Abstract

Nuclear factor kappa B (NF-κB) is a redox-associated transcription factor that is involved in the activation of survival pathways. We have previously shown that deoxycholate (DOC) activates NF-κB in hepatocytes and colon epithelial cells and that persistent exposure of HCT-116 cells to increasing concentrations of DOC results in the constitutive activation of NF-κB, which is associated with the development of apoptosis resistance. The mechanisms by which DOC activates NF-κB in colon epithelial cells, and whether natural antioxidants can reduce DOC-induced NF-κB activation, however, are not known. Also, it is not known if DOC can generate reactive oxygen species within mitochondria as a possible pathway of stress-related NF-κB activation. Since we have previously shown that DOC activates the NF-κB stress-response pathway in HCT-116 cells, we used this cell line to further explore the mechanisms of NF-κB activation. We found that DOC induces mitochondrial oxidative stress and activates NF-κB in HCT-116 cells through multiple mechanisms involving NAD(P)H oxidase, Na + /K + -ATPase, cytochrome P450, Ca ++ and the terminal mitochondrial respiratory complex IV. DOC-induced NF-κB activation was significantly ( P < 0.05) inhibited by pre-treatment of cells with CAPE, EGCG, TMS, DPI, NaN 3 , EGTA, Ouabain and RuR. The NF-κB-activating pathways, induced by the dietary-related endogenous detergent DOC, provide mechanisms for promotion of colon cancer and identify possible new targets for chemoprevention.

Introduction

Nuclear factor kappa B (NF-κB) is a redox-associated transcription factor ( 1 ) that is involved in the activation of survival pathways ( 2–4 ). NF-κB protects against apoptosis ( 5–8 ) and is constitutively elevated in many different types of cancer ( 9–13 ). NF-κB enhances tumor progression ( 14 , 15 ), in part, through the activation of inducible nitric oxide synthase and cyclooxygenase-2 ( 16 , 17 ) and the release of proliferative and anti-apoptotic cytokines ( 12 , 18 ). NF-κB can be activated through multiple mechanisms involving genotoxic stress ( 19 ), the activation of cell surface receptors ( 20–23 ), the generation of exogenous and endogenous reactive oxygen species (ROS) ( 22 , 24–27 ) and increased cytosolic levels of Ca ++ ( 27–35 ).

Persistent activation of NF-κB in the colon can lead to the production of pro-inflammatory molecules, resulting in colitis and, ultimately, colon cancer ( 7 , 16–18 ). The inflammatory diseases of the colon, Crohn's disease and ulcerative colitis, are associated with the constitutive activation of NF-κB in the pre-neoplastic colonic crypts and stroma ( 16 , 17 ). However, most cases of sporadic colon cancer are not associated with predisposing inflammatory conditions such as Crohn's disease and ulcerative colitis. From an epidemiologic standpoint, the consumption of a high-fat, low-fiber, low micronutrient Western-style diet is associated with increased colon cancer risk ( 36–39 ), which has been confirmed in an animal model in the absence of a carcinogen ( 40 ). One major factor associated with a high-fat, low-fiber diet is the endogenous production of bile acids from cholesterol ( 41 ). Primary conjugated bile acids are biotransformed by intestinal bacteria through a process of deconjugation and dehydroxylation to produce secondary, hydrophobic bile acids ( 42 ), the most prominent of which is deoxycholate (DOC).

DOC has pleiotropic effects on colon cells including the induction of oxidative stress, DNA damage and apoptosis [see recent review from our group ( 43 )]. We were the first to show that DOC activates NF-κB in hepatocytes ( 44 ), HCT-116 and HT-29 colon epithelial cells ( 45 , 46 ), which could be prevented by lazaroids ( 46 ), potent inhibitors of lipid peroxidation. We also reported that persistent exposure of HCT-116 cells to increasing concentrations of DOC results in the selection for cells having constitutive activation of NF-κB, which is associated with the development of apoptosis resistance ( 6 ). Apoptosis resistance is now considered to be an important condition that contributes to genomic instability and the initiation and progression of cancer ( 47–49 ).

What is not known are the mechanisms by which DOC activates NF-κB in colon epithelial cells and whether natural antioxidants can reduce DOC-induced NF-κB activation. Also, it is not known if DOC can generate ROS within mitochondria as a possible pathway of stress-related NF-κB activation in colon epithelial cells. In non-colon cell types, it has been shown that NF-κB can be activated by ROS-dependent and ROS–independent signaling pathways ( 1 , 24 , 50 ). Since DOC is known to cause membrane perturbation ( 51–53 ), we explored, in the present study, the activation of ROS-generating plasma membrane-associated enzymes by DOC, in addition to other hypothesis-driven mechanisms based on our studies ( 6 ) and those of others. We found that DOC activates NF-κB through multiple mechanisms involving NAD(P)H oxidase, Na + /K + -ATPase, cytochrome P450, Ca ++ and the terminal mitochondrial respiratory complex IV. These NF-κB-activating pathways, induced by the dietary-related endogenous detergent DOC, provide mechanisms for promotion of colon cancer and identify possible new targets for chemoprevention.

Materials and methods

Chemicals

Sodium azide (NaN 3 ) and the sodium salt of deoxycholic acid (NaDOC) were obtained from Sigma (St. Louis, MO). The following inhibitors, antioxidants and Ca ++ modulating agents were obtained from Calbiochem (La Jolla, CA): CAPE (caffeic acid phenylethyl ester), TMS [(E)-2,3′,4,5′-tetramethoxystilbene)], DPI (diphenyleneiodonium chloride), EGTA [ethyleneglycol- bis (β-aminoethyl)- N , N , N ′, N ′-tetraacetic acid], EGCG [(2R,3R)-2-(3,4,5-trihydroxyphenyl)-3,4,dihydro-1(H)-benzopyran-3,5,7-triol-3-(3,4,5-trihydroxybenzoate)], ouabain octahydrate (strophanthin G), RuR (non-permeable, ammoniated ruthenium oxychloride). MitoSOX™ Red mitochondrial superoxide indicator and Mitotracker Red ® (CMXRos) were obtained from Molecular Probes (Eugene, OR).

Cell culture

HCT-116 cells are derived from a colon carcinoma [American Type Culture Collection (ATCC), Rockville, MD; ATCC # CCL247], and serve as a useful model for studying NF-κB activation ( 50 ) and oxidative stress/DOC-induced apoptosis, as described previously ( 54 ). Persistent exposure of these cells to increasing concentrations of DOC also results in the constitutive activation of NF-κB ( 6 ). HCT-116 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal calf serum (Omega Scientific, Tarzana, CA), 1% MEM non-essential amino acids, 100 μg/ml streptomycin, 100 U/ml penicillin and 3.44 mg/ml l -glutamine. HCT-116 cells, growing in log phase, were plated into 24-well Falcon flat-bottom polystyrene plates (Becton Dickinson). For all treatments, only cells in approximate mid-logarithmic growth phase were used, so that comparisons between treatment groups would be valid and reproducible ( 55 ). Experiments were performed between passages 3 and 15, after dilution of the cells from stationary phase to 2 × 10 5 , and allowing cells to grow exponentially to 8 × 10 5 cells/ml. This density of exponentially growing cells placed the cells at 30–50% of their maximum stationary phase value. Media components were from Gibco BRL Life Technologies (Grand Island, NY). All experiments were repeated at least twice.

Assessment of oxidative stress within mitochondria

Mitochondrial stress within mitochondria was assessed using MitoSOX™ Red, a highly selective fluorescent probe for the detection of ROS generated within mitochondria. All incubations with DOC were performed at 37°C in a CO 2 incubator, and the fluorescent signal emitted from the oxidized MitoSOX™ Red reagent was detected using flow cytometry. HCT-116 cells were grown to mid-log phase, and dose–response curves were performed at 4 h incubation with varying concentrations of DOC (0.1–0.5 mM). The cells were then trypsinized and washed with fresh medium. MitoSOX™ Red was added to the washed cells at a final concentration of 5 μM in culture media and incubated at 37°C for 10 min. The cells were then washed with PBS. Fluorescence intensity was analyzed using a BD FACScan flow cytometer with excitation at 488 nm and emission at 620 nm. A minimum of 10 000 events was collected. Results are presented as single-parameter histograms or scattergrams of cellular events versus fluorescence intensity. The experiment was repeated twice, each time in triplicate. The flow cytometry data were evaluated using WinMDI 2.8 software. Statistical evaluation was assessed on triplicate values for control (untreated) and treatment groups using Student's t -test. The difference in the mean values between control and treatment groups was considered to be statistically significant at the 95% probability level.

Assessment of mitochondrial membrane potential (MMP) by flow cytometry

The lipophilic, cationic dye, Mitotracker Red ® (CMXRos) (Molecular Probes, Eugene, OR) was used to detect changes in level of MMP, as described previously ( 54 , 56 , 57 ). Control (untreated) cells and cells incubated with varying concentrations of DOC (0.1–0.5 mM) for 4 h were trypsinized and added to fresh media. CMXRos was added to the cell suspension at a final concentration of 100 nM and incubated for 30 min at 37°C. The cells were then analyzed by flow cytometry using excitation at 488 nm and emission at 600 nm. A minimum of 10 000 events was collected. Results are presented as single-parameter histograms. The experiment was repeated twice, each time in triplicate. The flow cytometry data were evaluated using WinMDI 2.8 software. Statistical evaluation was assessed on triplicate values for control (untreated) and treatment groups using Student's t -test. The difference in the mean values between control and treatment groups was considered to be statistically significant at the 95% probability level.

Assessment of non-lethal concentrations of inhibitors/antioxidants/Ca ++ modulators

Cells were pretreated for 24 h with a range of concentrations (obtained from the literature) of the particular inhibitor, antioxidant or Ca ++ modulating agent to be used in the NF-κB assay. Apoptosis was assessed using brightfield microscopy as described previously ( 6 , 58–60 ). After pretreatment, cells were spun onto glass slides using a Shandon Cytospin 3 centrifuge, fixed in 100% methanol for 3 min and air-dried. Slides were stained for 4 h with modified Giemsa stain (Sigma). One hundred cells were scored for the presence or absence of apoptotic cells. All apoptosis experiments were repeated at least twice. The morphological criteria used for determining the presence of apoptotic cells were condensed chromatin, nuclear fragmentation, cell shrinkage, increased cytoplasmic vacuolization and apoptotic body formation ( 6 , 58–60 ). The highest concentration that did not induce apoptosis (see criteria above) or necrosis (observed as pink-staining swollen cells after staining with Giemsa) was selected for use in the NF-κB assay.

NF-κB activation assay

Cells were seeded at 5 × 10 5 cells/ml in 100 mm tissue culture plates and allowed to attach overnight at 37°C with 5% CO 2 . After 24 h, fresh media with and without inhibitors/antioxidants/Ca ++ modulators (added individually) was added for an additional 24 h. DOC at a concentration of 0.5 mM DOC was added for 4 h. Untreated cells served as the control. Cells were then lysed and the nuclear fractions were collected using the Nuclear Extract kit (Active Motif; Carlsbad, CA) according to the manufacturer's instructions. Briefly, cells were washed with PBS supplemented with phosphatase inhibitors to limit further protein modifications (e.g. dephosphorylation), scraped from the plates and centrifuged for 5 min at 500 r.p.m. at 4°C. The supernatant was removed and the cell pellet was resuspended in hypotonic buffer and incubated on ice. Detergent was added and the solution was briefly vortexed and centrifuged at 14 000× g . The resulting supernatant containing the cytoplasmic fraction was discarded. The nuclear pellet was resuspended in lysis buffer supplemented with DTT and protease inhibitors and incubated on ice. After briefly vortexing, the solution was centrifuged at 14 000× g for 10 min at 4°C. The nuclear fraction supernatant was then stored at −80°C before use. Protein concentration was quantified using the Commassie Plus Assay kit from Pierce Biotechnology (Rockford, Illinois).

The TransAm assays (Active Motif; Carlsbad, CA) for NF-κB activation were performed according the manufacturer's instructions. Briefly, binding buffer was added to wells of an NF-κB-specific, oligonucleotide-coated microtiter plate followed by 2 μg of nuclear extract of each sample diluted in lysis buffer. Each sample was plated in triplicate. The plate was incubated for 1 h on a rocking platform at 100 r.p.m. at room temperature. The plate was washed with washing buffer, and the primary antibody (anti-p50 subunit or anti-p65 subunit), diluted 1 : 1000 in antibody binding buffer, was added for 1 h at room temperature with no agitation. The plate was washed again, and the secondary antibody (1 : 1000 dilution) was added for 1 h. After a final wash step, developing solution was added for 10–20 min at room temperature, and the reaction was terminated with stop solution. The plate was read immediately at 450 nm using a microtiter plate reader.

Statistical evaluation was assessed on triplicate values for control (untreated) and treatment groups using Student's t -test. The difference in the mean values between control and treatment groups was considered to be statistically significant at the 95% probability level.

Results

DOC induces mitochondrial oxidative stress and NF-κB activation in HCT-116 cells

First, we evaluated whether DOC could specifically induce the formation of ROS within mitochondria, using MitoSOX™ Red, a mitochondrial superoxide indicator. A dose–response experiment (DOC concentration versus MitoSOX™ Red fluorescence) was performed by incubating HCT-116 cells with 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mM DOC for 4 h, followed by incubation with MitoSOX™ Red. Treatment of cells with 0.4 and 0.5 mM DOC showed a statistically significant increase in ROS ( P < 0.05) compared with control, untreated cells using flow cytometry ( Figure 1 ). Although there was an increase in the percentage of cells that exhibited increased fluorescence of MitoSox after incubation with 0.3 mM DOC compared with untreated, control cells ( Figure 1 ), the difference between the mean values was not statistically significant.

Fig. 1

Representative dose–response experiment (DOC concentration versus mitochondrial oxidative stress) assessed in HCT-116 cells after 4 h of incubation using MitoSOX™ Red mitochondrial superoxide indicator in conjunction with flow cytometry. Asterisks above individual bars of the graph indicate a significant difference in the percentage of cells with increased MitoSOX fluorescence compared with untreated control cells.

Fig. 1

Representative dose–response experiment (DOC concentration versus mitochondrial oxidative stress) assessed in HCT-116 cells after 4 h of incubation using MitoSOX™ Red mitochondrial superoxide indicator in conjunction with flow cytometry. Asterisks above individual bars of the graph indicate a significant difference in the percentage of cells with increased MitoSOX fluorescence compared with untreated control cells.

A dose–response experiment (DOC concentration versus CMXRos fluorescence) was also performed to determine mitochondrial stress. Treatment of cells with DOC in the concentration range of 0.3–0.5 mM showed a statistically significant decrease in MMP ( P < 0.05) compared with control, untreated cells using flow cytometry ( Figure 2 ). Treatment of cells with DOC in the concentration range of 0.3–0.5 mM showed a statistically significant decrease in MMP ( P < 0.05) compared with control, untreated cells using flow cytometry ( Figure 2 ).

Fig. 2

Representative dose–response experiment (DOC concentration versus loss of MMP) assessed in HCT-116 cells after 4 h of incubation using Mitotracker Red ® (CMXRos). Asterisks above individual bars of the graph indicate a significant difference in the percentage of cells with low MMP compared with untreated control cells.

Fig. 2

Representative dose–response experiment (DOC concentration versus loss of MMP) assessed in HCT-116 cells after 4 h of incubation using Mitotracker Red ® (CMXRos). Asterisks above individual bars of the graph indicate a significant difference in the percentage of cells with low MMP compared with untreated control cells.

To determine the length of time required to produce the DOC-induced increase in mitochondrial ROS, cells were exposed to 0.5 mM DOC for 1, 2, 3 and 4 h. There was no increase in DOC-induced mitochondrial ROS for the first 3 h of incubation ( Figure 3 ). However, there was a marked increase in mitochondrial ROS after 4 h of incubation with 0.5 mM DOC ( Figure 3 ).

Fig. 3

Exposure of HCT-116 cells to 0.5 mM DOC for 1–4 h using MitoSOX™ Red mitochondrial superoxide indicator in conjunction with flow cytometry. The marked shift to the right induced at 4 h of incubation by DOC may be noted.

Fig. 3

Exposure of HCT-116 cells to 0.5 mM DOC for 1–4 h using MitoSOX™ Red mitochondrial superoxide indicator in conjunction with flow cytometry. The marked shift to the right induced at 4 h of incubation by DOC may be noted.

To demonstrate that DOC induces NF-κB activation at the cellular level, we previously used a monoclonal antibody that recognizes an epitope that spans the nuclear localization signal sequence of the p65 subunit of NF-κB that is normally masked by an inhibitor of NF-κB, IκB ( 45 ). In the present study, we evaluated the ability of the p50 and p65 subunits, comprising the classic NF-κB dimer, to bind to NF-κB oligonucleotide consensus sequences using nuclear lysates (TransAm assays) from HCT-116 cells treated with 0.5 mM DOC for 4 h. DOC-induced p50 and p65 nuclear translocations and binding to DNA, assessed in triplicate, were comparable and reproducible in a total of 20 experiments (10 experiments to assess p50 binding and 10 experiments to assess p65 binding) performed for this study. The mean fold increase of DOC-induced p50 oligonucleotide binding compared with untreated cells was 7.0, with a range of 3.3 to 11.2 in individual experiments. The mean fold increase of DOC-induced p65 oligonucleotide binding compared with untreated cells was 6.0 with a range of 2.9 to 9.8 in individual experiments.

Inhibition of DOC-induced NF-κB activation

A total of 43 different agents that affect various plasma membrane-initiated, organelle-related, Ca ++ -dependent and ROS-dependent signaling pathways were evaluated for ability to affect DOC-induced NF-κB activation. Nuclear translocation of p50 and p65 were evaluated on separate plates using nuclear lysates in triplicate, followed by an antibody against either the p50 or p65 subunit of NF-κB, as described in the Materials and methods section. Only those agents that caused statistically significant changes ( P < 0.05) on DOC-induced NF-κB activation and, when used alone, had no effect on constitutive levels of activated NF-κB are shown in Table I. (Note: if constitutive levels of activated NF-κB were altered by a specific agent, the DOC-induced NF-κB activation results would be difficult to interpret). Significant changes based on triplicate assays of either p50 or p65 were used as relevant end points in repeat experiments. There were eight agents that did not, when used alone, affect constitutive NF-κB activation but significantly inhibited DOC-induced NF-κB activation using either p50 or p65 nuclear translocation as the end point. The eight agents ( Table I ) that inhibited DOC-induced NF-κB activation included (i) two natural or dietary-related antioxidants (CAPE and EGCG); (ii) two inhibitors that affect plasma membrane-associated enzymes [one (ouabain) that affects ion transport by Na + /K + -ATPase and another (DPI) that affects the generation of superoxide by NAD(P)H oxidase]; (iii) an inhibitor (TMS) that affects the endoplasmic reticulum (ER) associated enzyme, cytochrome P450; (iv) an inhibitor (NaN 3 ) that affects mitochondrial electron transport; and (v) two agents that inhibit cellular Ca ++ uptake (EGTA and RuR). Of the eight agents that inhibited DOC-induced NF-κB activation, RuR consistently had the greatest effect (>40.0% reduction).

Table I

Statistically significant ( P < 0.05) effects of various agents on DOC-induced NF-κB activation

Agent (concentration) Function  % Inhibition DOC-induced NF-κB activation a 
Antioxidants   
    CAPE (100 μM)  NF-κB/ODC/LOX inhibitor b 17.0–32.7 
    EGCG (10 μM)  NOS2/ODC/telomerase inhibitor c 12.9–15.8 
Inhibitors   
    Ouabain (10 nM)  Na + /K + -ATPase inhibitor  14.7–20.2 
    DPI (30 nM) NAD(P)H Oxidase inhibitor 28.6–32.3 
    TMS (5 μM) Cytochrome P450 inhibitor 15.0–38.3 
    NaN 3 (10 μM)  Mitochondrial complex IV inhibitor 33.3–38.5  
Calcium modulators   
    EGTA (100 μM) Calcium chelator 8.9–44.0 
    RuR (100 μM)  Blocker of transmembrane Ca ++ fluxes  40.6–41.0 
Agent (concentration) Function  % Inhibition DOC-induced NF-κB activation a 
Antioxidants   
    CAPE (100 μM)  NF-κB/ODC/LOX inhibitor b 17.0–32.7 
    EGCG (10 μM)  NOS2/ODC/telomerase inhibitor c 12.9–15.8 
Inhibitors   
    Ouabain (10 nM)  Na + /K + -ATPase inhibitor  14.7–20.2 
    DPI (30 nM) NAD(P)H Oxidase inhibitor 28.6–32.3 
    TMS (5 μM) Cytochrome P450 inhibitor 15.0–38.3 
    NaN 3 (10 μM)  Mitochondrial complex IV inhibitor 33.3–38.5  
Calcium modulators   
    EGTA (100 μM) Calcium chelator 8.9–44.0 
    RuR (100 μM)  Blocker of transmembrane Ca ++ fluxes  40.6–41.0 

a p50/p65 nuclear translocation.

b ODC, ornithine decarboxylase; LOX, lipoxygenase.

c NOS2, inducible nitric oxide synthase.

Discussion

Since DOC is a multiple stress inducer ( 44 ), it is not surprising that DOC-induced NF-κB activation occurs through multiple mechanisms. In vivo , the persistent activation of NF-κB can lead to inflammation and the development of apoptosis resistance, two conditions that contribute to genomic instability. Thus, chemopreventive strategies that inhibit NF-κB activation should prove beneficial for the prevention of colon cancer.

The mechanisms by which DOC activates NF-κB pathways are diagrammed in Figure 4 . We previously reported that DOC induces NF-κB activation, using a monoclonal antibody that recognizes an epitope that spans the nuclear localization signal sequence of the p65 subunit of NF-κB that is normally masked by an inhibitor of NF-κB, IκB ( 45 ). We also showed that the unmasked p65 subunit translocates to the nucleus, using confocal laser scanning microscopy ( 45 ). This mechanism of NF-κB activation was complemented by the elegant studies of Muhlbauer et al . ( 61 ). These investigators showed that NF-κB activation could be blocked by using MG132, a proteasomal inhibitor, or by preventing IKK activity with a dominant-negative IKKβ delivered by adenovirus transfection. The signal-transduction/stress pathways depicted in Figure 4 are based on data obtained in the present study ( Figures 1–3 , Table I ) and published data [see Figure 3 of recent review by Bernstein et al . ( 43 )]. The DOC-induced mediation of NF-κB activation occurs through (i) the generation of oxidative stress; (ii) the activation of plasma membrane-associated proteins; (iii) action of xenobiotic metabolizing enzymes; (iv) mitochondrial perturbation; and (v) the modulation of Ca ++ fluxes. Each of these pathways will be discussed briefly with respect to colon carcinogenesis and the promoting effects of hydrophobic bile acids.

Fig. 4

Schematic of possible signal-transduction/stress pathways induced by DOC and resulting in NF-κB activation. These stress pathways are based on data generated in the present study and published data on the molecular and cellular effects of DOC treatment [see Figure 3 in review by Bernstein et al . ( 43 )]. Question marks indicate intriguing potential effects of K + ions on NF-κB activation for future investigation.

Fig. 4

Schematic of possible signal-transduction/stress pathways induced by DOC and resulting in NF-κB activation. These stress pathways are based on data generated in the present study and published data on the molecular and cellular effects of DOC treatment [see Figure 3 in review by Bernstein et al . ( 43 )]. Question marks indicate intriguing potential effects of K + ions on NF-κB activation for future investigation.

In the present study, we also showed that DOC generates mitochondrial oxidative stress ( Figures 1 and 3 ) and reduces MMP ( Figure 2 ). Mitochondrial perturbation can then result in increased NF-κB activation (present study) and apoptosis resistance ( 57 ). It is probable that high concentrations of the lumenal risk factor, DOC, may promote colon cancer, in part, through an increase in ROS-mediated mitochondrial genomic instability.

Generation of oxidative stress and role of antioxidants in colon carcinogenesis

We have previously shown that lazaroids, inhibitors of lipid peroxidation, prevented DOC-induced NF-κB activation ( 46 ). In the present study, we show that the natural or dietary-related antioxidants, CAPE and EGCG, protect against DOC-induced NF-κB activation. CAPE is a phenolic antioxidant derived from the propolis (‘bee glue’) of honeybee hives ( 62 ) and is a known inhibitor of NF-κB ( 63 ). Among all of the green tea phenolic compounds, EGCG is the most bioreactive and represents >50% of the polyphenolic fraction ( 64 ). EGCG has been reported to inhibit NF-κB activation ( 65–68 ). Here, we report that EGCG inhibits DOC-induced NF-κB activation. This is a potentially new mechanism for the protective effect of EGCG against the tumor-promoting effects of DOC on colon carcinogenesis.

Activation of plasma membrane-associated proteins and role in colon carcinogenesis

The inhibition of NAD(P)H oxidase with DPI and Na + /K + -ATPase with ouabain ( Table I ) attenuates DOC-induced NF-κB activation. The involvement of these two plasma membrane-associated proteins in DOC-induced NF-κB activation has not been described previously, but is consistent with DOC-induced membrane perturbation at the concentrations used in this study. Since the activation of NAD(P)H oxidase generates superoxide ( O2 ) ( 69 ), this enzyme may be an important source of ROS for the activation of NF-κB. The effect of DOC on the Na + /K + -ATPase may have central importance in colon carcinogenesis. The Na + /K + -ATPase is a key regulator of intracellular Na + and K + concentrations ( 70 ). A DOC-induced increase in intracellular K + may be involved in NF-κB activation through the modulation of established or novel kinase pathways ( Figure 4 ). A high concentration of intracellular K + ions may interfere with multimeric protein assembly as has been shown for the apoptosome ( 71 ). On the other hand, signaling from the Na + /K + -ATPase stimulates the mitochondrial production of ROS ( 72 ) that leads to the activation of NF-κB through this mitochondrial-to-cytosol signaling pathway. Since we have shown here that DOC stimulates the production of ROS within mitochondria, the activation of the Na + /K + -ATPase may contribute, in part, to DOC-induced mitochondrial damage.

Xenobiotic metabolizing enzymes and role in colon carcinogenesis

The mechanisms by which cytochrome P450 monooxygenases (CYT450) mediate DOC-induced NF-κB activation are most probably multifactorial. One potential mechanism could be the activation of NF-κB by ROS, since CYT450 represent a significant source of ROS and can damage mitochondria ( 73 , 74 ), culminating in massive ROS production from mitochondria ( 75 ). These findings could explain why inhibiting the distal electron transport chain at complex IV with sodium azide (see Table I ) could reduce DOC-induced NF-κB activation through a decrease in respiration, eventually resulting in less total ROS released by the mitochondrial electron transport chain. A second mechanism could involve the metabolism of arachidonic acid (released through DOC-induced phospholipase A 2 activation) to epoxyeicosatrienoic acids (EETs) by cytochrome P450 enzymes ( 76 ). EETs are known to activate voltage-dependent (L-type) Ca ++ channels ( 76 ) resulting from the release of Ca ++ from intracellular stores, an increase in cytosolic Ca ++ levels ( 77 ) and the activation of NF-κB. Since there are numerous subtypes of CYT450, we cannot determine the exact subtype that contributed to NF-κB activation in the present study. Although the tetramethoxystilbene inhibitor (TMS) used in this study shows specificity for the 1B1 subtype at low nanomolar concentrations in enzyme activity assays, the 5 μM concentration of TMS used to treat the HCT-116 cells may inhibit other subtypes within the intracellular milieu.

Mitochondrial perturbation and colon carcinogenesis

ROS released from mitochondria were shown to result in NF-κB activation in other cell types ( 27 , 78 , 79 ). We showed here that inhibition of electron transport using the complex IV inhibitor, NaN 3 , significantly reduced DOC-induced NF-κB activation. We previously reported that DOC induces mitochondrial damage ( 46 , 54 , 57 ). In the present study, we performed dose–response studies indicating that relatively high pathophysiological concentrations of DOC induce ROS within mitochondria and a loss of MMP. Thus, ROS released from damaged mitochondria with an active electron transport chain is a plausible mechanism for the activation of NF-κB in colon epithelial cells.

Modulation of Ca ++ fluxes and colon carcinogenesis

High cytosolic Ca ++ levels seem to be a major factor in DOC-induced NF-κB activation, since two agents (i.e. EGTA, RuR) used in the present study that affect calcium levels by different mechanisms each had a significant effect on DOC-induced NF-κB activation. EGTA does not cross cell membranes and chelates Ca ++ ions in the extracellular milieu, thereby preventing Ca ++ entry into the cell. RuR is an inorganic polycationic dye that inhibits Ca ++ influx through voltage-sensitive calcium channels ( 80 ) and blocks the release of Ca ++ from the ER ( 81 ). RuR also acts as an antioxidant through the quenching of ROS ( 81 , 82 ). Therefore, the inhibition of DOC-induced NF-κB activation by this agent may occur by RuR binding Ca ++ ions and/or preventing the generation of ROS.

Another mechanism by which increased intracellular Ca ++ levels can lead to the DOC-induced activation of NF-κB is through the activation of the calcium/calmodulin-dependent protein kinase (CaMK). The CaMKs are known to phosphorylate IκB proteins in other cell types ( 83–86 ) and could represent novel targets for chemopreventive strategies.

Conclusions

Understanding the mechanism by which bile acids activate NF-κB, an anti-apoptotic redox-sensitive transcription factor, may be crucial to the prevention of colon carcinogenesis. Our initial findings on DOC-induced NF-κB activation in colon epithelial cells ( 45 , 46 ) have been confirmed by others using colon epithelial cell lines ( 61 , 87 , 88 ) and primary human colorectal cells challenged ex vivo ( 61 ). In addition, DOC and other relevant bile acids (e.g. taurocholate, taurodeoxycholate, taurochenodeoxycholate, chenodeoxycholate, glycochenodeoxycholate) have been reported to induce NF-κB activation in other cell types of the GI tract (e.g. hepatocytes, esophageal cells, pancreatic acinar cells) ( 2 , 89–97 ). Our findings, therefore, may have global significance for the prevention of GI cancer, in general, by offering targets for reducing bile acid-induced activation of NF-κB.

Abbreviations

  • CAPE

    caffeic acid phenylethyl ester

  • DOC

    deoxycholate

  • EGTA

    ethyleneglycol- bis (β-aminoethyl)- N , N , N ′, N ′-tetraacetic acid

  • MMP

    mitochondrial membrane potential

  • NF-κB

    nuclear factor kappa B

  • ROS

    reactive oxygen species

  • TMS

    (E)-2,3′,4,5′-tetramethoxystilbene

This work was supported in part by NIH Institutional Core Grant #CA23074, NIHPPG #CA72008, Arizona Disease Control Research Commission Grants #10016 and #6002, VAH Merit Review Grant 2HG, NCI SPORE Grant 1 P50CA95060-01, NIH 1R21CA111513-01A1, 1R01CA119087-01A1 and Biomedical Diagnostics & Research, Inc., Tucson, AZ 85719.

Conflict of Interest Statement : None declared.

References

1
Schreck
R.
Albermmann
K.
Baeuerle
P.A.
Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review)
Free Radic. Res. Commun.
 , 
1992
, vol. 
17
 (pg. 
221
-
237
)
2
Rust
C.
Karnitz
L.M.
Paya
C.V.
Moscat
J.
Simari
R.D.
Gores
G.J.
The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kianse-dependent survival signaling cascade
J. Biol. Chem.
 , 
2000
, vol. 
275
 (pg. 
20210
-
20216
)
3
Mistry
P.
Deacon
K.
Mistry
S.
Blank
J.
Patel
R.
NF-κB promotes survival during mitotic cell cycle arrest
J. Biol. Chem.
 , 
2004
, vol. 
279
 (pg. 
1482
-
1490
)
4
Mattson
M.P.
NF-κB in the survival and plasticity of neurons
Neurochem. Res.
 , 
2005
, vol. 
30
 (pg. 
883
-
893
)
5
Colell
A.
Garcia-Ruiz
C.
Roman
J.
Ballesta
A.
Fernandez-Checa
J.C.
Ganglioside GD3 enhances apoptosis by suppressing the nuclear factor-κB-dependent survival pathway
FASEB J.
 , vol. 
15
 (pg. 
1068
-
1070
)
6
Crowley-Weber
C.L.
Payne
C.M.
Gleason-Guzman
M.
Watts
G.S.
Futscher
B.
Bernstein
C.
Garewal
H.
Bernstein
H.
Development and molecular characterization of colon cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate
Carcinogenesis
 , 
2002
, vol. 
23
 (pg. 
2063
-
2080
)
7
Richmond
A.
NF-κB, chemokine gene transcription and tumour growth
Nat. Rev. Immunol.
 , 
2002
, vol. 
2
 (pg. 
664
-
674
)
8
Monks
N.R.
Biswas
D.K.
Pardee
A.B.
Blocking anti-apoptosis as a strategy for cancer chemotherapy: NF-κB as a target
J. Cell. Biochem.
 , 
2004
, vol. 
92
 (pg. 
646
-
650
)
9
Bours
V.
Dejardin
E.
Goujon-Letawe
F.
Merville
M.O.
Castronovo
V.
The NF-kappa B transcription factor and cancer: high expression of NF-kappa B- and I kappa B-related proteins in tumor cell lines
Biochem. Pharmacol.
 , 
1994
, vol. 
47
 (pg. 
145
-
149
)
10
Smirnov
A.S.
Ruzov
A.S.
Budanov
A.V.
Prokhortchouk
A.V.
Ivanov
A.V.
Prokhortchouk
E.B.
High constitutive level of NF-κB is crucial for viability of adenocarcinoma cells
Cell Death Differ.
 , 
2001
, vol. 
8
 (pg. 
621
-
630
)
11
Niu
J.
Li
Z.
Peng
B.
Chiao
P.J.
Identification of an autoregulatory feedback pathway involving interleukin-1α in induction of constitutive NF-κB activation in pancreatic cancer cells
J. Biol. Chem.
 , 
2004
, vol. 
279
 (pg. 
16452
-
16462
)
12
Lu
T.
Sathe
S.S.
Swiatkowski
S.M.
Hampole
C.V.
Stark
G.R.
Secretion of cytokines and growth factors as a general cause of constitutive NFκB activation in cancer
Oncogene
 , 
2004
, vol. 
23
 (pg. 
2138
-
2145
)
13
Lind
D.S.
Hochwald
S.N.
Malaty
J.
Rekkas
S.
Hebig
P.
Mishra
G.
Moldawer
L.L.
Copeland
E.M.
III
Mackay
S.
Nuclear factor-kappa B is upregulated in colorectal cancer
Surgery
 , 
2001
, vol. 
130
 (pg. 
363
-
369
)
14
Sharma
H.W.
Narayanan
R.
The NF-kappaB transcription factor in oncogenesis
AntiCancer Res.
 , 
1996
, vol. 
16
 (pg. 
589
-
596
)
15
Lin
A.
Karin
M.
NF-kappaB in cancer: a marked target
Semin. Cancer Biol.
 , 
2003
, vol. 
13
 (pg. 
107
-
114
)
16
Schottelius
A.J.G.
Baldwin
A.S.
Jr
A role for transcription factor NF-κB in intestinal inflammation
Int. J. Colorect. Dis.
 , 
1999
, vol. 
14
 (pg. 
18
-
28
)
17
Schmid
R.M.
Adler
G.
NF-κB/Rel/IκB: implications in gastrointestinal diseases
Gastroenterology
 , 
2000
, vol. 
118
 (pg. 
1208
-
1228
)
18
Jobin
C.
Sartor
R.B.
The IκB/NF-κB system: a key determinant of mucosal inflammation and protection
Am. J. Physiol. Cell Physiol.
 , 
2000
, vol. 
278
 (pg. 
C451
-
C462
)
19
Wu
Z.-H.
Shi
Y.
Tibbetts
R.S.
Miyamoto
S.
Molecular linkage between the kinase ATM and NF-κB signaling in response to genotoxic stimuli
Science
 , 
2006
, vol. 
311
 (pg. 
1141
-
1146
)
20
Biswas
D.K.
Cruz
A.P.
Gansberger
E.
Pardee
A.B.
Epidermal growth factor-induced nuclear factor kappa B activation: a major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells
Proc. Natl Acad. Sci. USA
 , 
2000
, vol. 
97
 (pg. 
8542
-
8547
)
21
Habib
A.A.
Chatterjee
S.
Park
S.K.
Ratan
R.R.
Lefebvre
S.
Vartanian
T.
The epidermal growth factor receptor engages receptor interacting protein and nuclear factor-kappa B (NF-kappa B)-inducing kinase to activate NF-kappa B. Identification of a novel receptor-tyrosine kinase signalosome
J. Biol. Chem.
 , 
2001
, vol. 
276
 (pg. 
8865
-
8874
)
22
Asehnoune
K.
Strassheim
D.
Mitra
S.
Kim
J.Y.
Abraham
E.
Involvement of reactive oxygen species in toll-like receptor 4-dependent activation of NF-κB
J. Immunol.
 , 
2004
, vol. 
172
 (pg. 
2522
-
2529
)
23
Le Page
C.
Koumakpayi
I.H.
Lessard
L.
Mes-Masson
A.M.
Saad
F.
EGFR and Her-2 regulate the constitutive activation of NF-kappaB in PC-3 prostate cancer cells
Prostate
 , 
2005
, vol. 
65
 (pg. 
130
-
140
)
24
Janssen-Heininger
Y.M.W.
Macara
I.
Mossman
B.T.
Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-κB. Requirement of ras/mitogen-activated protein kinases in the activation of NF-κB by oxidants
Am. J. Respir. Cell Mol. Biol.
 , 
1999
, vol. 
20
 (pg. 
942
-
952
)
25
Kamata
H.
Manabe
T.
Oka
S.-I.
Kamata
K.
Hirata
H.
Hydrogen peroxide activates IκB kinases through phosphorylation of serine residues in the activation loops
FEBS Lett.
 , 
2002
, vol. 
519
 (pg. 
231
-
237
)
26
Pullar
J.M.
Winterbourn
C.C.
Vissers
M.C.M.
The effect of hypochlorous acid on the expression of adhesion molecules and activation of NF-κB in cultured human endothelial cells
Antioxid. Redox Signal.
 , 
2002
, vol. 
4
 (pg. 
5
-
15
)
27
Mogensen
T.H.
Melchjorsen
J.
Hollsberg
P.
Paludan
S.R.
Activation of NF-κB in virus-infected macrophages is dependent on mitochondrial oxidative stress and intracellular calcium: downstream involvement of the kinases TGF-β-activated kinase 1, mitogen-activated kinase/extrcellular signal-regulated kinase kinase 1, and IκB kinase
J. Immunol.
 , 
2003
, vol. 
170
 (pg. 
6224
-
6233
)
28
Pahl
H.L.
Baeuerle
P.A.
Activation of NF-κB by ER stress requires both Ca2+ and reactive oxygen intermediates as messengers
FEBS Lett.
 , 
1996
, vol. 
392
 (pg. 
129
-
136
)
29
Shatrov
V.A.
Lehmann
V.
Chouaib
S.
Sphingosine-1-phosphate mobilizes intracellular calcium and activates transcription factor NF-kappa B in U937 cells
Biochem. Biophys. Res. Comm.
 , 
1997
, vol. 
234
 (pg. 
121
-
124
)
30
Petranka
J.
Wright
G.
Forbes
R.A.
Murphy
E.
Elevated calcium in preneoplastic cells activates NF-κB and confers resistance to apoptosis
J. Biol. Chem.
 , 
2001
, vol. 
276
 (pg. 
37102
-
37108
)
31
He
D.
Sougioultzis
S.
Hagen
S.
Liu
J.
Keates
S.
Keates
A.C.
Pothoulakis
C.
Lamont
J.T.
Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation
Gastroenterology
 , 
2002
, vol. 
122
 (pg. 
1048
-
1057
)
32
Kim
Y.H.
Kim
J.M.
Kim
S.N.
Kim
G.S.
Baek
J.H.
P44/42 MAPK activation is necessary for receptor activator of nuclear factor-kappaB ligand induction by high extracellular calcium
Biochem. Biophys. Res. Comm.
 , 
2003
, vol. 
304
 (pg. 
729
-
735
)
33
Lilienbaum
A.
Israel
A.
From calcium to NF-kappa B signaling pathways in neurons
Mol. Cell. Biol.
 , 
2003
, vol. 
23
 (pg. 
2680
-
2698
)
34
Gukovskaya
A.S.
Hosseini
S.
Satoh
A.
Cheng
J.H.
Nam
K.J.
Gukovsky
I.
Pandol
S.J.
Ethanol differentially regulates NF-kappaB activation in pancreatic acinar cells through calcium and protein kinase C pathways
Am. J. Physiol. Gastrointest. Liver Physiol.
 , 
2004
, vol. 
286
 (pg. 
G204
-
G213
)
35
Berchtold
C.M.
Chen
K.-S.
Miyamoto
S.
Gould
M.N.
Perillyl alcohol inhibits a calcium-dependent constitutive nuclear factor-κB pathway
Cancer Res.
 , 
2005
, vol. 
65
 (pg. 
8558
-
8566
)
36
Cheah
P.Y.
Hypotheses for the etiology of colorectal cancer—an overview
Nutr. Cancer
 , 
1990
, vol. 
14
 (pg. 
5
-
13
)
37
Slattery
M.L.
Diet, lifestyle, and colon cancer
Semin. Gastrointest. Dis.
 , 
2000
, vol. 
11
 (pg. 
142
-
146
)
38
Wu
B.
Iwakiri
R.
Ootani
A.
Tsunada
S.
Fujise
T.
Sakata
Y.
Sakata
H.
Toda
S.
Fujimoto
K.
Dietary corn oil promotes colon cancer by inhibiting mitochondria-dependent apoptosis in azoxymethane-treated rats
Exp. Biol. Med.
 , 
2004
, vol. 
229
 (pg. 
1017
-
1025
)
39
Yang
K.
Yang
W.
Mariadason
J.
Velcich
A.
Lipkin
M.
Augenlicht
L.
Dietary components modify gene expression: implications for carcinogenesis
J. Nutr.
 , 
2005
, vol. 
135
 (pg. 
2710
-
2714
)
40
Newmark
H.L.
Yang
K.
Lipkin
M.
Kopelovich
L.
Liu
Y.
Fan
K.
Shinozaki
H.
A western-style diet induces benign and malignant neoplasms in the colon of normal C57B1/6 mice
Carcinogenesis
 , 
2001
, vol. 
22
 (pg. 
1871
-
1875
)
41
De Kok
T.M.
van Faassen
A.
Glinghammar
B.
Pachen
D.M.
Eng
M.
Rafter
J.J.
Baeten
C.G.
Engels
L.G.
Kleinjans
J.C.
Bile acid concentrations, cytotoxicity, and pH of fecal water from patients with colorectal adenomas
Dig. Dis. Sci.
 , 
1999
, vol. 
44
 (pg. 
2218
-
2225
)
42
Ridlon
J.M.
Kang
D.J.
Hylemon
P.B.
Bile salt transformations by human intestinal bacteria
J. Lipid Res.
 , 
2006
, vol. 
47
 (pg. 
241
-
259
)
43
Bernstein
H.
Bernstein
C.
Payne
C.M.
Garewal
H.
Bile acids as carcinogens in human gastrointestinal cancers
Mutat. Res.
 , 
2005
, vol. 
589
 (pg. 
47
-
65
)
44
Bernstein
H.
Payne
C.
Bernstein
C.
Beard
S.
Schneider
J.
Crowley
C.
Activation of the promoters of genes associated with DNA damage, oxidative stress, ER stress and protein malfolding by the bile salt, deoxycholate
Toxicol. Lett.
 , 
1999
, vol. 
108
 (pg. 
37
-
46
)
45
Payne
C.M.
Crowley
C.
Washo-Stultz
D.
Briehl
M.
Bernstein
H.
Bernstein
C.
Beard
S.
Holubec
H.
Warneke
J.
The stress-response proteins poly(ADP-ribose) polymerase and NF-κB protect against bile salt-induced apoptosis
Cell Death Different.
 , 
1998
, vol. 
5
 (pg. 
623
-
636
)
46
Washo-Stultz
D.
Crowley-Weber
C.L.
Dvorakova
K.
Bernstein
C.
Bernstein
H.
Kunke
K.
Waltmire
C.N.
Garewal
H.
Payne
C.M.
Role of mitochondrial complexes I & II, reactive oxygen species and arachidonic acid metabolism in deoxycholate-induced apoptosis
Cancer Lett.
 , 
2002
, vol. 
177
 (pg. 
129
-
144
)
47
Saintigny
Y.
Dumay
A.
Lambert
S.
Lopez
B.S.
A novel role for the Bcl-2 protein family: specific suppression of the RAD51 recombination pathway
EMBO J.
 , 
2001
, vol. 
20
 (pg. 
2596
-
2607
)
48
Nelson
D.A.
Tan
T.T.
Rabson
A.B.
Anderson
D.
Degenhardt
K.
White
E.
Hypoxia and defective apoptosis drive genomic instability and tumorigenesis
Genes Dev.
 , 
2004
, vol. 
18
 (pg. 
2095
-
2107
)
49
Mendez
O.
Fernandez
Y.
Peinado
M.A.
Moreno
V.
Sierra
A.
Anti-apoptotic proteins induce non-random genetic alterations that result in selecting breast cancer metastatic cells
Clin. Exp. Metastasis
 , 
2005
, vol. 
22
 (pg. 
297
-
307
)
50
Schmitz
M.L.
Mattioli
I.
Buss
H.
Kracht
M.
NF-κB: a multifaceted transcription factor regulated at several levels
Chem. Bio. Chem.
 , 
2004
, vol. 
5
 (pg. 
1348
-
1358
)
51
Vyvoda
O.S.
Coleman
R.
Holdsworth
G.
Effects of different bile salts upon the composition and morphology of a liver plasma membrane preparation. Deoxycholate is more membrane damaging than cholate and its conjugates
Biochim. Biophys. Acta.
 , 
1977
, vol. 
465
 (pg. 
68
-
76
)
52
Sugimoto
Y.
Saito
H.
Tabeta
R.
Kodama
M.
Nagata
C.
Itabashi
M.
Hirota
T.
Toyoshima
S.
Binding of bile acids with rat colon and resultant perturbation of membrane organization as studied by uptake measurement and 31P nuclear magnetic resonance spectroscopy
Gann
 , 
1984
, vol. 
75
 (pg. 
798
-
808
)
53
Akare
S.
Martinez
J.D.
Bile acid induces hydrophobicity-dependent membrane alterations
Biochim. Biophys. Acta.
 , 
2005
, vol. 
1735
 (pg. 
59
-
67
)
54
Crowley-Weber
C.L.
Dvorakova
K.
Crowley
C.
Bernstein
H.
Bernstein
C.
Garewal
H.
Payne
C.M.
Nicotine increases oxidative stress, activates NF-kappaB and GRP78, induces apoptosis and sensitizes cells to genotoxic/xenobiotic stresses by a multiple stress inducer, deoxycholate: relevance to carcinogenesis
Chem. Biol. Interact.
 , 
2003
, vol. 
145
 (pg. 
53
-
66
)
55
Washo-Stultz
D.
Crowley
C.
Payne
C.M.
Bernstein
C.
Marek
S.
Gerner
E.W.
Bernstein
H.
Increased susceptibility of cells to inducible apoptosis during growth from early to late log phase: An important caveat for in vitro apoptosis research
Toxicol. Lett.
 , 
2000
, vol. 
116
 (pg. 
119
-
207
)
56
Dvorakova
K.
Waltmire
C.N.
Payne
C.M.
Tome
M.E.
Briehl
M.M.
Dorr
R.T.
Induction of mitochondrial changes in myeloma cells by imexon
Blood
 , 
2001
, vol. 
97
 (pg. 
3544
-
3551
)
57
Payne
C.M.
Crowley-Weber
C.L.
Dvorakova
K.
Bernstein
C.
Bernstein
H.
Holubec
H.
Crowley
C.
Garewal
H.
Mitochondrial perturbation attenuates bile acid-induced cytotoxicity
Cell Biol. Toxicol.
 , 
2005
, vol. 
21
 (pg. 
215
-
231
)
58
Payne
C.M.
Bjore
C.G.
Jr
Schultz
D.A.
Change in the frequency of apoptosis after low- and high-dose X-irradiation of human lymphocytes
J. Leukoc. Biol.
 , 
1992
, vol. 
52
 (pg. 
433
-
440
)
59
Crowley
C.
Payne
C.M.
Bernstein
H.
Bernstein
C.
Roe
D.
The NAD + precursors, nicotinic acid and nicotinamide protect cells against apoptosis induced by a multiple stress inducer, deoxycholate
Cell Death Differ.
 , 
2000
, vol. 
7
 (pg. 
314
-
326
)
60
Payne
C.M.
Waltmire
C.N.
Crowley
C.
Crowley-Weber
C.L.
Dvorakova
K.
Bernstein
H.
Bernstein
C.
Holubec
H.
Garewal
H.
Caspase-6 mediated cleavage of guanylate cyclase α1 during deoxycholate-induced apoptosis: protective role of the nitric oxide signaling module
Cell Biol. Toxicol.
 , 
2003
, vol. 
19
 (pg. 
373
-
392
)
61
Muhlbauer
M.
Allard
B.
Bosserhoff
A.K.
Kiessling
S.
Herfarth
H.
Rogler
G.
Scholmerich
J.
Jobin
C.
Hellerbrand
C.
Differential effects of deoxycholic acid and taurodeoxycholic acid on NF-κB signal transduction and IL-8 gene expression in colonic epithelial cells
Am. J. Physiol. Gastrointest. Liver Physiol.
 , 
2004
, vol. 
286
 (pg. 
G1000
-
G1008
)
62
Grundberger
D.
Banerjee
R.
Eisinger
K.
Oltz
E.M.
Efros
L.
Caldwell
M.
Estevez
V
Nakanishi
K.
Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis
Experientia.
 , 
1988
, vol. 
44
 (pg. 
230
-
232
)
63
Natarajan
K.
Singh
S.
Burke
T.R.
Jr
Grunberger
D.
Aggarwal
B.B.
Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B
Proc. Natl Acad. Sci. USA
 , 
1996
, vol. 
93
 (pg. 
9090
-
9095
)
64
Chen
C.
Shen
G.
Hebbar
V.
Hu
R.
Owuor
E.D.
Kong
A.-N.T.
Epigallocatechin-3-gallate-induced stress signals in human colon adenocarcinoma cells
Carcinogenesis
 , 
2003
, vol. 
24
 (pg. 
1369
-
1378
)
65
Nomura
M.
Ma
W.
Chen
N.
Bode
A.M.
Dong
Z.
Inhibition of 12- O -tetradecanoylphorbol-13-acetate-induced NF-kappaB activation by tea polyphenols, (-)-epigallocatechin gallate and theaflavins
Carcinogenesis
 , 
2000
, vol. 
21
 (pg. 
1885
-
1890
)
66
Ahmad
N.
Gupta
S.
Mukhtar
H.
Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappaB in cancer cells versus normal cells
Arch. Biochem. Biophys.
 , 
2000
, vol. 
376
 (pg. 
338
-
346
)
67
Yang
F.
Oz
H.S.
Barve
S.
de Villiers
W.J.
McClain
C.J.
Varilek
G.W.
The green tea polyphenol (-)-epigallocatechin-3-gallate blocks nuclear factor-kappa B activation by inhibiting I kappa B kinase activity in the intestinal epithelial cell line IEC-6
Mol. Pharmacol.
 , 
2001
, vol. 
60
 (pg. 
528
-
533
)
68
Surh
Y.-J.
Chun
K.-S.
Cha
H.-H.
Han
S.S.
Keum
Y.-S.
Park
K.-K.
Lee
S.S.
Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-κB activation
Mutat. Res.
 , 
2001
, vol. 
480–481
 (pg. 
243
-
268
)
69
DeCoursey
T.E.
Ligeti
E.
Review. Regulation and termination of NADPH oxidase activity
Cell. Mol. Life Sci.
 , 
2005
, vol. 
62
 (pg. 
2173
-
2193
)
70
Jorgensen
P.L.
Hakansson
K.O.
Karlish
S.J.D.
Structure and mechanism of Na,K-ATPase: functional sites and their interactions
Annu. Rev. Physiol.
 , 
2003
, vol. 
65
 (pg. 
817
-
849
)
71
Cain
K.
Langlais
C.
Sun
X.M.
Brown
D.G.
Cohen
G.M.
Physiological concentrations of K+ inhibit cytochrome c-dependent formation of the apoptosome
J. Biol. Chem.
 , 
2001
, vol. 
276
 (pg. 
41985
-
41990
)
72
Xie
Z.
Cai
T.
Na + -K + -ATPase-mediated signal transduction: from protein interaction to cellular function
Mol.. Interv.
 , 
2003
, vol. 
3
 (pg. 
157
-
168
)
73
Haouzi
D.
Lekehal
M.
Moreau
A.
Moulis
C.
Feldman
G.
Robin
M.-A.
Letteron
P.
Fau
D.
Pessayre
D.
Cytochrome P450-generated reactive metabolites cause mitochondrial permeability transition, caspase activation, and apoptosis in rat hepatocytes
Hepatology
 , 
2000
, vol. 
32
 (pg. 
303
-
311
)
74
Gottlieb
R.A.
Minireview. Cytochrome P450: major player in reperfusion injury
Arch. Biochem. Biophys.
 , 
2003
, vol. 
420
 (pg. 
262
-
267
)
75
Davydov
D.R.
Microsomal monooxygenase in apoptosis: another target for cytochrome c signaling?
Trends Biochem. Sci.
 , 
2001
, vol. 
26
 (pg. 
155
-
160
)
76
Fang
X.
Weintraub
N.L.
Stoll
L.L.
Spector
A.A.
Epoxyeicosatrienoic acids increase intracellular calcium concentration in vascular smooth muscle cells
Hypertension
 , 
1999
, vol. 
34
 (pg. 
1242
-
1246
)
77
Rzigalinski
B.A.
Willoughby
K.A.
Hoffman
S.W.
Falck
J.T.
Ellis
E.F.
Calcium influx factor, further evidence it is 5, 6-epoxyeicosatrienoic acid
J. Biol. Chem.
 , 
1999
, vol. 
274
 (pg. 
175
-
182
)
78
Garcia-Ruiz
C.
Colell
A.
Morales
A.
Kaplowitz
N.
Fernandez-Checa
J.C.
Role of oxidative stress generated from the mitochondrial electron transport chain and mitochondrial glutathione status in loss of mitochondrial function and activation of transcriptional factor nuclear factor-κB: studies with isolated mitochondria and rat hepatocytes
Mol. Pharmacol.
 , 
1995
, vol. 
48
 (pg. 
825
-
834
)
79
Cassarino
D.S.
Halvorsen
E.M.
Swerdlow
R.H.
Abramova
N.N.
Parker
W.D.
Sturgill
T.W.
Bennett
J.P.
Jr
Interaction among mitochondria, mitogen-activated protein kinases, and nuclear factor-κB in cellular models of Parkinson's disease
J. Neurochem.
 , 
2000
, vol. 
74
 (pg. 
1384
-
1392
)
80
Tapia
R.
Velasco
I.
Ruthenium red as a tool to study calcium channels, neuronal death and the function of neural pathways
Neurochem. Int.
 , 
1997
, vol. 
30
 (pg. 
137
-
147
)
81
Kessel
D.
Castelli
M.
Reiners
J.J.
Jr
Ruthenium red-mediated suppression of Bcl-2 loss and Ca 2+ release initiated by photodamage to the endoplasmic reticulum: scavenging of reactive oxygen species
Cell Death Differ.
 , 
2005
, vol. 
12
 (pg. 
502
-
511
)
82
Meinicke
A.R.
Bechara
E.J.
Vercesi
A.E.
Ruthenium red-catalyzed degradation of peroxides can prevent mitochondrial oxidative damage induced by either tert-butyl hydroperoxide or inorganic phosphate
Arch. Biochem. Biophys.
 , 
1998
, vol. 
349
 (pg. 
275
-
280
)
83
Hughes
K.
Edin
S.
Antonsson
A.
Grundstrom
T.
Calmodulin-dependant kinase II mediates T cell receptor/CD3- and phorbol ester-induced activation of IkappaB kinase
J. Biol. Chem.
 , 
2001
, vol. 
276
 (pg. 
36008
-
36013
)
84
Choi
J.
Krushel
L.A.
Crossin
K.L.
NF-kappaB activation by N-CAM and cytokines in astrocytes is regulated by multiple protein kinases and redox modulation
Glia
 , 
2001
, vol. 
33
 (pg. 
45
-
56
)
85
Howe
C.J.
LaHair
M.M.
Maxwell
J.A.
Lee
J.T.
Robinson
P.J.
Rodriquez-Mora
O.
McCubrey
J.A.
Franklin
R.A.
Participation of the calcium/calmodulin-dependent kinases in hydrogen peroxide-induced IκB phosphorylation in human T lymphocytes
J. Biol. Chem.
 , 
2002
, vol. 
277
 (pg. 
30469
-
30476
)
86
Mishra
S.
Mishra
J.P.
Gee
K.
McManus
D.C.
LaCasse
E.C.
Kumar
A.
Distinct role of calmodulin and calmodulin-dependent protein kinase-II in lipopolysaccharide and tumor necrosis factor-alpha-mediated suppression of apoptosis and antiapoptotic c-IAP2 gene expression in human monocytic cells
J. Biol. Chem.
 , 
2005
, vol. 
280
 (pg. 
37536
-
37546
)
87
Glinghammar
B.
Inoue
H.
Rafter
J.J.
Deoxycholic acid causes DNA damage in colonic cells with subsequent induction of caspases, COX-2 promoter activity and the transcription factors NF-kB and AP-1
Carcinogenesis
 , 
2002
, vol. 
23
 (pg. 
839
-
845
)
88
Lee
D.K.
Park
S.Y.
Baik
S.K.
Kwon
S.O.
Chung
J.M.
Oh
E.S.
Kim
H.S.
Deoxycholic acid-induced signal transduction in HT-29 cells: role of NF-kappa B and interleukin-8. Korean
J. Gastroenterol.
 , 
2004
, vol. 
43
 (pg. 
176
-
185
)
89
Vaquero
E.
Gukovsky
I.
Zaninovic
V.
Gukovskaya
A.S.
Pandol
S.J.
Localized pancreatic NF-kappaB activation and inflammatory response to taurocholate-induced pancreatitis
Am. J. Physiol. Gastrointest. Liver Physiol.
 , 
2001
, vol. 
280
 (pg. 
G1197
-
G1208
)
90
Kim
J.Y.
Kim
K.H.
Lee
J.A.
Namkung
W.
Sun
A.Q.
Ananthanarayanan
M.
Suchy
F.J.
Shin
D.M.
Muallem
S.
Lee
M.G.
Transporter-mediated bile acid uptake causes Ca2+-dependent cell death in rat pancreatic acinar cells
Gastroenterology
 , 
2002
, vol. 
122
 (pg. 
1941
-
1953
)
91
Chu
S.H.
Lee-Kang
J.
Lee
K.-H.
Lee
K.
Roles of reactive oxygen species, NF-kB, and peroxiredoxins in glycochenodeoxycholic acid-induced rat hepatocytes death
Pharmacology
 , 
2003
, vol. 
69
 (pg. 
12
-
19
)
92
Jenkins
G.J.S.
Harries
K.
Doak
S.H.
Wilmes
A.
Griffiths
A.P.
Baxter
J.N.
Parry
J.M.
The bile acid deoxycholic acid (DCA) at neutral pH activates NF-kB and induces IL-8 expression in oesophageal cells in vitro
Carcinogenesis
 , 
2004
, vol. 
25
 (pg. 
317
-
323
)
93
Abdel-Latif
M.M.
O'Riordan
J.
Windle
H.J.
Carton
E.
Ravi
N.
Kelleher
D.
Reynolds
J.V.
NF-kappaB activation in esophageal adenocarcinoma: relationship to Barrett's metaplasia, survival, and response to neoadjuvant chemoradiotherapy
Ann. Surg.
 , 
2004
, vol. 
239
 (pg. 
491
-
500
)
94
Toledo
A.
Yamaguchi
J.
Wang
J.Y.
Bass
B.L.
Turner
D.J.
Strauch
E.D.
Taurodeoxycholate stimulates intestinal cell proliferation and protects against apoptotic cell death through activation of NF-kappaB
Dig. Dis. Sci.
 , 
2004
, vol. 
49
 (pg. 
1664
-
1671
)
95
Wong
N.A.
Wilding
J.
Bartlett
S.
Liu
Y.
Warren
B.F.
Piris
J.
Maynard
N.
Marshall
R.
Bodmer
W.F.
CDX1 is an important molecular mediator of Barrett's metaplasia
Proc. Natl. Acad. Sci. USA
 , 
2005
, vol. 
102
 (pg. 
7565
-
7570
)
96
Meng
Y.
Ma
Q.Y.
Kou
X.P.
Xu
J.
Effect of resveratrol on activation of nuclear factor kappa-B and inflammatory factors in rat model of acute pancreatitis. World
J. Gastroenterol.
 , 
2005
, vol. 
11
 (pg. 
525
-
528
)
97
Kazumori
H.
Ishihara
S.
Rumi
M.A.
Kadowaki
Y.
Kinoshita
Y.
Bile acids directly augment caudal related homeobox gene Cdx2 expression in oesophageal keratinocytes in Barrett's epithelium
Gut
 , 
2006
, vol. 
55
 (pg. 
16
-
25
)