulation of bone morphogenetic protein-2 expression in endothelial cells: role of nuclear factor-kappaB activation by tumor necrosis factor-alpha, H2O2, and high intravascular pressure. Circulation 111, 2364–2372, 2005. Dolcet, X., Llobet, D., Encinas, M., Pallares, J., Cabero, A., Schoenenberger, J.A., Comella, J.X., Matias-Guiu, X. Proteasome inhibitors induce death but activate NF-kappaB on endometrial carcinoma cell lines and primary culture explants. J. Biol. Chem. 281, 22118–22130, 2006. Fitzpatrick, L.R., Wang, J., Le, T. Caffeic acid phenethyl ester, an inhibitor of nuclear factor-kappaB, attenuates bacterial peptidoglycan polysaccharide-induced colitis in rats. J. Pharmacol. Exp. Ther. 299, 915–920, 2001.
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Advances in Space Research 44 (2009) 907–916 www.elsevier.com/locate/asr
Up-stream events in the nuclear factor jB activation cascade in response to sparsely ionizing radiation Christine E. Hellweg *, Britta Langen, Galina Klimow, Roland Ruscher, Claudia Schmitz, Christa Baumstark-Khan, Gu¨nther Reitz Division of Radiation Biology, Institute of Aerospace Medicine, German Aerospace Center (DLR), Linder Hoehe, 51147 Koeln, Germany Received 10 October 2008; received in revised form 13 July 2009; accepted 13 July 2009
Abstract Radiation is a potentially limiting factor for manned long-term space missions. Prolonged exposure to galactic cosmic rays may shorten the healthy life-span after return to Earth due to cancer induction. During the mission, a solar flare can be life threatening. For better risk estimation and development of appropriate countermeasures, the study of the cellular radiation response is necessary. Since apoptosis may be a mechanism the body uses to eliminate damaged cells, the induction by cosmic radiation of the nuclear antiapoptotic transcription factor nuclear factor jB (NF-jB) could influence the cancer risk of astronauts exposed to cosmic radiation by improving the survival of radiation-damaged cells. In previous studies using a screening assay for the detection of NF-jB-dependent gene induction (HEK-pNF-jB-d2EGFP/Neo cells), the activation of this transcription factor by heavy ions was shown [BaumstarkKhan, C., Hellweg, C.E., Arenz, A., Meier, M.M. Cellular monitoring of the nuclear factor kappa B pathway for assessment of space environmental radiation. Radiat. Res. 164, 527–530, 2005]. Studies with NF-jB inhibitors can map functional details of the NF-jB pathway and the influence of radiation-induced NF-jB activation on various cellular outcomes such as survival or cell cycle arrest. In this work, the efficacy and cytotoxicity of four different NF-jB inhibitors, caffeic acid phenethyl ester (CAPE), capsaicin, the proteasome inhibitor MG-132, and the cell permeable peptide NF-jB SN50 were analyzed using HEK-pNF-jB-d2EGFP/Neo cells. In the recommended concentration range, only CAPE displayed considerable cytotoxicity. CAPE and capsaicin partially inhibited NF-jB activation by the cytokine tumor necrosis factor a. MG-132 completely abolished the activation and was therefore used for experiments with X-rays. NF-jB SN-50 could not reduce NF-jB dependent expression of the reporter destabilized Enhanced Green Fluorescent Protein (d2EGFP). MG-132 entirely suppressed the X-ray induced NF-jB activation in HEK-pNF-jB-d2EGFP/Neo cells. In conclusion, the degradation of the inhibitor of NF-jB (IjB) in the proteasome is essential for X-ray induced NF-jB activation, and MG-132 will be useful in studies of the NF-jB pathway involvement in the cellular response to heavy ion exposure and other space-relevant radiation qualities. Ó 2009 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: Nuclear factor jB inhibitors; Ionizing radiation; Human cells; Proteasome; Capsaicin; Caffeic acid phenethyl ester
1. Introduction During space missions, astronauts are exposed to not only greater amounts of natural radiation than they receive
*
Corresponding author. Tel.: +49 2203 601 3243; fax: +49 2203 61970. E-mail addresses: [email protected] (C.E. Hellweg), claudia. [email protected] (C. Schmitz), [email protected] (C. BaumstarkKhan), [email protected] (G. Reitz).
on earth but also to a differing radiation quality, which can result in immediate and long-term risks. The cellular radiation response is essential to the development of acute and chronic radiation exposure sequels. A possible modulator of the cellular radiation response might be the ubiquitously expressed transcription factor nuclear factor jB (NF-jB), which has been shown to play a role in apoptosis regulation, mostly by inducing the expression of anti-apoptotic proteins in many cell types (Baichwal and Baeuerle, 1997; Chen and Greene, 2003; Kucharczak et al., 2003).
0273-1177/$36.00 Ó 2009 COSPAR. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.asr.2009.07.009
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In order to examine the role of NF-jB in the cellular response to space-relevant radiation qualities, a stably transfected human embryonic kidney cell line (HEKpNF-jB-d2EGFP/Neo cells) indicating NF-jB activation by increased expression of the reporter destabilized Enhanced Green Fluorescent Protein (d2EGFP, Chalfie et al., 1994; Li et al., 1998) was constructed and evaluated (Hellweg et al., 2003). HEK cells are a useful tool to study many different processes in human cells, as they can easily be transiently and stably transfected. This allows reporter gene studies or functional studies with overexpression of the proteins in question (examples: Sung et al., 2009; Lodeiro et al., 2009). Many studies, including own investigations, have shown that the NF-jB pathway is functional in HEK cells (examples: Muroi and Tanamoto, 2008; Rajagopal et al., 2008; Ait-Ghezala et al., 2007; Muscat et al., 2007; Simon and Samuel, 2007; Bladh et al., 2005; Matsuda et al., 2003; Hellweg et al., 2006). The HEK-pNF-jB-d2EGFP/Neo cell line demonstrates the translocation of NF-jB containing p65 to the cell nucleus, its binding to NF-jB-dependent promoters and subsequent increased mRNA transcription by an increase in production of the reporter protein d2EGFP. The dose effect relationship for NF-jB activation in HEK cells in response to several known NF-jB activators such as tumor necrosis factor a (TNF-a), interleukin 1b (IL-1b), phorbol ester and lipopolysaccharide was previously examined (Hellweg et al., 2006), revealing the functionality of the NF-jB sub-pathways which start at membrane receptors (IL-1 and TNF-a receptors). NF-jB activation by DNA damaging agents was also detected using this stably transfected cell line, showing the functionality of the NF-jB subpathway which starts in the cell nucleus from DNA double strand breaks induced by the topoisomerase I inhibitor camptothecin. Camptothecin induces the NF-jB dependent d2EGFP expression in this cell line with slower kinetics compared to cytokines (Hellweg et al., 2006). Different radiation qualities such as heavy ions with a medium linear energy transfer (LET 270 keV/lm), X-rays and Pm-147, a b-emitter, were shown to activate the NF-jB pathway in this cell line (Baumstark-Khan et al., 2005; Hellweg and BaumstarkKhan, 2007; Hellweg et al., 2007). Activation of the NFjB pathway is supposed to play a role in the negative regulation of apoptosis in many cell types, which in turn would favor survival of cells with residual DNA damage. This again would result in survival of mutation-prone cells. Furthermore, the production of NF-jB-regulated inflammatory cytokines could initiate a tissue reaction; production of NF-jB dependent degrading enzymes could facilitate tumor progression and metastasis of cancer cells. Therefore, further investigation of the NF-jB pathway activated in response exposure to ionizing radiation might reveal possible pharmacological targets for modulation of the cellular radiation response. As the NF-jB pathway was identified to be a central element in the pathogenesis of inflammatory diseases and also in therapy resistance of
tumors (Chastel et al., 2004), many different NF-jB inhibitors with varying specificity and efficiency are described. A detailed overview of the involvement of NF-jB in various diseases and a list of agents that inhibit NF-jB activation is given by Aggarwal et al. (2006). Bhattacharjee (2008) describes the influence of NF-jB modulators on radiation resistance. Caffeic acid phenethyl ester (CAPE), a component of propolis from honeybee hives, belongs to the group of natural chemopreventive agents which are also potent NF-jB inhibitors, such as resveratrol and curcumin (Aggarwal et al., 2006). It has been used in traditional medicine due to its anti-inflammatory and anti-oxidative potential. The efficacy of CAPE in inhibiting NF-jB activation and proinflammatory production has been demonstrated in various animal models of inflammation (Fitzpatrick et al., 2001; Orban et al., 2000). CAPE was shown to suppress the binding of the p50–p65 complex to the DNA (Natarajan et al., 1996). The NF-jB suppressive effect of CAPE was also present in vivo in irradiated rats and resulted in a reduced inflammatory response (Linard et al., 2004). Capsaicin, a homovanillic acid derivative, is an active component of the red pepper of the genus Capsicum and has been found to protect against experimentally induced mutagenesis and tumorigenesis (Surh et al., 1998; Watson et al., 1988). In vitro, capsaicin has been found to inhibit the growth of various immortalized and malignant cells (Morre et al., 1995; Takahata et al., 1999; Zhang et al., 2003) and induce apoptosis in transformed cells (Macho et al., 1999; Wolvetang et al., 1996). In addition, capsaicin is also found to be a potent inhibitor of TNF-mediated NF-jB activation in human myeloid ML-1a cells (Singh et al., 1996). Many inflammatory mediators (e.g. TNF) trigger NFjB activation by causing the phosphorylation of the inhibitor of NF-jB (IjB), which leads to its rapid ubiquitination and degradation. The proteasomal degradation of IjB proteins is an essential step in the activation of NF-jB not only in the canonical NF-jB pathway, but also in the atypical pathway which is activated in response to DNA double strand breaks (Habraken and Piette, 2006). Pharmacological inhibitors of the proteasome, which is responsible for degradation of cytosolic proteins, are available since 1994 (Lee and Goldberg, 1998). The activity of the 26S proteasome can be suppressed by the peptide aldehydes, such as Cbz-leu-leu-leucinal (MG-132) (Lee et al., 1998; Schenkein, 2002), and therefore, degradation of IjB proteins is blocked, leading to the accumulation of IjB in a phosphorylated form. Degradation of IjB allows the liberated p50– p65 complex to translocate into the nucleus and activate gene transcription. Other inhibitors are synthetic peptides interacting with the agonist induced nuclear translocation of NF-jB (Lin et al., 1995). The cell permeable peptide NF-jB SN50 carries the p50 nuclear localization sequence (NLS) bound to the hydrophobic region of the signal peptide of Kaposi fibroblast growth factor which interacts with lipid bilayers.
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Thus, the recognition mechanism for the p50 NLS is blocked and translocation of p50 containing NF-jB dimers is inhibited (Lin et al., 1995). These inhibitors can suppress radiation-induced NF-jB activation, especially the strong activation which was observed after exposure of HEK cells to high-LET radiation (Baumstark-Khan et al., 2005). Studies with NF-jB inhibitors targeting different components of the pathway can reveal the architecture of the radiation-induced NFjB pathway. Furthermore, the influence of radiationinduced NF-jB activation on various cellular outcomes such as survival or cell cycle arrest can be assessed by NF-jB inhibition. The cytotoxicity and potency of these inhibitors in suppressing heavy-ion-radiation induced NF-jB activation is unknown. As TNF-a is a strong NFjB activator, and the down-stream pathway of the TNFa receptor and the DNA double strand induced NF-jB pathway share many components – IjBa, IjB kinase (IKK) consisting of NEMO, IKK-a, IKK-b and ELKS, the 26S proteasome and the NF-jB subunits p50 and p65 (Habraken and Piette, 2006) – the potency of the inhibitors can be monitored in TNF-a treated HEK cells. In this study, the potency and cytotoxicity of several chemical inhibitors (Table 1) of different components of the NF-jB pathway was determined in the stably transfected NF-jB reporter cell line. Reduction in vitality after incubation with the inhibitors was measured using the MTT assay. Concerning the inhibitory effects, first their interference with TNF-a activated NF-jB was determined. Inhibitors which effectively blocked this pathway were applied in experiments with ionizing radiation. X-rays (150 kV) were used as model radiation.
2. Materials and methods 2.1. Cell line and growth conditions Human Embryonic Kidney (293/HEK) cells (CRL1573) were obtained from the American Type Culture Collection, Manassas, VA, USA. Construction of the plasmid
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pNF-jB-d2EGFP/Neo and stable transfection of HEK cells was already delineated in detail (Hellweg et al., 2003). The vector contains a synthetic promoter consisting of four j enhancer elements (jB4 = 4 NF-jB response elements) and the thymidine kinase minimal promoter for controlling expression of d2EGFP in response to NF-jB activation after challenging the cells with chemicals or radiation. Cells were grown under standard conditions in 80 cm2 flasks (Nunc, Wiesbaden, Germany) in a-MEMmedium (modified MEM, Biochrom KG, Berlin, Germany), with 10% fetal bovine serum (FBS) at 37 °C, saturated humidity and in a 5% CO2/95% air atmosphere. Cultures were split (1:20) every 7 days using standard detachment procedures (0.05% trypsin containing 0.02% EDTA solution, Biochrom KG, Berlin, Germany). Medium was changed after 4 days. Culture vessels were coated with poly-D-lysine for 5 min at room temperature and washed with PBS three times prior to seeding of HEK cells.
2.2. Inhibition of the NF-jB pathway NF-jB signaling pathways can be inhibited at different points by various agents. An overview of the inhibitors used and their modes of action are given in Table 1. Capsaicin was prepared freshly before use and dissolved in ethanol, then mixed into complete media. Ethanol concentration in the medium was maintained below 0.1 vol.% and this concentration was not found to influence cell growth. CAPE and MG-132 were dissolved in DMSO, aliquoted and stored at 20 °C until use. SN50 was dissolved in sterile deionised water and stored at 20 °C. The IC50 of MG-132 is a few micromolar for the inhibition of proteolysis in cultured cells (Lee et al., 1998). Toxicity of inhibitors was tested using the MTT test (Section 2.3) and the efficacy of NF-jB inhibition was analyzed in HEK-pNF-jB-d2EGFP/Neo L2 cells treated with the respective inhibitor and the NF-jB activator TNF-a. Therefore, HEK cells were seeded in a density of 3 104 cells per cm2 with 1 ml complete a-medium in poly-D-lysine coated 24-well plates. After 2 days, cells reached 30–40%
Table 1 Inhibitors of the NF-jB signaling pathway. Inhibitor
Mode of action
CAPE (caffeic acid phenylester)a
Specific p65 inhibitor, decreases NF-jB DNA binding and promoter activity (Joo et al., 2003; Natarajan et al., 1996) Decreases phosphorylated IjBa, which is marked for degradation, and inhibition of p65 translocation and its subsequent binding to jB sites (Zhang et al., 2003) Proteasome inhibitor (Munshi et al., 2004) which prevents the degradation of IjB proteins by the proteasome
Capsaicin (trans-8-methyl-N-vanillyl-6nonenamide)a MG-132 (carbobenzoxy-L-leucyl-Lleucyl-L-leucinal)b NF-jB SN50c
a b c
Cell permeable peptide carrying the p50 nuclear localization sequence (NLS) bound to the hydrophobic region of the signal peptide of Kaposi fibroblast growth factor which interacts with lipid bilayers – the recognition mechanism for the p50 NLS is blocked and translocation p50 containing NF-jB dimers is inhibited (Lin et al., 1995)
Sigma–Aldrich, Taufkirchen, Germany. Calbiochem, San Diego, CA, USA. Biomol GmbH, Hamburg, Germany.
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confluence. The NF-jB activation potential was shown to be maximal in this confluence range (Hellweg et al., 2006). The inhibitors were added in concentration ranges recommended by the literature and as determined by the cytotoxicity test. The solvents (ethanol, DMSO, sterile deionised water) were added to controls. After pre-incubation with the inhibitor for 1 h, TNF-a stock solution (10 ng/ll in PBS) was added to a final concentration of 0.6 nmol/l. Cells were incubated for another 20 h and prepared for FACS analysis (Section 2.5). 2.3. MTT test HEK-pNF-jB-d2EGFP/Neo L2 cells were seeded into polylysin-coated 96-well plates at a density of 3 104 cells per cm2 in 200 ll a-medium per well. 48 h later, inhibitors in increasing concentration were added. After 20 h, cells in four wells were killed by adding pure DMSO to generate the blank, medium was exchanged in all wells by serum free medium containing 0.5 mg/ml MTT reagent (3-(4,5dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma M5655, Taufkirchen, Germany) and cells were incubated at 37 °C for one hour. Medium was removed and the formazan crystals were dissolved in DMSO containing 0.6% v/v acetic acid and 10% w/v sodium dodecyl sulphate (SDS) by mixing for 20 min. Absorption was measured at 562 nm in a microplate reader (Lambda Fluoro 320 plus, MWG Biotech, Ebersberg, Germany). Relative absorption was calculated according to the following formula: relative absorption ¼
ODtreated ODblank ODuntreated ODblank
ð1Þ
OD = optical density at 562 nm. 2.4. Irradiation of human cells For radiation challenging, cells were seeded into poly-Dlysine-coated (Section 2.1) petri dishes (diameter 3 cm) in a density of 3 104 cells per cm2 and were incubated for 2 days resulting in 30–40% confluence. Cells were preincubated with the selected inhibitors (2 lmol/l MG-132) as described in Section 2.2. Cells were irradiated at 37 °C on a warming device coupled to a thermostat (mgw Lauda RDS RC20, Lauda-Ko¨nigshofen, Germany) with X-rays generated by an X-ray unit (Mu¨ller Typ MG 150, MCN 165, Philips, Hamburg, Germany) operated at 150 kV and 19 mA yielding a dose rate of 2 Gy/min (focus-object distance: 312 mm). TNF-a treated cells served as positive control of NF-jB activation. After incubation for 20 h, cells were prepared for FACS analysis (Section 2.5). 2.5. Flow cytometry Distribution patterns of fluorescent protein expressing cells were analyzed using a fluorescence activated cell scanner (FACS, Becton–Dickinson; San Jose, CA, USA) equipped with an argon laser (488 nm) as excitation source
for d2EGFP. The samples’ forward and sideward scatter and d2EGFP fluorescence in fluorescence channel FL1 were determined and analyzed using the CellQuest software (version 1.2, Becton–Dickinson). Samples of cells treated with different agents were prepared as follows: cells were detached from the growth surface using trypsine and fixed with cold 3.5% formaldehyde in PBS for 30 min at 4 °C. The fixative was diluted with PBS (1:3) and cells were stored at 4 °C. Before FACS analysis, cells were centrifuged and resuspended in PBS. Cells (2 104) were analyzed at a rate of 200–600 cells per second. The markers M1 (EGFP()) and M2 (EGFP(+)) were set by means of untreated and TNF-a treated HEK-pNF-jB-d2EGFP/ Neo cells. The population fractions displaying fluorescence intensities in the zone of marker M2 (EGFP(+)-cells) were used as a measure of induction. 2.6. Measurement of NF-jB activation Cells were seeded into petri dishes (diameter 6 cm), incubated for 2 days, and treated with 0.6 nmol/l TNF-a or exposed to X-rays (Section 2.4). After incubation for up 4 h, petri dishes were washed with ice-cold PBS, and cells were scraped in 3 ml ice-cold PBS, centrifuged, lysed using the lysis buffer provided by the TransAMTM supplier and stored at 80 °C until analysis. Protein concentration was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Mu¨nchen, Germany), based on the Bradford dye-binding procedure. Activated NF-jB in the cell lysates (adjusted for 7.5 lg total protein) was measured using the NF-jB p65 Transcription Factor Assay Kit (TransAMTM, Active Motif Europe, Rixensart, Belgium), containing an olignucleotide with a NF-jB consensus binding site for p65 dimers (50 -GGGACTTTCC-30 ). Results were expressed as optical density measured at 450 nm per lg protein. As internal controls, lysates were incubated with mutated and wildtype oligonucleotides. The experiments were repeated three times. 2.7. Statistics Each experiment was repeated up to five times with one to three replicates each. Means, standard errors and significance levels in the t test were calculated with MicrosoftÒ Office Excel 2003. 3. Results As activation of the NF-jB pathway represents one important cellular stress response with anti-apoptotic functions which can be involved in the cellular radiation response, we evaluated several inhibitors of different steps in the NF-jB pathway. For that purpose, cytotoxicity and potential of suppression of NF-jB activation were tested using a fluorescent screening assay for NF-jB activation dependent gene expression, based on recombinant
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human cell lines. Using this HEK-pNF-jB-d2EGFP/Neo clone, NF-jB activation in presence of the inhibitors was measured by flow cytometric analysis of d2EGFP in cells treated with TNF-a, or exposed to X-rays. 3.1. Up-stream events in the NF-jB pathway To investigate a crucial step in the pathway leading finally to the increased d2EGFP expression, the activation of NF-jB by X-rays was verified by means of an oligonucleotide based NF-jB-ELISA (Fig. 1). Incubation with TNF-a resulted in an increased NF-jB binding to the oligonucleotide (jB binding sequence) 1–4 h after addition of the cytokine. Controls incubated with a mutated jB site oligonucleotide showed no competition in p65 binding in treated and untreated cells, while addition of an excess amount of wildtype oligonucleotide nearly completely abolished p65 binding. Exposure of HEK cells to 150 kV X-rays resulted in a dose-dependent p65 translocation 2 and 4 h after irradiation. Even after exposure to a high radiation dose of 16 Gy, activation was less pronounced compared to TNF-a-treated cells.
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3.2. Chemical inhibition of the NF-jB pathway at different levels 3.2.1. Cytotoxicity of the inhibitors and of the cytokine TNF-a In order define the concentration range in which the inhibitors under investigation suppress NF-jB-dependent gene expression at an acceptable cell viability level (>80%), the cytotoxicity of the inhibitors was assessed by means of the MTT test. For functional testing, combined treatment of cells with the NF-jB activator TNF-a and the inhibitor was necessary. Therefore, cytotoxicity of both was assessed using the MTT test 20 h after addition of the agents (Fig. 2). This test measures the activity of mitochondrial dehydrogenases and is used as marker of cell viability. In a wide concentration range, TNF-a elicits no cytotoxicity in HEK cells (Fig. 2A). The inhibitor of NF-jB translocation SN50 even enhances cellular viability, only at the highest tested concentration, viability is slightly reduced to 90%. E-capsaicin has no significant effect on cell viability even at the highest concentrations tested. Incubation of HEK cells with MG-132 with concentrations up to 64 lmol/l affected the viability moderately, but significantly. CAPE displays the strongest cytotoxic effect in the examined concentration range, with a reduction to 20% at the highest dose (Fig. 2B). 3.2.2. Inhibition of TNF-a induced NF-jB activation CAPE is a partial inhibitor of TNF-a induced NF-jB activation in the examined dose range, with concentrations above 12.5 lmol/l, the inhibition ranges around 50%. Further dose increase up to 300 lmol/l does not enhance the inhibitory effect (Fig. 3 CAPE). The inhibitory effect of capsaicin is also only partial (Fig. 3 Capsaicin), already with the lowest tested concentration (1 lmol/l) the percentage of d2EGFP-expressing cells drops from 60% to 30%, with no further enhancement with dose increase. There is a slight increase of d2EGFP expression in control cells with increasing CAPE and capsaicin concentrations. With doses of 2 lmol/l and higher, MG-132 is a potent inhibitor of TNF-a induced NF-jB activation, resulting in nearly total abolishment of d2EGFP expression to the control level (Fig. 3 MG-132). Incubation with SN50 increases d2EGFP to a higher level, while control cells remain unaffected (Fig. 3 SN50).
Fig. 1. Translocation of the NF-jB subunit p65 in the nucleus after exposure to X-rays (TransAMTM p65 Oligo ELISA of nuclear extracts). HEK-pNF-jB-d2EGFP/Neo clone L2 cells were seeded into petri dishes in medium containing 10% FBS, grown for 2 days, and irradiated with 150 kV X-rays at 37 °C. TNF-a (10 ng/ml) was used as positive control for NF-jB activation. For control of binding specificity, nuclear extracts of solvent and of TNF-a-treated cells were incubated with excess wild type oligonucleotide with p65 binding specificity (wt Oligo), or with a mutated oligonucleotide without p65 binding capacity (mut Oligo). Bars show the standard deviation of three independent experiments (2 and 4 h).
3.2.3. Inhibition of X-ray induced NF-jB activation As MG-132 was shown to be the most potent NF-jB inhibitor in HEK cells, its effectiveness in suppression of radiation-induced NF-jB activation was examined. After exposure to X-rays, an increase in NF-jB activation was seen with high doses (P8 Gy) and comprises a small part of the cell population (Fig. 4). During proteasome inhibition with 2 lmol/l MG-132, this response is suppressed. MG-132 incubation results in a slight, but not significant increase of d2EGFP expression in mock irradiated cells.
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pathway in the cellular radiation response. Initially, it was shown that exposure to X-rays results in nuclear translocation of the NF-jB subunit p65. 4.1. Translocation after X-ray exposure In the canonical pathway, here initiated by TNF-a, through a series of intracellular steps, the activation of the receptor promotes the phosphorylation and subsequent dissociation of the inhibitor protein IjB from the inactive NF-jB complex, allowing liberated NF-jB to translocate to the nucleus. Once active and inside the nucleus, NFjB binds to the j enhancer element on the DNA and activates transcription of several apoptosis-related, cell growth-dependent, and B-cell-proliferation genes. Activation of NF-jB by ionizing radiation is thought to occur via ATM, which is activated in response to DNA double strand breaks. The pathway involves shuttling of NEMO between the cytoplasm and the nucleus and occurs with lower intensity and slower kinetics compared to the canonical pathway (Habraken et al., 2006). 4.2. Chemical inhibition of the NF-jB activation by TNF-a
Fig. 2. Effect of TNF-a (A) and inhibitors (B) on HEK cell viability as determined by the MTT test. TNF-a was used as positive control for NFjB activation in HEK-pNF-jB-d2EGFP/Neo cells in further experiments. The cells were seeded into microtiter plates in medium with 10% FBS, treated with TNF-a or inhibitors 2 days later, incubated for 20 h after addition, and treated with MTT reagent for one hour. Bars show standard errors of three independent experiments. t Tests were performed with TNF-a or inhibitor treated versus solvent treated cells. significant (p < 0.05), highly significant (p < 0.01), no symbol – not significant.
4. Discussion Low cytotoxicity and high inhibitory efficiency of the proteasome inhibitor MG-132 suggest this peptide aldehyde as a useful tool in studying the role of the NF-jB
In mammalian systems, the NF-jB signal transduction pathway is induced by stimulation of the TNF or IL-1 (or other) lymphokine receptors by their respective ligands. In the cellular monitoring system used here, the HEKpNF-jB-d2EGFP/Neo cells show increased NF-jB-dependent d2EGFP expression in response to the known NF-jB activator TNF-a. The inhibitors differ strongly in the specificity for NF-jB pathway inhibition, which might partially explain the differences in their cytotoxicity. The proteasomal inhibitor MG132 disturbs the metabolism of all proteins and therefore has a potentially higher toxicity than the highly specific peptide SN50, which was even shown to have a paradoxical effect in HEK cells. Capsaicin shows no cytotoxic effect in the examined dose range. CAPE was shown to strongly induce apoptosis in cells of the immune system (Orban et al., 2000), which might explain its cytotoxic effect in HEK cells. In this work, CAPE was shown to be a partial inhibitor of TNF-a induced NF-jB activation with considerable cytotoxicity. Linard et al. (2004) show that CAPE treatment after irradiation did not completely inhibit the NFjB p65 DNA-binding activity, although it had some effects on proinflammatory cytokine production. The selective blockade of the NF-jB pathway did not inhibit the radiation-induced increase in TNF-a expression, and it reduced IL-1b expression only slightly. Similar observations have been already reported in alveolar epithelial cells, where CAPE did not totally abrogate lipopolysaccharide-mediated TNF-a biosynthesis (Haddad and Land, 2001). A total blockade of TNF-induced NF-jB activation was achieved in the human monocyte cell line U937, with a maximal effect at 90 lmol/l (Natarajan et al., 1998;
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Fig. 3. Inhibition of NF-jB activation by TNF-a in HEK-pNF-jB-d2EGFP/Neo cells. The cells were seeded into 24-well plates in medium with 10% FBS. Two days later, they were preincubated with increasing inhibitor concentrations (CAPE, Capsaicin, MG-132, NF-jB SN-50) for 1 h, treated with TNF-a, and collected for FACS analysis of d2EGFP expression 20 h after treatment. Bars show the standard deviation of up to five independent experiments. t Tests were performed with TNF-a treated versus untreated cells, both without inhibitor (dose 0 lmol/l), to show the effect of TNF-a alone. t Tests demonstrating the inhibitor effectiveness were operated with inhibitor treated cells versus cells without inhibitor, for each TNF-a treated (black ) and control cells (gray ). significant (p < 0.05), highly significant (p < 0.01), no symbol – not significant.
Natarajan et al., 1996). As human embryonic kidney cells are epithelial cells and not closely related to cells of the immune system, cell-type specific differences in the TNF response might be responsible for the differences in NFjB inhibition by CAPE. In HEK cells, capsaicin was shown to be a partial inhibitor of TNF-induced NF-jB activation. A possible mechanism is the decrease of phosphorylated IjBa, which is
marked for degradation, and inhibition of p65 translocation and subsequent binding to jB sites (Zhang et al., 2003). MG-132 was an effective inhibitor of TNF-a and X-ray induced NF-jB activation in HEK cells. In interpreting these results, it should be considered that this proteasome inhibitor is not a specific NF-jB inhibitor. As the degradation of many cellular proteins occurs at the proteasome, its inhibition by MG-132 influences many pathways in
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Fig. 4. NF-jB-dependent expression of the reporter protein d2EGFP after X-ray exposure in presence of the proteasome inhibitor MG-132 (FACS analysis of d2EGFP expression). The HEK-pNF-jB-d2EGFP/Neo clone L2 cells were seeded into petri dishes in medium containing 10% FBS, grown for 2 days, and irradiated with 150 kV X-rays at 37 °C. TNF-a (10 ng/ml) was used as positive control for NF-jB activation. For proteasome inhibition during and after irradiation, 2 lmol/l MG-132 was added one hour before irradiation. Bars show the standard deviation of four independent experiments. t Tests were performed with irradiated treated versus mock irradiated cells and with TNF-a treated versus mock treated cells, to show the effect of X-ray exposure or TNF-a treatment on NF-jB activation in absence of MG-132 (black ). T tests demonstrating the inhibitor effectiveness (no significant NF-jB activation in presence of the inhibitor) were operated with inhibitor treated and irradiated cells versus in absence of MG-132 mock irradiated/TNF-a treated cells (gray ). significant (p < 0.05), highly significant (p < 0.01), no symbol – not significant.
mammalian cells by altering the level of signaling molecules, transcription factors and splicing regulatory proteins (Katzenberger et al., 2009). For example, the stability of complexes formed in response to double strand breaks consisting of ATM, CHK2 and p53 or of ATR and CHK1 is controlled by the 26S proteasome. Therefore, treatment of tumor cells with MG-132 prior to irradiation radiosensitizes them (Choudhury et al., 2006). This effect might be explained also by the NF-jB inhibition elicited by MG132. High MG-132 concentrations (100 lmol/l) are not only toxic to tumor cells, but also to normal peripheral neutrophil granulocytes, resulting in 100% apoptosis after 20 h incubation (Ward et al., 1999). Short-term viability of HEK cells was not strongly decreased in this study. The long-term viability of cells treated with MG-132 may be reduced as other transcription factors than NF-jB, cyclins and enzymes are degraded by the proteasome (Lee et al., 1998). In other cases, proteasome inhibition induces the heat-shock response in mammalian cells (Lee et al., 1998) and may therefore result in an adaptive response to various stressors including ionizing radiation.
The paradoxical NF-jB activation by MG-132 in control cells after 20 h was also found by other researchers in some cell lines after prolonged incubation: The authors demonstrate an NF-jB dependent luciferase expression 14–16 h after treatment of a human endometrial adenocarcinoma cell line (Ishikawa cells) carrying a NF-jB-luciferase reporter construct with 0.5 lmol/l MG-132 (Dolcet et al., 2006). Eighteen hours after treatment of human colon cancer HT-29 cells with 0.3–10 lmol/l MG-132, interleukin-8 (IL-8) and growth-related oncogene protein a (Gro-a) expression increased NF-jB activation (Nemeth et al., 2004). Although the mechanism leading to NF-jB activation after treatment of the cells with proteasome inhibitors is unclear, Nemeth et al. (2004) proposed the following explanations: Proteasome inhibitors can activate stress kinases, including c-Jun terminal kinase, p42/p44 mitogen-activated protein kinase or p38. These pathways are known to mediate NF-jB signaling. Also, the FasL– Fas pathway could be involved, since it can be activated by proteasome inhibitors and FasL–Fas interaction can activate the IKK complex and hence NF-jB. The NF-jB inhibitor SN50 should prevent the translocation of NF-jB dimers containing p50 by binding to its NLS (Lin et al., 1995). In myeloma cell lines, 20 lmol/l SN50 slightly reduced cell viability, as determined by the MTT assay (Goel et al., 2005). In rat endothelial cells, 18 lmol/l SN-50 effectively suppressed TNF-a induced (via NF-jB) expression of bone morphogenetic protein 2 (BMP-2) (Csiszar et al., 2005). In HEK cells, SN50 was ineffective; it even enhanced NF-jB activation. One possible explanation for the ineffectiveness of SN50 is that redundant processes could allow HEK cells to escape from NF-jB inhibition. 4.3. Chemical inhibition of the NF-jB Activation by sparsely ionizing radiation Activation of the transcription factor NF-jB has been shown to play a key role in the anti-apoptotic pathway of apoptosis in X-irradiated cells (Piette et al., 1997). High doses are required to induce nuclear translocation of p65 and subsequent increased reporter gene expression in HEK cells. This activation can be suppressed using the proteasome inhibitor MG-132. As NF-jB activates prosurvival pathways in many cell types, this suppression is not only relevant for long-term manned space missions, resulting in exposure to radiation qualities much more effective in NF-jB activation (Baumstark-Khan et al., 2005), but also for radiotherapy. Proteasome inhibitors can induce apoptosis in tumor cells or sensitize them to radiation or chemotherapy (Schenkein, 2002). 4.4. Outlook The study presented here revealed the potency of several chemical NF-jB inhibitors in human embryonic kidney cells. CAPE and capsaicin, which interfere with p65
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DNA binding and IjBa phosphorylation, respectively, partially inhibited the pathway, or complete inhibition was reached only at highly cytotoxic concentrations. The proteasome inhibitor MG-132 most effectively suppressed NF-jB activation, having the drawback of inhibiting other processes in the cell. Therefore, this work is a considerable contribution to the understanding of the pharmacological targets for modulation of the NF-jB pathway activated in response to exposure to ionizing radiation. In future experiments, the proteasome inhibitor MG-132 will be applied in experiments with heavy ions to analyze the involvement of the NF-jB pathway in the cellular response to densely ionizing radiation. Acknowledgement The authors thank Susanne Bogner for technical support. References Aggarwal, B.B., Sethi, G., Nair, A., Ichikawa, H. Nuclear factor-kappaB: A holy grail in cancer prevention and therapy. Current Signal Transduction Therapy 1, 25–52, 2006. Ait-Ghezala, G., Volmar, C.H., Frieling, J., Paris, D., Tweed, M., Bakshi, P., Mullan, M. CD40 promotion of amyloid beta production occurs via the NF-kappaB pathway. Eur. J. Neurosci. 25, 1685–1695, 2007. Baichwal, V.R., Baeuerle, P.A. Activate NF-kappa B or die? Curr. Biol. 7, R94–R96, 1997. Baumstark-Khan, C., Hellweg, C.E., Arenz, A., Meier, M.M. Cellular monitoring of the nuclear factor kappa B pathway for assessment of space environmental radiation. Radiat. Res. 164, 527–530, 2005. Bhattacharjee, Y. Medicine. Drug bestows radiation resistance on mice and monkeys. Science 320, 163, 2008. Bladh, L.G., Liden, J., Pazirandeh, A., Rafter, I., Dahlman-Wright, K., Nilsson, S., Okret, S. Identification of target genes involved in the antiproliferative effect of glucocorticoids reveals a role for nuclear factor-(kappa)B repression. Mol. Endocrinol. 19, 632–643, 2005. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C. Green fluorescent protein as a marker for gene expression. Science 263, 802– 805, 1994. Chastel, C., Jiricny, J., Jaussi, R. Activation of stress-responsive promoters by ionizing radiation for deployment in targeted gene therapy. DNA Repair (Amst.) 3, 201–215, 2004. Chen, L.F., Greene, W.C. Regulation of distinct biological activities of the NF-kappaB transcription factor complex by acetylation. J. Mol. Med. 81, 549–557, 2003. Choudhury, A., Cuddihy, A., Bristow, R.G. Radiation and new molecular agents part I: targeting ATM-ATR checkpoints, DNA repair, and the proteasome. Semin. Radiat. Oncol. 16, 51–58, 2006. Csiszar, A., Smith, K.E., Koller, A., Kaley, G., Edwards, J.G., Ungvari, Z. Regulation of bone morphogenetic protein-2 expression in endothelial cells: role of nuclear factor-kappaB activation by tumor necrosis factor-alpha, H2O2, and high intravascular pressure. Circulation 111, 2364–2372, 2005. Dolcet, X., Llobet, D., Encinas, M., Pallares, J., Cabero, A., Schoenenberger, J.A., Comella, J.X., Matias-Guiu, X. Proteasome inhibitors induce death but activate NF-kappaB on endometrial carcinoma cell lines and primary culture explants. J. Biol. Chem. 281, 22118–22130, 2006. Fitzpatrick, L.R., Wang, J., Le, T. Caffeic acid phenethyl ester, an inhibitor of nuclear factor-kappaB, attenuates bacterial peptidoglycan polysaccharide-induced colitis in rats. J. Pharmacol. Exp. Ther. 299, 915–920, 2001.
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