|Year : 2016 | Volume
| Issue : 1 | Page : 364-373
Factors regulating nuclear factor-kappa B activation in esophageal cancer cells: Role of bile acids and acid
Mohamed Mahmoud M Abdel-Latif1, Hiroyasu Inoue2, Dermot Kelleher3, John V Reynolds4
1 Department of Clinical Pharmacy, Faculty of Pharmacy, Assiut University, Assiut, Egypt; Department of Surgery, Trinity Centre for Health Sciences, St. James's Hospital, Dublin 8, Ireland
2 Department of Pharmacology, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka, Japan
3 Department of Clinical Medicine, Trinity Centre for Health Sciences, St. James's Hospital, Dublin 8, Ireland
4 Department of Surgery, Trinity Centre for Health Sciences, St. James's Hospital, Dublin 8, Ireland
|Date of Web Publication||13-Apr-2016|
John V Reynolds
Department of Surgery, Trinity Centre for Health Sciences, St. James's Hospital, Dublin 8
Source of Support: None, Conflict of Interest: None
Aims: Gastroesophageal reflux disease is considered to be a major risk in the development of esophageal adenocarcinoma. Nuclear factor-kappa B (NF-κB) plays important roles in the regulation of several genes coding for cytokines, cell proliferation, and apoptosis. To understand the role of bile and acid in the causation of esophageal cancer, we have examined the effects of bile acids and acid on NF-κB activation in the esophageal epithelial cells OE33 and SKGT-4 qualitatively and quantitatively.
Materials and Methods: Analysis of NF-κB activation in esophageal epithelial cells in response to bile acids and acid was performed by electrophoretic mobility shift assay, Western blotting and the translocation NF-κB was assessed by high content analysis (HCA). Cyclooxygenase-2 (COX-2) promoter activity was assessed by transient transfection assays.
Results: This study demonstrated that bile acids and acid activated NF-κB in a dose- and time-dependent manner. HCA analysis was an invaluable method in quantifying NF-κB translocation at the single cell population level following bile or acid treatment. Furthermore, deoxycholic acid (DCA) and acid-induced COX-2 promoter activity, and a mutation in the NF-κB and activator protein-1 (AP-1) binding sites remarkably reduced the reporter gene activity induced by DCA or acid.
Conclusions: Our data demonstrate that bile and acid induce NF-κB activation in esophageal cells qualitatively and quantitatively. The induction of COX-2 promoter activity by DCA and acid was mediated via NF-κB and AP-1 transcription. The activation of NF-κB signaling pathway in esophageal cells may contribute to the development of esophageal cancer, and, therefore, modulating of NF-κB pathway may uncover new therapeutic strategies.
Keywords: Acid, bile acids, cyclooxygenase-2, deoxycholic acid, esophageal cancer, high content analysis, nuclear factor-kappa B
|How to cite this article:|
Abdel-Latif MM, Inoue H, Kelleher D, Reynolds JV. Factors regulating nuclear factor-kappa B activation in esophageal cancer cells: Role of bile acids and acid. J Can Res Ther 2016;12:364-73
|How to cite this URL:|
Abdel-Latif MM, Inoue H, Kelleher D, Reynolds JV. Factors regulating nuclear factor-kappa B activation in esophageal cancer cells: Role of bile acids and acid. J Can Res Ther [serial online] 2016 [cited 2020 Feb 24];12:364-73. Available from: http://www.cancerjournal.net/text.asp?2016/12/1/364/174525
| > Introduction|| |
The incidence of esophageal cancer has increased rapidly worldwide during the last two decades. Esophageal adenocarcinoma is the eighth most common malignancy and the sixth most common cause of cancer-related death because of it's extremely aggressive nature and poor survival rate.,, The prognosis is poor, and approximately 15% of patients survive 5 years.,, Esophageal carcinoma affects more than 450,000 people worldwide, and the incidence is rapidly increasing.
The factors underlying this increase are unclear but may be related to Barrett's esophagus which is associated with a 30–125-fold increase in the risk of developing adenocarcinoma of the esophagus., Gastroesophageal reflux of bile acids and acid are the major factors linked to the Barrett's metaplasia and the development of esophageal adenocarcinoma. Bile acids are known endogenous promoters of gastrointestinal cancer and enhance cell transformation., Bile acids activate cytoplasmic protein kinase cascades and play a role in the regulation of cellular processes, including growth, differentiation, and apoptosis, suggesting that they mediate their effects by altering cell signaling pathways.
Chronic reflux of duodenal bile and gastric acid into the esophagus cause chronic injury and inflammation, which activate signaling that promotes carcinogenesis in Barrett's esophagus.,, Acid directly affects cell proliferation and differentiation of Barrett's epithelium and acid damage to the esophagus is followed by re-epithelialization of the esophagus with the columnar epithelium of esophageal origin., Fitzgerald et al. have suggested that both cultured intestinal cells and Barrett's epithelial cells may be induced to change their phenotype on culture in conditions of low pH.
Nuclear factor-kappa B (NF-κB) resides in the cytoplasm in an inactive form as a heterodimer consisting of p50 and p65 subunits complexed to the inhibitory molecule IκB. Following a range of stimuli in many cell types, NF-κB translocates to the nucleus and binds to its specific DNA site and subsequently up-regulates gene expression involved in the control of cell proliferation, apoptosis, cytokines production, and the regulation of cell adhesion molecules., The previous report from our laboratory demonstrated an increased expression of NF-κB in 40% of patients with Barrett's esophagus and esophageal tumors compared with normal esophageal epithelium. Moreover, incubation of esophageal epithelial cells with deoxycholic acid (DCA) or acidic pH-induced NF-κB expression. It has also shown that DCA induced the NF-κB target gene expression IκB and interleukin-8 (IL-8). Cyclooxygenase-2 (COX-2) expression has been correlated with resistance to apoptosis, inflammation, and cancer. Accumulating evidence suggests that COX-2 is up-regulated in Barrett's esophagus and esophageal adenocarcinoma., Looby et al. has demonstrated that DCA-induced activator protein-1 (AP-1) DNA binding activity and substantial induction of COX-2 expression in esophageal cells. The regulation of COX-2 expression by NF-κB is well-described in colonic cells, however, the relationship between NF-κB and AP-1 transcription factors and COX-2 expression in esophageal cancer has not been addressed. Therefore, the analysis of NF-κB and COX-2 expression could identify some of the molecular mechanisms underlying the outcome of esophageal cancer.
High content analysis (HCA) permits the analysis of cellular events in a high throughput system and allows the capability of measuring multiple cellular characteristics in a nonbiased fashion. HCA uses an imaging analysis to quantify cellular events such as transcription factor translocation accurately and quickly in multiple cells at the cell population level. Translocation of NF-κB is a critical step in the transcriptional activation of specific target genes. The translocation is calculated by measuring the average intensity of “difference” of the NF-κB protein between the cytoplasmic region and nuclear region (cyto-nuc difference) using automated fluorescent microscopy. We have previously demonstrated that HCA method was a very useful approach in studying early growth response gene 1 (Egr-1) and extracellular-signal-regulated kinase translocation in gastric epithelial cells.
The aim of this study was to investigate the factors regulating NF-κB activation in esophageal cancer cells following exposure to bile acids or acid. HCA was used to quantify NF-κB activation using Cellomics KineticScan HCS in multiple cells population and single cell level. NF-κB translocation and changes of fluorescence intensity in the nuclear and cytoplasmic area were quantified to analyze the kinetics of NF-κB translocation in esophageal cells. Here, we demonstrate that bile acids or acid exposure activates NF-κB-binding activity and nuclear translocation in esophageal epithelial cells. We have also demonstrated a role for NF-κB and AP-1 transcription factors in the regulation of bile- and acid-induced COX-2 promoter activity.
| > Materials and Methods|| |
NF-κB consensus oligonucleotides were obtained from Promega (Promega Corp., Madison, WI, USA). Polyclonal antibodies to IκB-α and anti-p50 (sc-114X), anti-p65 (sc-109X), and anti-c-Rel (sc-70X) for gel supershift assays were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). (γ32 P) adenosine triphosphate (ATP) (35 pmol, 3000 Ci/mmol) were from Amersham International (Aylesbury, UK). Poly(dI-dC) was obtained from Pharmacia (Biosystems, Milton Keynes, UK). Bile acids (DCA, chenodeoxycholic acid [CDCA], ursodeoxycholic acid [UDCA], and cholic acid [CA]) were obtained from Sigma (Poole, Dorset, UK).
The esophageal epithelial cell line OE33 (derived from the adenocarcinoma of the lower esophagus; Barrett's metaplasia) was obtained from the European Collection of Animal Cell Cultures, ECACC (Porton Down, Salisbury, UK). SKGT-4 cells were a gift from Dr. David S. Schrump (Thoracic Oncology Section, Surgery Branch, National Cancer Institute, NIH, Bethesda, Maryland, USA). OE33 and SKGT-4 cells were grown in RPMI 1640 medium supplemented with 10% filtered fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine.
OE33 and SKGT-4 cells were removed from flasks by trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and seeded at a density of 5 × 105 cells/ml. To adjust media to the required pH value, 0.1 M HCl was added to the cell culture medium and titrated to the required pH. We have chosen of pH 6.8 for subsequent studies, this pH range has been previously shown to induce NF-κB and Egr-1 expression.,, Cells were treated with the bile acids; DCA, CDCA, CA and UDCA, as indicated in figure legends. Bile acids were dissloved in dimethyl sulfoxide (DMSO) and appropriate dilutions were made in cell culture medium just before use. The final amount of DMSO did not exceed 0.1% (v/v) in our experiments.
Preparation of total cell extracts
Cells were collected by centrifugation at 1400 rpm for 5 min. The pellet of cells was resuspended in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% (w/v) sodium dodecyl sulfate, 150 mM NaCl, 1 mM ethyleneglycoltetraacetic acid, 1 mM EDTA, 0.5 mM phenylmethylsulfonylfluoride and leupeptin (10 µg/ml) and then the cells were solubilized by boiling for 5 min.
Western blot analysis
Whole cell extracts (50 µg of protein/lane) were resolved by electrophoresis through polyacrylamide gels using 10% separating gels according to the method of Laemmli. Proteins were electrotransfered onto polyvinylidene difluoride membrane using a semidry blotting apparatus (Atto). Blots were blocked with 5% (w/v) dried skim milk in phosphate buffered saline for 1 h at room temperature and then incubated for 1 h at room temperature with the appropriate primary antibody (anti-IκB-α) at a dilution of 1:1000). Blots were then incubated with the appropriate horseraddish peroxidase-conjugated secondary antibody (1:1000) for 1 h at room temperature. Immunodetection was performed by enhanced chemiluminesence.
Electrophoretic mobility shift assay
Nuclear extracts were prepared from the cells as described previously. The protein concentration was determined on nuclear extracts by the method of Bradford. Nuclear extracts (4 µg protein) were incubated with 10000 cpm of the 32 P-labeled NF-κB oligonucleotide (that had been previously labeled with (γ32 P) ATP (10 mCi/mmol) at the 5'-ends with T4 polynucleotide kinase in the presence of 2 mg poly(dI-dC) as nonspecific competitor. The DNA-protein complexes were separated on 5% polyacrylamide gels at 150 V for 1–2 h. After electrophoresis, the gels were dried and autoradiographed at −70°C for 24–36 h with intensifying screens. In supershift and competition assays, 0.5 µl of anti-p50, anti-p65, anti-C-rel antibodies or 100-fold molar excess of unlabeled oligonulcelotide was preincubated with nuclear extract for 30 min at room temperature prior to the addition of the labeled probe.
Nuclear factor-kappa B staining for high content analysis
SKGT-4 or OE33 cells (5 × 104 cells/ml) grown in 200 µl in Packard 96 view microplates were treated with bile acids or incubated in media of different pH values ranging from pH 7.4 to pH 3.0 for different periods of time. At the end of treatment, the cells were stained with NF-κB (according to Cellomics manufacturer's instructions) and the cells were scanned in the Cellomics KineticScan HCS Reader and subjected to image analysis. Images were acquired on Nikon TE 300 inverted microscope equipped with Leica DC-100 color digital camera. The system is equipped with emission and excitation filters for selectively imaging fluorescent signals emitted by Hoechst 34442 and Alexa Fluor 488, a CCD camera with a frame grabber, and a Pentium PC computer and applications software. Data are expressed as the mean ± standard deviation (SD). The nuclear translocation of NF-κB in esophageal cells was examined using the Cytoplasm to Nucleus Translocation BioApplication as described previously. The most used parameters in the analysis are mean nuclear intensity (NucInten), mean cytoplasmic intensity (CytInten) and mean cytoplasmic nuclear difference intensity (CytNucDiff).
The COX-2 promoter constructs (−1432/+59, −327/+59, −52/+59) and the mutant promoters (KBM, CRM) were from Dr. Hiroyasu Inoue (Department of Pharmacology, Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka, Japan). The human COX-2 promoter containing −1840 bases upstream and +123 downstream of the transcriptional start site, as described previously. The full-length COX-2 promoter construct (−1432/+59), containing sites for SP1, GRE, GATA, NF-κB, PEA3, SP1, AP-2, C/EBP and c-AMP response element (CRE; AP-1). The construct (−327/+59), containing sites for SP1, NF-κB, AP-2, C/EBP, CRE. The shortest deleted construct (−52/+59), lacking all response elements. The COX-2 promoter contains the consensus site for NF-κB binding located at −223/−214 and the consensus site for AP-1 at −59/−53 within the promoter, and the mutations in NF-κB and AP-1 binding were made at these sites. The wild-type and mutated promoters were transfected into esophageal epithelial cells.
Transfection and luciferase assay
SKGT-4 cells were transiently transfected with COX-2 luciferase constructs using Lipofectamine 2000, as described by the manufacturer (Sigma). Twenty-four hours before transfection, cells were seeded in 24-well plates at a density of 50,000 cells per well. The following day, the cells were transfected with the reporter plasmids (5 µg) and exposed to DCA 300 μM or pH 6.8 for 15 h. Luciferase and β-galactosidase activities were measured in cell lysates (50 µl) in a nontransparent 96-well plate using a luciferase reporter assay kit (Promega) and a β-galactosidase assay kit (Promega) according to the manufacturer's instructions, and readout was quantified by luminescence. Transfection efficiency was determined by normalizing luciferase activity of each sample to β-galactosidase activity and the results are presented as means ± SD.
All values were expressed as mean ± SD. Statistical differences between means were calculated by Student's t-test. P < 0.05 was considered statistically significant.
| > Results|| |
Bile acids induce nuclear factor-kappa B in esophageal epithelial cells
It has been clearly shown that different bile acids exert distinct biological effects. Exposure of OE33 cells to different bile acids resulted in distinct effects on NF-κB DNA-binding activity as demonstrated by gel shift assays. [Figure 1]a shows that exposure of OE33 cells to 300 µM DCA and CDCA induced NF-κB DNA-binding activity. However, UDCA and CA had no effect on NF-κB DNA-binding. Treatment of OE33 cells with 300 µM DCA and CDCA induced degradation of the 37 kDa band of IκB-α protein [Figure 1]b. This effect was not seen with UDCA or CA. The decrease in IκB-α levels is coincident with NF-κB activation as demonstrated in gel shift assays. An HCA was used to quantitate the nuclear translocation of NF-κB in cells by measuring changes in fluorescence intensities of NF-κB between the nuclear (Circ) and cytoplasmic (Ring) regions. HCA showed that the bile acids DCA and CDCA induced NF-κB translocation in OE33 [Figure 1]c and SKGT-4 [Figure 1]d cells, whereas UDCA or CA did not.
|Figure 1: Bile acids induce nuclear factor-kappa B in esophageal epithelial cells. (a) Effect of bile acids on nuclear factor-kappa B DNA-binding activity. OE33 cells were treated with bile acids deoxycholic acid, cholic acid, chenodeoxycholic acid, lithocholic acid and ursodeoxycholic acid at 300 μM for 2 h, nuclear extracts were prepared and analyzed by gel shift assay (as described under Experimental Procedures Section). (b) Effect of bile acids on IκB-α protein levels. OE33 cells were treated with 300 μM deoxycholic acid, cholic acid, chenodeoxycholic acid, lithocholic acid or ursodeoxycholic acid and Western blot analysis for IκB-α detection were performed. β-actin was used as a loading control. Experiments were performed 3 times with similar results and a representative gel is shown. High content analysis of nuclear factor-kappa B nuclear translocation by bile acids in OE33 cells (c) and SKGT-4 cells (d). SKGT-4 or OE33 cells were treated with different bile acids (deoxycholic acid, cholic acid, chenodeoxycholic acid, cholic acid or ursodeoxycholic acid) at 300 mM for 2 h. Mean from 3 wells of the difference in nuclear factor-kappa B fluorescence intensities (CircRingAvgIntenDiffCh2) is shown|
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The Molecular Translocation BioApplication running on the Cellomics KineticScan HCS Reader automatically quantitate changes in fluorescence intensities of NF-κB in cells to provide individual cell and well level data. The BioApplication uses two fluorescent labels (one for cell identification; blue and one for the target NF-κB; green) for measuring translocation events. Primary objects (nuclear stained cells) were selected based on average intensity, total intensity, and morphological features such as area, shape, and size. The BioApplication quantifies translocation events by measuring intensity difference and ratio between the nuclear (Circ) and cytoplasmic (Ring) regions. Collectively, these results demonstrate that the bile acids DCA and CDCA induce NF-κB activation and nuclear translocation.
Deoxycholic acid induces nuclear factor-kappa B in a dose- and time-dependent manner
Treatment of OE33 cells with DCA-induced NF-κB DNA-binding in a dose- and time-dependent manner. Maximal induction of NF-κB was observed using 300 µM DCA [Figure 2]a. Higher concentrations of DCA did not induce pronounced NF-κB. Exposure to DCA resulted in a reduction in the levels of IκB-α protein [Figure 2]b. Time-course experiments showed a time-dependent increase in the levels of NF-κB on exposure to DCA. Detectable increases in NF-κB levels were seen from 15 min to 4 h [Figure 2]c. Western blotting with an antibody against IκB-α demonstrated that DCA treatment also resulted in a reduction in the levels of IκB-α and this occurred in a time-dependent manner [Figure 2]d. To identify the composition of the NF-κB DNA-complex induced by DCA, a panel of antibodies directed against various NF-κB subunits (p50, p65, c-Rel) were preincubated with nuclear extracts from OE33 cells stimulated with 300 µM DCA. Antibodies to p50 and p65 recognized this NF-κB DNA complex while anti-C-rel had no effect in the supershift assay [Figure 2]e. Moreover, competition assays with a 100-fold molar excess of unlabeled NF-κB oligonucleotide confirmed the specificity of NF-κB DNA-complex induced by DCA.
|Figure 2: Dose- and time-dependence of nuclear factor-kappa B activation by deoxycholic acid. (a) Dose-response of nuclear factor-kappa B activation by deoxycholic acid. OE33 cells were treated with different concentrations of deoxycholic acid (300 μM) as indicated. Nuclear extracts were prepared and gel shift assay for nuclear factor-kappa B binding activity were performed using a radiolabeled nuclear factor-kappa B probe. (b) Effect of deoxycholic acid doses on IκB-α protein levels. OE33 cells were treated with deoxycholic acid at the indicated concentrations, and Western blotting for IκB-α was performed. (c) Time-course of nuclear factor-kappa B induction by deoxycholic acid. OE33 cells were treated with deoxycholic acid (300 μM) for different periods of time as shown. Nuclear extracts were prepared and gel shift assays for nuclear factor-kappa B binding activity were performed using a radiolabeled nuclear factor-kappa B probe. (d) Time effect of deoxycholic acid exposure on IκB-α protein levels. OE33 cells were treated with deoxycholic acid for different periods of time, and Western blotting for IκB-α was performed. (e) Supershift assay and competition assays. Supershift assay was performed using 0.5 μl of rabbit antisera to p50, lane 2, p65, lane 3, and c-Rel, lane 4. Competition assay for nuclear factor-kappa B was also performed using 100-fold molar excess of unlabeled nuclear factor-kappa B, lane 5|
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High content analysis of kinetics and dose-dependence of nuclear factor-kappa B activation by deoxycholic acid
SKGT-4 or OE33 cells were treated with different doses of DCA (100–400 µM) for 2 h or with 300 µM DCA for different periods of time (0–4 h) and stained for NF-κB. The difference in NF-κB fluorescence intensities between the nuclear and cytoplasmic regions were measured and plotted. Cytoplasmic (RingAvgInten), nuclear (CircAvgInten) and cytoplasmic nuclear difference (CircRingAvgIntenDiff) are shown in the same cells of SKGT-4 cells. The analysis shows that stimulation by DCA resulted in the activation of NF-κB translocation in a time- and dose-dependent manner [Figure 3]a and b]. NF-κB translocation was detected from 15 min to 4 h following treatment in a dose-dependent manner. CircRingAvgIntenDiffCh2 of NF-κB translocation is also shown in OE33 cells [Figure 3]c and [Figure 3]d. Nuclear translocation of NF-κB by DCA in esophageal epithelial cells with respect to kinetics and dose-dependence was clearly correlated with increased NF-κB DNA-binding activity and the rapid depletion of IκB-α.
|Figure 3: High content analysis of time- and dose-dependence of nuclear factor-kappa B activation by deoxycholic acid in esophageal cells. SKGT-4 cells (a and b) or OE33 cells (c and d) were treated with 300 μM deoxycholic deoxycholic for doses of acid acid different periods of time (0-4 h) or with different (100-400 μM) for 2 h and then stained for nuclear factor-kappa B. The difference in nuclear factor-kappa B fluorescence intensities between the nuclear and cytoplasmic regions of the same cells were measured and plotted. RingAvgInten, CircAvgInten and CircRingAvgIntenDiffCh2 in SKGT-4 cells or CircRingAvgIntenDiffCh2 in OE33 cells are shown|
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Acid induces nuclear factor-kappa B in esophageal epithelial cells
OE33 cells were incubated in media of different acidic pH values for 1 h. Following the incubation, cells were harvested, and nuclear extracts were prepared. The DNA-binding activity of NF-κB was then assayed by electrophoretic mobility shift assay (EMSA). Exposure of OE33 cells to acidic pH resulted in a pronounced activation of NF-κB DNA-binding activity. The DNA-binding activity increased as the pH of the extracellular medium was reduced from pH 7.4 to pH 4 [Figure 4]a. However, exposure of OE33 cells to very acidic conditions (≤ pH 3) resulted in a marked reduction in cell viability as assessed by fluorescence staining. In the subsequent experiments, we have selected pH 6.8 and esophageal cells were exposed to that pH value for 1 h and considered as acidic pH. To investigate the effect of acidic pH on IκB-α, OE33 cells were exposed to increasingly acidic environment ranging from pH 7.4 to pH 4. Total cell extracts were prepared and subjected to immunoblotting with antiserum against IκB-α. In cell extracts from resting OE33 cells, anti-IκB-α detected a single 37 kDa species on Western blots [Figure 4]b. As the pH decreased from pH 7.4 to pH 4, there was a concomitant decrease in IκB-α protein levels. This event is coincident with the appearance of activated NF-κB, as demonstrated in gel retardation assays. The EMSA analysis also showed that incubation of OE33 cells at pH 6.8 for different periods of time (0–4 h) resulted in increased NF-κB DNA-binding activity in a time-dependent manner. NF-κB activation was observed from 15 min to 4 h following acid exposure, pH 6.8 [Figure 4]c. Accordingly, a decrease in IκB-α protein levels were seen as the time of acid exposure increased from 15 min to 4 h [Figure 4]d.
|Figure 4: Acid induces nuclear factor-kappa B in esophageal epithelial cells. (a) Effect of acid on nuclear factor-kappa B binding activity. OE33 cells were incubated in media of different pH values ranging from pH 7.4 to pH 4.0. The pH of the medium was adjusted by adding 0.1 M HCl. OE33 cells were exposed to low pH for 1 h and nuclear extracts were prepared and assayed for nuclear factor-kappa B binding activity by electrophoretic mobility shift assay. (b) Effects of acid on IκB-α protein levels. OE33 cells were exposed to increasingly acidic conditions as indicated and total cell extracts were prepared and analyzed by Western blotting for IκB-α proteins using specific antisera. (c) Time-course of nuclear factor-kappa B induction by acid. OE33 cells were incubated in media of pH 6.8 for different periods of time as shown. Nuclear extracts were prepared and gel shift assays for nuclear factor-kappa B binding activity were performed using a radiolabeled nuclear factor-kappa B probe. (d) Time effect of acid exposure on IκB-α protein levels. OE33 cells were incubated in media of pH 6.8 for different periods of time as shown. The cells were collected for the preparation of total cell extracts. Western blots for IκB-α were performed on total cell extracts. A representative gel of three independent experiments with similar results is shown. (e) Supershift and competition assays were carried out on nuclear extracts from OE33 cells stimulated at pH 6.8. The binding reaction was performed after 30 min incubation with or without 0.5 μl of rabbit antisera to p50, lane 2, p65, lane 3, c-Rel, lane 4 and 100-fold molar excess of unlabeled nuclear factor-kappa B, lane 5. Each experiment was repeated 3 times with similar results and a representative gel is shown|
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The specificity of the DNA-bound complex of activated NF-κB under acidic conditions was confirmed by competition assays. A 100-fold molar excess of unlabeled NF-κB oligonucleotide was added to the EMSA-binding reaction containing nuclear extracts prepared from OE33 cells incubated at pH 6.8. Supershift studies were performed to identify the components of the activated NF-κB heterodimer found under acidic conditions using antibodies directed against various NF-κB subunits (p50, p65). The reaction mixture containing nuclear extracts were preincubated with anti-p50 or anti-p65 for 30 min before gel electrophoresis. Antibodies to p50 and p65 induced a supershift of the NF-κB DNA-complex, which confirms the presence of both p50 and p65 in the NF-κB DNA-complex [Figure 4]e. The addition of unlabeled NF-κB oligonucleotide completely abolished NF-κB-DNA complex formation. A HCA showed that exposure of esophageal cells to different low pH values from pH 7.4 to pH 3.0 for 1 h resulted in activation of NF-κB in OE33 cells [Figure 5]a or SKGT-4 cells [Figure 5]b. Nuclear translocation of NF-κB was detected from 15 min to 4 h following to pH 6.8 in OE33 cells [Figure 5]c or SKGT-4 cells [Figure 5]d. CytNucDiff showed maximal NF-κB translocation at 1–2 h. Immunofluorescence staining demonstrated a nuclear translocation of NF-κB in SKGT-4 cells-treated with DCA (300 µM) or pH 6.8 compared to untreated control cells [Figure 6].
|Figure 5: High content analysis of time- and pH-dependence of nuclear factor-kappa B activation by acid. OE33 cells (a and c) or SKGT-4 cells (b and d) were incubated in media of different low pH values ranging from pH 7.4 to pH 3.0 for 60 min or with pH 6.8 for different periods of time (0–4 h) and stained for nuclear factor-kappa B. Mean from 3 wells of the results for CircRingAvgIntenDiffCh2 for nuclear factor-kappa B staining was plotted|
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|Figure 6: Nuclear factor-kappa B immunofluorescence. SKGT-4 cells were treated with deoxycholic acid (300 μM) for 2 h or incubated at pH 6.8 for 1 h, and cells were visualized by confocal microscopy. Unstimulated cells show no basal nuclear factor-kappa B stain in the nucleus and deoxycholic acid or acid exposure increased nuclear factor-kappa B stain in the nucleus. Green: Nuclear factor-kappa B staining, blue: Nuclei. Representative fields are shown (×40)|
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The role of nuclear factor-kappa B and activator protein-1 in mediating cyclooxygenase-2 promoter activity
To understand the role of NF-κB and AP-1 transcription factors in regulating COX-2 expression in response to DCA and acid treatment, SKGT-4 cells were transiently transfected with the wild-type COX-2 promoter plasmids (−1432/+59, −327/+59) or the deletion construct for 24 h, followed by exposure to DCA (300 µM) or pH 6.8 for 15 h. The wild-type COX-2 promoter constructs (−1432/+59, −327/+59), containing sites for NF-κB, and CRE (CRE; AP-1) induced greater luciferase activity above control [Figure 7]a. The deleted construct (−52/+59), lacking all response elements shows a remarkable reduction in response compared to untreated cells. In subsequent transfection experiments, we used the wild-type (−327/+59) plasmid and mutated forms of the (−327/+59) plasmids (KBM plasmid; NF-κB site mutated, CRE plasmid; AP-1 site mutated or ILM plasmid; C/EBP site mutated) to elucidate the role of NF-κB and AP-1 in mediating COX-2 promoter activity. Using the KBM plasmid (−327/+59, NF-κB site mutated), the CRM plasmid (CRE mutation; AP-1) or the ILM plasmid (C/EBP mutation) resulted in a significant reduction in luciferase response compared with wild-type (−327/+59) in response to DCA treatment or acid exposure [Figure 7]b.
|Figure 7: (a) Deoxycholic acid and acid induce cyclooxygenase-2 promoter activity in esophageal cells. SKGT-4 cells were transfected with 5 μg of wild-type cyclooxygenase-2 promoter (−1432/+59, −327/+59) with intact nuclear factor-kappa B and activator protein-1 sites or the cyclooxygenase-2 plasmid lacking the response elements and exposed to deoxycholic acid 300 μM or pH 6.8 for 15 h. (b) Effect of nuclear factor-kappa B and activator protein-1 mutations of the cyclooxygenase-2 promoter plasmid on deoxycholic acid- and acid-induced cyclooxygenase-2 promoter activity. Deletion mutants of the cyclooxygenase-2 promoter plasmid were transfected into SKGT-4 cells and exposed to deoxycholic acid 300 μM or pH 6.8 for 15 h. KBM, represents the −327/+59 cyclooxygenase-2 plasmid, with a mutation in the nuclear factor-kappa B site; CRM represents the −327/+59 cyclooxygenase-2 plasmid, with a mutation in the CRE site and ILM, represents the −327/+59 cyclooxygenase-2 plasmid, with a mutation in the C/EBP site. Luciferase activity from cells transfected with the constructs was normalized to luciferase activity from the cotransfected cells. Bars represent mean ± standard deviation|
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| > Discussion|| |
Gastroesophageal reflux disease is a major risk factor for both the precancerous lesion Barrett's esophagus and esophageal adenocarcinoma.,, The chronic inflammation causes cellular changes and molecular alterations and creates an environment that is conducive to the development of cancer in the esophagus.,,, The underlying disease mechanisms remain unclear, but various cellular and molecular events seem to cause the malignant transformation of Barrett's esophagus and its associated cancer. The present study demonstrated that bile acids and acid-induced NF-κB activation and nuclear translocation in esophageal epithelial cells as well as changes in apoptotic features. We have used a cell-based imaging analysis to quantitate the activation of NF-κB in esophageal epithelial cells. The analysis showed that bile acids and acid resulted in activation of NF-κB in a dose- and time-dependent manner. NF-κB DNA-binding activity measured by EMSA and Western blotting, with respect to kinetics and dose-dependence.
NF-κB transcription factors have an essential role in inflammation and immunity, regulating the transcriptional activity of pro-inflammatory cytokines, adhesion molecules, apoptosis, cell proliferation, and cell surface receptors., Accumulating evidence suggests the involvement of NF-κB in mucosal inflammation of the stomach and small intestine., With respect to the esophagus, we have demonstrated a progressive expression of NF-κB from the normal esophagus through Barrett's epithelium and esophageal adenocarcinoma and reported that NF-κB could be activated by low pH or bile salts. Jenkins et al. also demonstrated that the bile acid DCA at neutral pH activates NF-κB and induces IL-8 expression in esophageal cells in vitro, suggesting that the activation of NF-κB by bile may play a role in Barrett's tumorigenesis. Certain bile acids appear to exert different effects on NF-κB activation. We have shown that bile acids such as DCA, CDCA, and lithocholic acid induce NF-κB DNA-binding activity and nuclear translocation while CA or UDCA had little or no effect on NF-κB activity. DCA activates NF-κB in a dose- and time-dependent manner.
Incubation of the esophageal cells OE33 and SKGT-4 cells in media of low pH-induced NF-κB DNA-binding activity and nuclear translocation and the induction was increased as the pH was reduced from pH 7.4 to pH 5. A small decrease in pH appeared to be an important factor in regulating the activity of NF-κB and subsequently NF-κB-regulated genes. Consistent with our findings, NF-κB activation by DCA also occurred at neutral pH. At acidic pH bile acids are nonionized and can penetrate the cell membrane. Once inside the cell the bile acids become reionized and are unable to exit the cell, leading to a gradual intracellular accumulation of ionized bile acids., Therefore, it appears that variations in acid and bile exposure may contribute to the molecular and cellular alterations that are observed in Barrett's tumorigenesis. Kaur et al. have demonstrated that a 1 h pulse of a bile acid mixture combined with acid caused suppression of proliferation. Consistent with these studies, Hopwood et al. have shown that low concentrations of bile acids were damaging only at high acid levels and that damage to the epithelium did not occur when the pH of the gastric juice had been raised.
We have demonstrated a quantitative method for determining NF-κB activation in esophageal cells in response to bile acids and acid using the HCA approach of NF-κB translocation between the cytoplasm and the nucleus in intact cells. The present study uses HCA method to quantify NF-κB activation in esophageal cells by measuring the immunofluorescently labeled NF-κB in both cytoplasm as well as the nucleus at a single cell population as well as a well population. Cellular analysis of NF-κB translocation showed that exposure of esophageal cells to the bile acid DCA or acid-induced NF-κB translocation in a time- and dose-dependent manner. Maximal NF-κB translocation was observed at 1–2 h. A decline in NF-κB activation to basal levels beyond 4 h is likely to reflect the cell adaptation mechanisms following exposure of esophageal cells to bile or acid.
NF-κB and AP-1 transcription factors regulate several genes involved in inflammation and carcinogenesis and play a synergistic role in many biological processes such as cell proliferation and gene regulation during the development of esophageal cancer. We have also demonstrated a role for NF-κB and AP-1 transcription factors in the regulation of bile- and acid-induced COX-2 expression in esophageal epithelial cells. We observed that the COX-2 promoter (−1432/+59, −327/+59) caused a significant increase in reporter gene activity induced by DCA or acid exposure. The Use of the COX-2 deletion plasmid construct, (−52/+59), which only contains a TATA box, resulted in no induction by DCA or acid compared with untreated cells. Using mutant (−327/+59) plasmids, we demonstrated that a mutation in the NF-κB binding site (KBM) or AP-1 binding site (CRE) significantly reduced the reporter gene activity compared with the wild-type (−327/+59). The results indicate that DCA induction of the COX-2 gene involves multiple transcription factors binding to NF-κB and AP-1. Zhang et al. showed that DCA treatment of esophageal adenocarcinoma cells resulted in activation of the COX-2 promoter activity and consequent increases in prostaglandin production. Furthermore, Our results in esophageal cell model are in agreement with those of Glinghammar et al. who demonstrated that DCA treatment of colonic cells resulted in activation of the COX-2 promoter activity and the transcription factors plays a critical role in mediating the COX-2 promoter activity.
| > Conclusion|| |
We demonstrated a role for bile acids and acid in activating NF-κB and inducing apoptosis. We have also shown for the first time a quantitative method for measurement of NF-κB activation in esophageal cells in response to bile acids and acid at multiple and single cell population level. We have also reported that bile acids and acid-induced COX-2 reporter gene activity, and NF-κB and AP-1 transcription factors play a significant role in regulating bile- and acid-induced COX-2 promoter activity in esophageal epithelial cells. We propose that the activation of NF-κB transcription may result in regulation of many genes during the inflammatory process, and therefore, correction of reflux and exposure to bile acids might protect against further epithelial injury and mucosal inflammation. Further studies of analysis of NF-κB activation could identify some of the molecular mechanisms underlying the outcome of esophageal cancer and subsequently the treatment of esophageal cancer.
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Conflicts of interest
There are no conflicts of interest.
| > References|| |
Umar SB, Fleischer DE. Esophageal cancer: Epidemiology, pathogenesis and prevention. Nat Clin Pract Gastroenterol Hepatol 2008;5:517-26.
Mao WM, Zheng WH, Ling ZQ. Epidemiologic risk factors for esophageal cancer development. Asian Pac J Cancer Prev 2011;12:2461-6.
Zhang Y. Epidemiology of esophageal cancer. World J Gastroenterol 2013;19:5598-606.
Brown CS, Ujiki MB. Risk factors affecting the Barrett's metaplasia-dysplasia-neoplasia sequence. World J Gastrointest Endosc 2015;7:438-45.
O'Sullivan KE, Phelan JJ, O'Hanlon C, Lysaght J, O'Sullivan JN, Reynolds JV. The role of inflammation in cancer of the esophagus. Expert Rev Gastroenterol Hepatol 2014;8:749-60.
Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med 2003;349:2241-52.
Pennathur A, Gibson MK, Jobe BA, Luketich JD. Oesophageal carcinoma. Lancet 2013;381:400-12.
Blot WJ. Esophageal cancer trends and risk factors. Semin Oncol 1994;21:403-10.
McCann J. Esophageal cancers: Changing character, increasing incidence. J Natl Cancer Inst 1999;91:497-8.
Cohen BI, Raicht RF. Effects of bile acids on colon carcinogenesis in rats treated with carcinogens. Cancer Res 1981;41(9 Pt 2):3759-60.
Debruyne PR, Bruyneel EA, Li X, Zimber A, Gespach C, Mareel MM. The role of bile acids in carcinogenesis. Mutat Res 2001;480-481:359-69.
Blobe GC, Obeid LM, Hannun YA. Regulation of protein kinase C and role in cancer biology. Cancer Metastasis Rev 1994;13:411-31.
Kavanagh ME, O'Sullivan KE, O'Hanlon C, O'Sullivan JN, Lysaght J, Reynolds JV. The esophagitis to adenocarcinoma sequence; the role of inflammation. Cancer Lett 2014;345:182-9.
Maley CC. Multistage carcinogenesis in Barrett's esophagus. Cancer Lett 2007;245:22-32.
Gillen P, Keeling P, Byrne PJ, West AB, Hennessy TP. Experimental columnar metaplasia in the canine oesophagus. Br J Surg 1988;75:113-5.
Fitzgerald RC, Omary MB, Triadafilopoulos G. Dynamic effects of acid on Barrett's esophagus. An ex vivo
proliferation and differentiation model. J Clin Invest 1996;98:2120-8.
Kopp EB, Ghosh S. NF-kappa B and rel proteins in innate immunity. Adv Immunol 1995;58:1-27.
Baeuerle PA, Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 1994;12:141-79.
Abdel-Latif MM, O'Riordan J, Windle HJ, Carton E, Ravi N, Kelleher D, et al.
NF-kappaB activation in esophageal adenocarcinoma: Relationship to Barrett's metaplasia, survival, and response to neoadjuvant chemoradiotherapy. Ann Surg 2004;239:491-500.
Jenkins GJ, Harries K, Doak SH, Wilmes A, Griffiths AP, Baxter JN, et al.
The bile acid deoxycholic acid (DCA) at neutral pH activates NF-kappaB and induces IL-8 expression in oesophageal cells in vitro
. Carcinogenesis 2004;25:317-23.
Shirvani VN, Ouatu-Lascar R, Kaur BS, Omary MB, Triadafilopoulos G. Cyclooxygenase 2 expression in Barrett's esophagus and adenocarcinoma: Ex vivo
induction by bile salts and acid exposure. Gastroenterology 2000;118:487-96.
Zimmermann KC, Sarbia M, Weber AA, Borchard F, Gabbert HE, Schrör K. Cyclooxygenase-2 expression in human esophageal carcinoma. Cancer Res 1999;59:198-204.
Looby E, Abdel-Latif MM, Athié-Morales V, Duggan S, Long A, Kelleher D. Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells. BMC Cancer 2009;9:190.
Glinghammar B, Inoue H, Rafter JJ. 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;23:839-45.
Ding GJ, Fischer PA, Boltz RC, Schmidt JA, Colaianne JJ, Gough A, et al.
Characterization and quantitation of NF-kappaB nuclear translocation induced by interleukin-1 and tumor necrosis factor-alpha. Development and use of a high capacity fluorescence cytometric system. J Biol Chem 1998;273:28897-905.
Abdel-Latif MM, Windle HJ, Davies A, Volkov Y, Kelleher D. A new mechanism of gastric epithelial injury induced by acid exposure: The role of Egr-1 and ERK signaling pathways. J Cell Biochem 2009;108:249-60.
O'Toole D, Abdel-Latif MM, Long A, Windle HJ, Murphy AM, Bowie A, et al.
Low pH and Helicobacter pylori
increase nuclear factor kappa B binding in gastric epithelial cells: A common pathway for epithelial cell injury? J Cell Biochem 2005;96:589-98.
Duggan SP, Gallagher WM, Fox EJ, Abdel-Latif MM, Reynolds JV, Kelleher D. Low pH induces co-ordinate regulation of gene expression in oesophageal cells. Carcinogenesis 2006;27:319-27.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.
Osborn L, Kunkel S, Nabel GJ. Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc Natl Acad Sci U S A 1989;86:2336-40.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.
Wild CP, Hardie LJ. Reflux, Barrett's oesophagus and adenocarcinoma: Burning questions. Nat Rev Cancer 2003;3:676-84.
Picardo SL, Maher SG, O'Sullivan JN, Reynolds JV. Barrett's to oesophageal cancer sequence: A model of inflammatory-driven upper gastrointestinal cancer. Dig Surg 2012;29:251-60.
Abdel-Latif MM, Duggan S, Reynolds JV, Kelleher D. Inflammation and esophageal carcinogenesis. Curr Opin Pharmacol 2009;9:396-404.
Van Den Brink GR, ten Kate FJ, Ponsioen CY, Rive MM, Tytgat GN, van Deventer SJ, et al.
Expression and activation of NF-kappa B in the antrum of the human stomach. J Immunol 2000;164:3353-9.
Neurath MF, Pettersson S. Predominant role of NF-kappa B p65 in the pathogenesis of chronic intestinal inflammation. Immunobiology 1997;198:91-8.
Triadafilopoulos G. Acid and bile reflux in Barrett's esophagus: A tale of two evils. Gastroenterology 2001;121:1502-6.
Bremner CG, Mason RJ. 'Bile' in the oesophagus. Br J Surg 1993;80:1374-6.
Kaur BS, Ouatu-Lascar R, Omary MB, Triadafilopoulos G. Bile salts induce or blunt cell proliferation in Barrett's esophagus in an acid-dependent fashion. Am J Physiol Gastrointest Liver Physiol 2000;278:G1000-9.
Hopwood D, Bateson MC, Milne G, Bouchier IA. Effects of bile acids and hydrogen ion on the fine structure of oesophageal epithelium. Gut 1981;22:306-11.
Zhang F, Subbaramaiah K, Altorki N, Dannenberg AJ. Dihydroxy bile acids activate the transcription of cyclooxygenase-2. J Biol Chem 1998;273:2424-8.
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