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Cancer Research 67, 7266, August 1, 2007. doi: 10.1158/0008-5472.CAN-07-1206
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Antiapoptotic Effects of Progastrin on Pancreatic Cancer Cells Are Mediated by Sustained Activation of Nuclear Factor-{kappa}B

William Rengifo-Cam1, Shahid Umar2, Shubhashish Sarkar1 and Pomila Singh1

Departments of 1 Neuroscience and Cell Biology and 2 Internal Medicine-Gastroenterology, University of Texas Medical Branch, Galveston, Texas

Requests for reprints: Pomila Singh, Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 10.104 Medical Research Building, Route 1043, Galveston, TX 77555-1043. Phone: 409-772-4842; Fax: 409-772-3222; E-mail: posingh{at}utmb.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progastrin (PG) exerts proliferative and antiapoptotic effects on intestinal epithelial and colon cancer cells via Annexin II (ANX-II). In here, we show that ANX-II similarly mediates proliferative and antiapoptotic effects of PG on a pancreatic cancer cell line, AR42J. The role of several signaling molecules was examined in delineating the biological activity of PG. PG (0.1–1.0 nmol/L) caused a significant increase (2- to 5-fold) in the phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt (Thr308), p38 mitogen-activated protein kinase (MAPK; Thr180/Tyr182), extracellular signal-regulated kinases (ERK; Thr202/Tyr204), I{kappa}B kinase {alpha}/ß (IKK{alpha}/ß; Ser176/180), I{kappa}B{alpha} (Ser32), and p65 nuclear factor-{kappa}B (NF-{kappa}B; Ser536). Inhibition of p44/42 ERKs (PD98059), p38 MAPK (SB203580), Akt, and PI3K (LY294002), individually or combined, partially reversed antiapoptotic effects of PG. The kinetics of phosphorylation of IKK{alpha}/ß in response to PG matched the kinetics of phosphorylation and degradation of I{kappa}B{alpha} and correlated with phosphorylation, nuclear translocation, and activation of p65 NF-{kappa}B. NF-{kappa}B essential modulator–binding domain peptide (an inhibitor of IKK{alpha}/ß) effectively blocked the activity of p65 NF-{kappa}B in response to PG. Activation of p65 NF-{kappa}B, in response to PG, was 70% to 80% dependent on phosphorylation of MAPK/ERK and PI3K/Akt molecules. Down-regulation of p65 NF-{kappa}B by specific small interfering RNA resulted in the loss of antiapoptotic effects of PG on AR42J cells. These studies show for the first time that the canonical pathway of activation of p65 NF-{kappa}B mediates antiapoptotic effects of PG. Therefore, targeting PG and/or p65 NF-{kappa}B may be useful for treating cancers, which are dependent on autocrine or circulating PGs for their growth. [Cancer Res 2007;67(15):7266–74]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The full-length progastrin1-80 peptide (PG) is the precursor peptide of gastrin (G1-G17). G17 plays an important role in gastric acid secretion via CCK2R (1). Additionally, PG and gastrin peptides exert growth effects on normal and cancerous intestinal epithelial cells (IEC) via PG-preferring receptors (2–5). We now know that colorectal cancers (CRC) express autocrine PG (2). Besides CRCs, pancreatic cancer (6), lung cancer (7), and ovarian cancer (8) also express the gastrin gene and PGs. It is possible that PG-like peptides play an equally important role in the growth and survival of many of these PG-expressing cancers as reported for PG-dependent CRCs (2, 9). Therefore, understanding the mechanisms by which PG-like peptides mediate growth and survival effects on cancer cells will be extremely useful in developing a targeted approach for treating PG-dependent cancer as previously discussed (2, 9, 10). The high-affinity PG/gastrin-binding sites are distinct from CCK1R and CCK2R and bind PG-like peptides in the order of PG>Gly gastrin>G17>CCK8 (3). Annexin II (ANX-II) was recently discovered as one such molecule and is required for mediating growth factor effects of PG/gastrin peptides on intestinal epithelial and colon cancer cells (11). Although high-affinity binding sites for PG-like peptides are present on normal IECs, and on gastrin-responsive CRCs, it is not known if high-affinity PG-binding sites are present on other cancer cell types as well. We used a rat pancreatic cancer cell line, AR42J, as the in vitro pancreatic cancer cell model. AR42J cells, unlike human pancreatic cancer cell lines, do not express autocrine growth factors and/or elevated levels of growth-promoting factors [such as nuclear factor-{kappa}B (NF-{kappa}B) or mutant Ras] and are responsive to exogenous G17/Gly gastrin (reviewed in ref. 2). In initial studies, we confirmed if AR42J cells were positive for high-affinity PG-binding sites and if the cells expressed ANX-II. Next, we confirmed if AR42J cells respond to proliferative and antiapoptotic effects of PG and if ANX-II mediates growth effects of PG on AR42J cells.

Activation of c-Src kinase is required for mediating proliferative effects of PG on IECs (12). In mice overexpressing PG (hGAS mice), significant increases in the relative levels of phosphorylated c-Src, phosphatidylinositol 3-kinase (PI3K)/Akt, Janus-activated kinase (JAK) 2, signal transducers and activators of transcription (STAT) 3, extracellular signal-regulated kinases (ERK), and transforming growth factor-{alpha} in colonic mucosa were reported (13), suggesting that one or more of these signaling molecules may be involved in mediating proliferative and/or antiapoptotic effects of PG. In fact, several of these molecules, including p38 mitogen-activated protein kinase (MAPK)/ERK p44/42 and PI3K/Akt, have been shown to play an important role in proliferation and survival of pancreatic cancer cells (14–16). Additionally, activation of NF-{kappa}B has been reported to be one of the most frequent molecular alterations in pancreatic cancer cells (14) and is required for the proliferation and survival of the cells (14, 17). Importantly, down-regulation of NF-{kappa}B sensitizes the pancreatic cancer cells to apoptotic death via anticancer agents (14, 18, 19). We therefore focused on examining the role of the above-listed signaling molecules in mediating the observed survival effects of PG on AR42J cells. As a result of our studies, we report for the first time that PG exerts potent antiapoptotic effects on AR42J cells, which are mediated by the phosphorylation of I{kappa}B kinase (IKK) {alpha}/ß, resulting in the degradation of I{kappa}B{alpha} and activation of p65 NF-{kappa}B; activation of NF-{kappa}B pathway seems to be 70% to 80% dependent on the phosphorylation (activation) of p38 MAPK/ERK and PI3K/Akt.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Leupeptin, aprotinin, benzamidine, phenylmethylsulfonyl fluoride, sodium, orthovanadate, EDTA, NP40, octyl-D glucoside, ß-mercaptoethanol, Tris, HEPES, sodium chloride, sodium fluoride, glycerol, camptothecin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Co. Polyclonal anti-caspase-3 and anti-caspase-9 antibodies were from BD PharMingen. Polyclonal anti-I{kappa}B{alpha} (C-21), anti-p50 NF-{kappa}B (H-119), anti–phosphorylated Akt1/2/3 (Thr308)-R, anti-Akt1/2 (H-136), anti-PI3K p85{alpha} (Z-8), anti-actin (I-19), anti-CCK2R (C-18), and anti-ANX-II (H-50) antibodies were from Santa Cruz Biotechnology. Monoclonal anti-lamin B (101-B7) antibody was from Calbiochem. Monoclonal anti–phosphorylated p65 NF-{kappa}B (Ser536; 7F1), anti–phosphorylated I{kappa}B{alpha} (Ser32/36; 5A5), polyclonal anti–phosphorylated stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK; Thr183/Tyr185), anti-MAPK p38, anti-ERK p44/42, anti-phosphotyrosine p85 PI3K, anti–phosphorylated MAPK p38 (Thr180/Tyr182), anti–phosphorylated ERK p44/42 (Thr202/Tyr204), anti-IKK{alpha}/ß, and anti–phosphorylated IKK{alpha}/ß (Ser176/180) antibodies were from Cell Signaling Technology. Monospecific polyclonal anti-PG antibodies were generated in house and used as described previously (3, 20, 21). Kinase inhibitors, LY294002, Akt inhibitor, SB203580, PD98059, and NF-{kappa}B essential modulator (NEMO)-binding domain peptide were from Calbiochem. Stock solutions (1 mmol/L) of all the inhibitors were prepared in 100% DMSO (Sigma) and stored at –70°C. Recombinant human PG (rhPG) was generated in our laboratory as described previously (3) and radiolabeled with Na125I by our published methods (3). G17 and CCK8 were purchased from Bachem.

Cell culture. The rat pancreatic cancer cell line AR42J was propagated in culture as described previously (22).

In vitro growth assays. The effect of increasing concentrations of rhPG, in the presence or absence of specific antibodies against ANX-II, PG, or CCK2R, on the growth response of AR42J cells was measured either in a cell count assay or in a MTT assay as described previously (4). To examine the effect of anti-PG antibodies, 0.1 nmol/L rhPG was preincubated with the antibody for 1 h before adding to the cells. In preliminary studies, we first confirmed the presence of significant levels of ANX-II protein in AR42J cells by Western blot analysis; significant levels of ANX-II were measured (data not shown) as previously reported for IEC and colon cancer cells (11). To examine the effect of receptor antibodies (including ANX-II), cells plated in the wells were preincubated with either anti-ANX-II or anti-CCK2R antibodies for 1 to 2 h; control cells were treated with an equal amount of nonimmune IgG followed by the addition of 0.1 nmol/L rhPG for 48 to 72 h.

Treatment of AR42J cells for measuring surrogate markers of apoptosis. Cells were cultured in 35-mm dishes as described previously (5). For measuring surrogate markers of apoptosis (relative levels of activated caspase-3 and caspase-9), cells were additionally treated for 5 h with the proapoptotic agent (camptothecin) as described (5). At the end of the treatment, cells were processed for measuring relative levels of activated caspase-3 and caspase-9 by Western immunoblot analysis as described (5). In a few experiments, cells were preincubated for 1 h with PP3 (20 µmol/L), LY294002 (20 µmol/L), Akt inhibitor (20 µmol/L), SB203580 (10 µmol/L), PD98059 (20 µmol/L), NEMO (200 µmol/L), and caspase-3 inhibitor I (200 µmol/L) before stimulating with rhPG as described in the figure legends.

Preparation of cellular lysates and crude nuclear extracts for Western blot analysis. Crude nuclear extracts were prepared from AR42J cells on the indicated days of cell culture using Nuclear Extract kit from Active Motif as per the instructions of the manufacturer. Cellular lysates and nuclear extracts were prepared from control and PG-treated AR42J cells as described previously (12). The samples were processed for Western immunoblot analysis with the indicated antibodies by our published methods (5, 12). All the primary antibodies were used at a dilution of 1:1,000. The antigen-antibody complexes were detected using chemiluminescence reagent kit (Amersham Pharmacia Biotech). To confirm equivalent loading, membranes were stripped and reprobed with either anti-ß-actin antibody or an antibody that detects the total amount of the indicated kinase as internal controls. The relative density of the bands was densitometrically analyzed.

Binding affinity and relative binding affinity of peptides for 125I rhPG-binding sites. The full-length rhPG was radiolabeled with Na125I (Amersham), and intact 125I-rhPG was purified by high-performance liquid chromatography as described previously (3). The binding affinity was obtained from binding assay data as described previously (3, 4). The relative binding affinity (RBA) of gastrin-like peptides for displacing specific binding of rhPG was determined as described previously (3, 4). 125I-Bolton Hunter-CCK8 (125I-BH-CCK8; Amersham Biosciences) was also used as the radiolabeled ligand, and the RBA of gastrin-like peptides for displacing the binding of 125I-BH-CCK8 to AR42J cells was examined by our published methods (4).

TransAM p65 NF-{kappa}B Chemi/Transcription Factor Assay to confirm the binding of activated p65 NF-{kappa}B to DNA. Activation of p65 NF-{kappa}B was examined using TransAM p65 NF-{kappa}B Chemi Transcription Factor Assay kit from Active Motif as per the instructions of the manufacturer. Briefly, 30 µL of complete binding buffer were added into each well coated with immobilized oligonucleotide containing the NF-{kappa}B consensus site (5'-GGGACTTTCC-3'). Nuclear proteins (20 µL), prepared from cells incubated with or without rhPG for different times, were added to the wells in 80 µL complete lysis buffer and incubated for 1 h at room temperature. The wells were washed and incubated with anti-p65 NF-{kappa}B antibody (1:1,000 dilution) for 1 h and washed thrice again followed by incubation with horseradish peroxidase–conjugated second antibody (1:10,000; 50 µL) for 1 h. The wells were washed four times followed by the addition of the chemiluminescent working solution. The resulting chemiluminescence, proportional to the levels of nuclear p65 NF-{kappa}B, bound to the consensus sequence, was measured with a luminometer (Dinex Technologies) by our published methods (23).

Promoter-reporter assay to verify the transcriptional activity of p65 NF-{kappa}B. A promoter-reporter plasmid (2 mg), which had three copies of the interleukin-8 (IL-8) promoter fragment containing RelA/NF-kB1–binding site, cloned upstream of an inert TATA box driving the expression of firefly luciferase (IL-8{kappa}BE)3-p59rAT/LUC (Invitrogen Life Technologies), was transfected in the cells by our published methods (23, 24). Twenty-four hours after transfection, the cells were treated with or without rhPG (0.1 nmol/L) for 6 h (optimal for measuring maximum activity of nuclear NF-{kappa}B as determined from previous experiments). Cells were lysed to measure luciferase levels by our published methods (23, 24).

Down-regulation of p65 NF-{kappa}B by small interfering RNA for examining the role of p65 NF-{kappa}B in mediating the antiapoptotic effects of PG on AR42J cells. p65 NF-{kappa}B small interfering RNA (siRNA; from Dharmacon) was used. AR42J cells were seeded in 35-mm dishes and incubated at 37°C for 24 h followed by growth in serum- and antibiotic-free medium for 24 h. The cells were then transfected with either 100 nmol/L p65 NF-{kappa}B siRNA construct or a negative control siRNA construct (Dharmacon) using 4 µL DharmaFECT transfection reagent 4 (Dharmacon). After 48 h, the siRNA-transfected cells were treated with or without 0.1 nmol/L PG for 48 h. The cells were treated with 10 µmol/L camptothecin for 4 h for inducing apoptosis. Total cellular proteins were extracted from camptothecin-treated cells, and relative levels of p65 NF-{kappa}B and activated caspase-3 were measured by Western immunoblot analysis as described above.

Cell death assay to measure the loss of antiapoptotic effects of PG in the presence of kinase inhibitors. A Cell Death Detection ELISAPLUS kit (Roche) was used for these studies as published (5). Briefly, AR42J cells were seeded in 35-mm dishes (0.5 x 104) and incubated at 37°C for 24 h followed by growth in serum-free medium for 24 h. Specific kinase inhibitors were added either alone or in combinations for 1 h followed by the addition of 0.1 nmol/L PG for 48 h. The proapoptotic agent camptothecin (10 µmol/L) was added at the end of the incubation for 4 h. The cells were processed as per the instructions of the supplier. The relative levels of cell death in the various samples were obtained from spectrophotometric readings of the wells at 405 nm.

Statistical analysis. Data are presented as the mean ± SE of values obtained from four to eight samples/two to three experiments. To test for significant differences between means, the nonparametric Mann-Whitney test was used using StatView 4.1 (Abacus Concepts); P values were considered to be statistically significant if <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth and antiapoptotic effects of PG on AR42J cells: presence of high-affinity binding sites (ANX-II) on AR42J cells. Dose-dependent effects of PG (0.1–1,000 nmol/L) were examined on the growth of AR42J cells in vitro. A biphasic response was measured, wherein the growth of AR42J cells was significantly increased (by >60–70%) in response to low concentrations (0.1–1.0 nmol/L) of PG, whereas a loss or no change in growth was measured at higher concentrations of 10 nmol/L to 1.0 µmol/L (Fig. 1A ). In previous studies, we have similarly reported biphasic effects of PG and gastrin peptides on the growth of IECs (3). We therefore used 0.1 nmol/L PG as an optimal dose for all further studies.


Figure 1
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  		Figure 1. A, dose-dependent (0.1 nmol/L to 1.0 µmol/L) growth effects of PG on AR42J cells. Growth in response to 1% FCS as a positive control (C). Total number of cells/35-mm dish at the end of the assay is presented. Columns, mean of six to nine separate measurements from three experiments; bars, SE. The control dishes were treated with the vehicle alone. Bi and Bii, antiapoptotic effects of an optimal dose of PG (0.1 nmol/L) were measured on AR42J cells in terms of relative levels of activated caspase-3 (Bi) and caspase-9 (Bii) in control (nontreated) versus PG-treated cells. Cells grown in the presence or absence (control) of 0.1 nmol/L PG for 48 to 72 h were treated with a potent proapoptotic agent camptothecin (10 µmol/L) for 4 h. At the end of the assay, relative levels of activated caspase-3 (Bi) and caspase-9 (Bii) were measured by Western blot analysis. Top, Western blot data from a representative experiment of a total of three experiments for caspase-3 and caspase-9. The Western blot membranes were stripped and reprobed for ß-actin levels as an internal control. The ratio of the densitometric readings for activated caspases versus ß-actin in control samples was arbitrarily designated 100% in (Bi) and (Bii). The ratio of activated caspases versus ß-actin in PG-treated samples is presented as a % of that measured in control samples. C, RBA of PG, G17, and CCK8 for displacing the binding of either 125I-rhPG (Ci) or 125I-BH-CCK8 (Cii) to AR42J cells. AR42J cells were incubated with the indicated radiolabeled ligand in the presence or absence of increasing concentrations of either CCK8 (cholecystokinin), G17, or rhPG. The RBA of the gastrin-like peptides for the specific binding sites for rhPG (Ci; ANX-II) and CCK8 (Cii) to CCK2R was calculated as described in Materials and Methods. Points, mean values of triplicate measurements from a single experiment and representative of two similar experiments. The intraexperimental variation for each data point was <5% to 10%. X axis, nanomolar concentration of each peptide used presented in a log scale. Parentheses, excess unlabeled peptide used for displacing the binding of the radiolabeled ligand. Y axis, percentage loss in the relative binding of the radiolabeled ligand in the presence of the increasing concentrations of the indicated nonlabeled peptide. D, the effect of anti-PG, anti-ANX-II, and anti-CCK2R antibodies (Ab) on the growth response of 0.1 nmol/L PG on AR42J cells was determined. Cells were treated with antibodies and PG and growth was determined in a MTT assay as described in Materials and Methods. At the end of the assay, the colorimetric reaction was read at 540 nm. IgG, nonimmune IgG. Columns, mean of six to eight measurements from an experiment measured in duplicate; bars, SE. SFM, serum-free medium.

 
Previously, we showed potent antiapoptotic effects of PG on IEC cells (5) and colon cancer cells (25). In the current studies, PG was found to similarly exert potent antiapoptotic effect on AR42J cells; camptothecin-induced activation of caspase-3 and caspase-9 was significantly reduced by >70% to 80% in the presence of 0.1 nmol/L PG (Fig. 1Bi and Bii).

Several investigators have shown that PG peptides exert growth effects on target cells via novel binding sites that are distinct from CCK2R (2). Because CCK2R are expressed at high concentrations on AR42J cells (4, 26), in the current study, we established the RBA of gastrin-like peptides (PG, G17, and CCK) for the high-affinity CCK receptors and the high-affinity PG-binding receptors. PG dose dependently displaced the binding of radiolabeled rhPG (Fig. 1Ci) but was largely ineffective in displacing the binding of radiolabeled 125I-BH-CCK8 to CCK2R on AR42J cells (Fig. 1Cii). On the other hand, CCK and G17, both of which bind CCK2R with high affinity (4, 26), showed a negligible binding affinity for PG-binding sites (Fig. 1Ci) but completely displaced binding of 125I-BH-CCK8 (Fig. 1Cii). Recently, we discovered that ANX-II molecules serve the role of high-affinity PG-binding sites (11). To confirm the role of ANX-II in mediating growth effects of PG on AR42J cells, we examined the effect of anti-ANX-II and anti-CCK2R antibodies (Fig. 1D). Anti-PG antibodies and anti-ANX-II antibodies significantly reduced growth of PG-stimulated AR42J cells to control levels, confirming a direct role of ANX-II and PG in the observed effects (Fig. 1D); treatment of cells with anti-CCK2R antibodies had statistically insignificant effects on PG-stimulated growth of AR42J cells (Fig. 1D). The binding affinity of rhPG for AR42J cells was determined by Scatchard analysis to be in the range of 0.1 to 0.5 nmol/L (data not shown) as previously reported for IEC cells (3).

Phosphorylation of PI3K, Akt, p38 MAPK, and p44/42 ERK in response to rhPG in AR42J cells. Specific antibodies against the indicated phosphorylated kinases were used. In the case of PI3K, we used a phosphotyrosine p85 PI3K-binding motif antibody that binds to tyrosine-phosphorylated YXXM motif, is recognized by p85, and docks PI3K to the membrane resulting in activation of the kinase. As can be seen from Supplementary Fig. S1A to E, phosphorylation of all the indicated kinases examined in this study was significantly increased by approximately 2- to 5-fold in AR42J cells in response to PG. In all cases, the relative levels of phosphorylated kinases were corrected for the total levels of the corresponding kinase. The phosphorylation moieties examined are detailed in the legend of Supplementary Fig. S1.

Loss of antiapoptotic effects of PG on AR42J cells in response to kinase inhibitors. To assess the role of phosphorylated (activated) kinases in mediating the antiapoptotic effects of PG, specific inhibitors of the kinases were used as detailed in Materials and Methods and in the legend of Supplementary Fig. S2. Activation of caspase-3 was used as a surrogate marker for loss of survival effects of PG in the presence of the indicated inhibitors in response to the proapoptotic agent camptothecin (Supplementary Fig. S2). Treatment with inhibitors of ERKs, MAPK, Akt, and PI3K significantly reversed the antiapoptotic effects of PG by ~70%, ~60%, ~45%, and ~30%, respectively, indicating that MAPK/ERK and PI3K/Akt kinases are involved in mediating antiapoptotic effects of PG. Interestingly, inhibition of MAPK or ERK activity was more effective than inhibition of PI3K/Akt in reversing the antiapoptotic effects of PG (Supplementary Fig. S2).

PG stimulates phosphorylation of IKK{alpha}/ß, I{kappa}B{alpha}, NF-{kappa}B, and translocation of NF-{kappa}B into the nucleus of AR42J cells. Because activation of MAPK/ERKs and PI3K/Akt is reported to result in activation of NF-{kappa}B (27), we next examined if NF-{kappa}B is activated in response to PG via the canonical pathway involving phosphorylation of IKK{alpha}/ß and degradation of I{kappa}B{alpha} (27–30). Treatment of AR42J cells with 0.1 nmol/L PG resulted in a significant increase in the phosphorylated levels of IKK{alpha}/ß, after 6 to 36 h of stimulation, with a peak at 6 h (Supplementary Table S1A). The relative levels of phosphorylated I{kappa}B{alpha} were also significantly increased by approximately 2- to 3-fold after 6 h of PG stimulation, and these levels remained elevated until 36 h after stimulation (Supplementary Table S1B). As expected, increased phosphorylation of I{kappa}B{alpha} was associated with a significant decline in the relative levels of total I{kappa}B{alpha} by 6 h of PG stimulation, and the levels of total I{kappa}B{alpha} remained significantly low until ~48 h after PG stimulation (Fig. 2A ). In association with the degradation of I{kappa}B{alpha}, a significant increase in phosphorylation of cellular p65 NF-{kappa}B was measured in response to PG, which once again peaked at 6 h after PG stimulation (Fig. 2B). A significant increase in total nuclear NF-{kappa}B (Supplementary Table S1C) and in relative levels of phosphorylated NF-{kappa}B (Fig. 2C) was measured in the nuclear fraction of AR42J cells after 6 h of stimulation with 0.1 nmol/L PG; the increase in nuclear NF-{kappa}B remained elevated until ~48 h after PG treatment, suggesting a sustained phosphorylation of p65 NF-{kappa}B in response to PG (Fig. 2C). In the presence of NEMO-binding peptide, which inhibits the activation of IKK{alpha}/ß (31), phosphorylation of p65 NF-{kappa}B in response to PG at 6 h was reduced to control levels (Fig. 2D).


Figure 2
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  		Figure 2. A–D, PG treatment stimulates degradation of I{kappa}B{alpha} and increases phosphorylation of NF-{kappa}B p65 in the cellular and nuclear compartments. AR42J cells were treated with 0.1 nmol/L PG for increasing times from 3 to 48 h. At the end of the treatment, cells were processed for Western blot analysis with specific antibodies. A, samples were probed with antibodies against total I{kappa}B{alpha} and reprobed with anti-ß-actin antibodies. Top, representative autoradiographs of Western blot data from one of three separate experiments with antibodies against I{kappa}B{alpha}; bottom, densitometric readings at 0 h for total I{kappa}B{alpha} versus ß-actin was arbitrarily assigned a 100% value, and the ratio at all other time points is presented as a % change from control values. *, P < 0.05 versus 0 h values in (A). B and C, samples were processed for measuring phosphorylated NF-{kappa}B (Ser536) in either the cellular lysates (B) or the nucleus (C). For nuclear samples, relative levels of ß-actin in the nucleus were measured as an internal control. A set of samples in (B) was additionally pretreated with or without NEMO (an inhibitor of IKK{alpha}/ß) for 1 h as indicated in Fig. 2D. B–D, top, representative autoradiographs from one of three separate experiments. The signal intensity of the bands was determined densitometrically for each blot. For samples in (B), the ratio of the readings for phosphorylated NF-{kappa}B versus total NF-{kappa}B in the cellular lysate samples was arbitrarily assigned 100% value for 0 time point. The ratios for all other time points are presented as a % change from the 0 time point values. For samples in (C), the densitometric readings for phosphorylated NF-{kappa}B (p65536) in the nucleus versus nuclear ß-actin levels was arbitrarily assigned a 100% value at 0 time point. Ratios at all other time points are presented as a % change from 0 time point values and were significantly different (P < 0.05) versus control (0 h) values at 6, 12, 24, 36, and 48 h. Columns, mean of four blots from two separate experiments; bars, SE. *, P < 0.05 versus 0 time point values. Cells in (D) were treated for 6 h with PG in the presence or absence of NEMO. The ratio of the densitometric readings for phosphorylated NF-{kappa}B versus total NF-{kappa}B for the untreated samples (first column) was arbitrarily assigned a 100% value, and the ratios of the readings for the samples treated with PG ± NEMO are presented as a % change from control nontreated samples.

 
PG stimulates the activation of p65 NF-{kappa}B as measured in DNA-binding assays. To confirm functional activation of nuclear p65 NF-{kappa}B, binding of nuclear proteins to {kappa}B sites in an in vitro DNA-binding assay was conducted as described in Materials and Methods. A significant increase in the binding of nuclear protein (NF-{kappa}B) was measured at 6 h after PG stimulation, which was ~4-fold higher than that in control samples and ~1/2 of that measured with the potent stimulator tumor necrosis factor-{alpha} (TNF-{alpha}; Fig. 3 ). Importantly, activation of NF-{kappa}B in terms of DNA binding remained elevated until ~36 h after PG stimulation (Fig. 3), once again providing evidence that PG stimulation results in sustained activation of NF-{kappa}B. In transient transfection assays, using a promoter-reporter construct, a significant increase in the expression of luciferase was once again measured in AR42J cells stimulated by PG for 6 to 36 h (data not shown).


Figure 3
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  		Figure 3. PG induces activation of NF-{kappa}B p65 as measured in a DNA binding assay. AR42J cells in culture were treated with 0.1 nmol/L PG for the indicated times. At the end of the treatment period, the cells were processed for the preparation of nuclear protein extracts. The presence of activated NF-{kappa}B p65 in the nuclear extracts was examined with the TransAM NF-{kappa}B p65 Chemi Transcription Factor Assay kit. The readout for the DNA binding assay was measured in chemiluminescence units, and the readings for control nontreated samples at 0 time point was arbitrarily assigned a 100% value. As a positive control, nuclear extracts were also prepared from AR42J cells stimulated with 25 ng TNF-{alpha}/mL medium for 30 min. The chemiluminescence units measured for samples treated with either TNF-{alpha} or with PG for increasing time points are presented as a % change from the control 0.0 values. Columns, mean of data from three separate experiments; bars, SE. *, P < 0.05 versus untreated 0.0 values in the first lane.

 
Phosphorylation (activation) of p65 NF-{kappa}B is downstream of PI3K/Akt/MAPK/ERKs. In the next set of experiments, we examined the role of individual kinases in mediating phosphorylation of the indicated kinases/transcription factor in response to PG. Surprisingly, inhibition of either Akt (via Akt inhibitor) or PI3K (via LY294002 compound) had no effect on the PG-stimulated phosphorylation of p44/42 ERKs (Supplementary Fig. S3A), or the phosphorylation of p38 MAPK (Supplementary Fig. S3B), suggesting that phosphorylation of MAPK/ERK in response to PG stimulation may not be dependent on phosphorylation of PI3K/Akt at the time point examined. On the other hand, inhibition of either MAPK or ERKs resulted in a significant inhibition in relative levels of phosphorylated PI3K (Supplementary Fig. S3C) and phosphorylated Akt (Supplementary Fig. S3D) to control levels, suggesting that phosphorylation of PI3K/Akt molecules may be downstream of phosphorylation of MAPK/ERK kinases in response to PG at time points examined in this study.

The role of MAPK/ERKs and PI3K/Akt in the phosphorylation and activation of NF-{kappa}B in response to PG was next examined as described in the legend of Fig. 4 . Inhibition of either MAPK or ERKs resulted in an almost complete loss in the relative levels of phosphorylated p65 NF-{kappa}B (Fig. 4A), whereas inhibition of either PI3K or Akt was less effective (Fig. 4B versus Fig. 4A). In transient transfection studies, using a promoter-reporter assay, a similar pattern was obtained in response to the specific kinase inhibitors (Fig. 4C). Inhibition of either MAPK or ERKs was more effective than inhibition of either PI3K or Akt in reversing the activation of NF-{kappa}B in response to PG (Fig. 4C).


Figure 4
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  		Figure 4. Phosphorylation and activation of NF-{kappa}B p65 is downstream of phosphorylation (activation) of MAPK/ERK and PI3K/Akt. AR42J cells in culture were pretreated with the indicated inhibitor followed by stimulation with 0.1 nmol/L PG as described in Materials and Methods. At the end of the treatment, the cells were either processed for preparation of extranuclear (cellular) extracts (A and B) or processed for the preparation of nuclear extracts (C). The samples in (A) and (B) were further processed for Western blot analysis using specific antibodies against either phosphorylated p65 NF-{kappa}B (Ser536) or total NF-{kappa}B p65. A and B, top, representative autoradiographs from one of three separate experiments. The signal intensity of the bands was densitometrically analyzed, the ratio of the readings for phosphorylated NF-{kappa}B p65 (Ser536) versus the corresponding relative levels of total NF-{kappa}B was arbitrarily assigned a 100% value, and the ratios for all other treated samples are presented as a % of the control values in (A) and (B). SB, SB203580; LY, LY294002; PD, PD98059. Columns, mean of data from three separate experiments; bars, SE. *, P < 0.05 versus control values (first column); {dagger}, P < 0.05 versus PG alone treated samples (second column). The abbreviation and the concentration of the indicated inhibitors are the same as described in the legend of Supplementary Fig. S2. For data presented in (C), a promoter-reporter assay was used for measuring the relative activation of NF-{kappa}B in response to 0.1 nmol/L PG in the presence or absence of the indicated kinase inhibitors. Cells were transiently transfected with the promoter-reporter plasmids as described in Materials and Methods. The transfected cells were then pretreated with the indicated kinase inhibitors followed by stimulation with 0.1 nmol/L PG. Control samples were not treated with either PG or the inhibitors. At the end of the treatment, cells were processed for measuring the relative levels of luciferase, and the luciferase units measured in the control samples were arbitrarily assigned a 0 value. The luciferase levels measured in all other treated samples are presented as a % of control values. A–C, columns, mean of data from two to three separate experiments. *, P < 0.05 versus control values (first column); {dagger}, P < 0.05 versus PG alone treated samples (second column).

 
NF-{kappa}B is required for mediating antiapoptotic effects of PG on AR42J cells. We next examined if NF-{kappa}B was required for mediating antiapoptotic effects of PG on AR42J cells. NF-{kappa}B siRNA was used for down-regulating expression of NF-{kappa}B. As can be seen in Fig. 5A , cells transfected with NF-{kappa}B siRNA showed a loss of ~75% of NF-{kappa}B protein (Fig. 5A, lane 5), whereas treatment of cells with a negative control siRNA in the presence or absence of PG or camptothecin had no effect on relative levels of NF-{kappa}B (Fig. 5A, lanes 1–4). Loss of NF-{kappa}B as a result of treatment of cells with NF-{kappa}B siRNA resulted in a complete loss of antiapoptotic effects of PG (in terms of relative levels of activated caspase-3) in response to proapoptotic agent camptothecin (Fig. 5B, lane 5). Results in Fig. 5 thus confirmed for the first time that NF-{kappa}B was required for mediating protective (survival) effects of PG on pancreatic cancer cells.


Figure 5
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  		Figure 5. A and B, p65 NF-{kappa}B siRNA reverses antiapoptotic effects of PG on AR42J cells. AR42J cells in culture were transiently transfected either with a control negative (Neg) siRNA or with a specific NF-{kappa}B p65 siRNA construct as described in Materials and Methods. The transfected cells were treated with or without 0.1 nmol/L PG for 48 to 72 h as indicated in (A) and (B) followed by treatment with camptothecin (10 µmol/L) for 4 h before the end of the treatment. The cells were processed for Western blot analysis with specific antibodies against either total p65 NF-{kappa}B (A) or activated caspase-3 (B). The blots were reprobed with anti-ß-actin antibodies as an internal control. Treatment with the control (negative) siRNA or PG (0.1 nmol/L) or camptothecin (10 µmol/L) had no effect on the relative levels of total NF-{kappa}B (A, lanes 1–4), but treatment with specific NF-{kappa}B p65 siRNA (A, lane 5) resulted in approximately 70% to 75% loss in the relative levels of total NF-{kappa}B. Similarly, the antiapoptotic effects of PG in response to camptothecin (lanes 3 and 4) were not affected in cells transfected with the negative (control) siRNA (B, lane 4) but were completely reversed in the presence of specific NF-{kappa}B p65 siRNA (B, lane 5). C, effect of kinase inhibitors on reversing the antiapoptotic effects of PG in response to camptothecin. AR42J cells in culture were treated with the indicated kinase inhibitors followed by treatment with PG as described in Materials and Methods. Apoptosis was induced by camptothecin. In one set of experiments (lane 9), the cells were treated with the caspase-3 inhibitor (Caspase-3-I; 1 µmol/L for 2 h). The explanation for the abbreviations for the inhibitors and concentrations used are the same as described in the legend of Supplementary Fig. S2. At the end of the treatment, the cells were processed for measuring the relative levels of cell death using a cell death assay kit as described in Materials and Methods. An increase in absorbance (A) readings at 405 nm reflects the relative increase in cell death of the samples being examined. Columns, mean of data from four separate observations from two experiments; bars, SE. {dagger}, P < 0.05 versus lane 1 values; *, P < 0.05 versus lane 2 values.

 
Relative role of MAPK/ERKs/PI3K/Akt and p65 NF-{kappa}B in mediating antiapoptotic effects of PG on AR42J cells. In the final set of experiments, rather than using surrogate markers of apoptosis, we used the cell death assay kit for measuring the role of various kinases in mediating antiapoptotic effects of PG as described in the legend of Fig. 5C. Inhibiting the activation of NF-{kappa}B (via NEMO peptide) was most effective in completely reversing protective effects of PG on cell death of AR42J cells in response to camptothecin (Fig. 5C, lane 4 versus lane 3). Inhibitors of MAPK and ERKs were also quite effective (70–80%) in reversing protective effects of PG (Fig. 5C, lanes 5 and 6 versus lane 3); inhibitors of PI3K and Akt were somewhat less effective (55–65%; Fig. 5C, lanes 7 and 8 versus lane 3). Inhibiting the activation of caspase-3 (Fig. 5C, lane 9) significantly reduced proapoptotic effects of camptothecin (Fig. 5C, lane 2). Various combinations of kinase inhibitors as shown in Fig. 5C (lanes 10–16) were not any more effective than inhibiting activation of NF-{kappa}B with NEMO peptide alone (Fig. 5C, lane 4); these results suggest that activation of NF-{kappa}B may represent the ultimate pathway for mediating antiapoptotic effects of PG on pancreatic cancer cells.

Based on the results presented in Figs. 1–5, a hypothetical model is presented in Fig. 6 , describing the upstream versus downstream effects of PG on activation of various kinases.


Figure 6
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  		Figure 6. Diagrammatic representation of receptor subtypes and intracellular kinases that are likely to be involved in mediating antiapoptotic effects of PG on AR42J cells. Solid lines, pathways that were confirmed in the current study; broken arrows, pathways that need to be confirmed. The results in Fig. 1D confirm a role for ANX-II (PG/gastrin receptor) in mediating the growth effects of PG. Other receptor subtypes, other than CCK2R, may also be involved in mediating the growth factor effects of PG as stipulated previously (49). Arrows up within the boxes, an increase in the phosphorylation (activation) status of the indicated kinase; arrows down in the boxes for caspase-3 and caspase-9, loss in the activation of these proapoptotic enzymes; sideway arrow in the box for p38 MAPK and ERK, possible coregulatory role of these kinases in mediating the up-regulation of the phosphorylation (activation) status of NF-{kappa}B.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we show for the first time that NF-{kappa}B plays a critical role in mediating antiapoptotic effects of PG on a pancreatic cancer cell line. There are five members of the mammalian NF-{kappa}B family, RelA (p65), RelB, c-Rel, p50/p105 (NF-{kappa}B1), and p52/p100 (NF-{kappa}B2), which exist in unstimulated cells as homodimers or heterodimers bound to I{kappa}B family proteins (27, 28). Binding of I{kappa}B to NF-{kappa}B prevents NF-{kappa}B-I{kappa}B complex from translocating to the nucleus, thereby maintaining NF-{kappa}B in an inactive state. In the classic (canonical) pathway of NF-{kappa}B activation, on stimulation by a cytokine/growth factor, the signaling pathways lead to activation of ß subunit of IKK, which phosphorylates I{kappa}B proteins, leading to their polyubiquitination and degradation (28). The freed NF-{kappa}B dimers translocate to the nucleus, where they bind to specific sequences in the promoter or enhancer regions of target genes. In AR42J cells, PG stimulated phosphorylation of IKK{alpha}/ß, resulting in phosphorylation and degradation of I{kappa}B{alpha} followed by phosphorylation, nuclear translocation, and activation of NF-{kappa}B p65.

The IKK complex contains two kinase subunits (IKK{alpha} and IKKß) and a regulatory subunit, NEMO (also called IKK{gamma}; ref. 31). In the classic NF-{kappa}B signaling pathway, IKKß is both necessary and sufficient for phosphorylating I{kappa}B{alpha} and I{kappa}Bß. We confirmed a critical role of IKKß and NEMO in experiments with NEMO peptide (a selective inhibitor of IKKß). NEMO peptide attenuated PG-stimulated phosphorylation of NF-{kappa}B p65 and abrogated the antiapoptotic effects of PG on AR42J cells; this critical role of NF-{kappa}B was further confirmed in experiments with specific siRNA against NF-{kappa}B p65 transcripts. Results of our studies thus suggest that NF-{kappa}B activation by the canonical pathway is primarily involved in mediating growth effects of PG on AR42J cells.

Multiple signaling pathways lead to phosphorylation and activation of IKK complexes. IKK kinases include members of the MAPK kinase family, such as MAPK/ERK kinase kinase (MEKK) 1, 2, and 3 (27, 31). Other putative IKK kinases include members of the atypical protein kinase C (PKC) family, Akt, and two proteins related to IKK{alpha}/ß, IKKI and NF-{kappa}B–activating kinase B (27). Our studies show that NF-{kappa}B activation in response to PG is downstream of MAPKs/ERKs and PI3K/Akt signaling pathways. A potent inhibitor of histone deacetylase, trichostatin A was also reported to activate NF-{kappa}B via PI3K and MAPK signaling pathways (32).

In many cells, Akt acts upstream of NF-{kappa}B (33). However, in a few cases, NF-{kappa}B activation was reported to be independent of Akt function (34). Whereas one group has reported that PI3K/Akt does not contribute toward constitutive NF-{kappa}B activation in the pancreatic cancer cells (19), another group reported that constitutive NF-{kappa}B–mediated transcription in CRC cell lines was dependent on activation of PI3K/Akt (35). These discrepancies may be due to noncoordinate expression of IKKs, which was postulated to play a role in determining the cell type–specific role of PI3K/Akt in NF-{kappa}B activation (33). Cells with high proportion of IKK{alpha} (activated by Akt) to IKKß are more sensitive to PI3K inhibitors and vice versa (33). The results of our studies suggest that inhibition of ERKs and/or p38 MAPK almost completely attenuated activation of NF-{kappa}B in response to PG, whereas inhibition of PI3K/Akt was less effective. Similarly, adiponectin, an obesity-related factor, activated NF-{kappa}B via increased phosphorylation of ERKs and p38 MAPKs (36). However, a cell-specific role of MAPKs versus PI3K/Akt has been shown. For example, acetyl-11-keto-ß-boswellic acid suppressed activation of IKK through inhibition of Akt and p38 MAPK but not p44/p42 MAPK (37), whereas both ERK1/2 and Akt pathways mediate inhibitory effects of green tea polyphenol (–)-epigallocatechin-3-gallate on NF-{kappa}B activation in colon cancer cells (38). These studies show that signaling pathways leading toward activation of NF-{kappa}B are cell and/or stimulus specific.

NF-{kappa}B can be additionally activated by IKK-independent pathways. For example, protein kinase A binds p65 and phosphorylates p65 at Ser276 independent of I{kappa}B degradation (31). Akt and PKC{zeta} also phosphorylate p65 at distinct residues and increase transcriptional activation of NF-{kappa}B without involving I{kappa}B degradation (31). {alpha}2-Macroglobulin, bound to surface-associated GRP 78 on prostrate cancer cells, was reported to activate NF-{kappa}B by both canonical and noncanonical pathways (39). However, a role of noncanonical pathways in response to PG in AR42J cells seems less likely because NEMO almost completely attenuated antiapoptotic effects of PG on AR42J cells.

In the current studies, we measured a delayed activation of NF-{kappa}B in response to PG in AR42J cells. A small peak of activation was measured at 30 min (Fig. 2B) followed by a more robust peak at 6 h, which was sustained for ~36 h. NF-{kappa}B regulates its own activity by transcriptional activation of I{kappa}B{alpha}, which represents a negative feedback loop and drives oscillations in levels of activated NF-{kappa}B (40). Etoposide (a topoisomerase inhibitor) induces lower amplitude oscillations than TNF-{alpha}, with a delayed peak of NF-{kappa}B activation at 300 min (40). The pattern of peak timing and amplitude/oscillation is not only stimulus specific but also cell specific (40). We measured peak levels of phosphorylated I{kappa}B{alpha} and phosphorylated NF-{kappa}B p65Ser536 (translocated to the nucleus) at 6 h compared with a rapid induction of peak levels in response to TNF-{alpha} (Fig. 2C). Activation of NF-{kappa}B has similarly been reported to be delayed but sustained in response to lipopolysaccharide (LPS; ref. 41). NF-{kappa}B activation induced by cigarette smoke condensate is similarly persistent (42). In the case of PG, we have measured activation of several signaling kinases, including MAPK/ERKs/JNK and PI3K/Akt (current studies) and ß-catenin.3 It is thus possible that activation of multiple signaling pathways may have resulted in the observed delayed and sustained activation of NF-{kappa}B in response to PG in AR42J cells as reported for LPS, etoposide, and cigarette smoke condensate.

Because IKK is one of the many kinases implicated in direct phosphorylation of p65 at Ser536 (29), we examined Ser536 phosphorylation of p65 in response to PG. We have also measured Ser276 phosphorylation of p65 in AR42J cells in response to PG,3 suggesting that NF-{kappa}B is likely phosphorylated at both Ser276 and Ser536 in response to PG stimulation in AR42J cells. Whereas almost all pancreatic cancers have high levels of constitutively active NF-{kappa}B (14), because AR42J cells are relatively differentiated, levels of activated NF-{kappa}B are very low in unstimulated AR42J cells (Fig. 3). We therefore measured a significant increase in the proliferation of AR42J cells in response to 0.1 to 1.0 nmol/L PG, which correlated with potent antiapoptotic effects of PG (Fig. 1).

Several receptor subtypes mediate the growth factor effects of gastrin/PG peptides on target cells (2, 26). However, we and others have shown that the growth factor effects of PG are mediated by novel receptor mechanisms that are distinct from CCK2R (2, 43, 44). In the current study, we confirmed that PG had a relatively poor binding affinity for CCK2R (Fig. 1C). On the other hand, the binding of radiolabeled PG to AR42J cells was not inhibited by either CCK or G17 (Fig. 1C). In cell lines that do not express CCK2R (such as IEC 18 cells), G17 showed a significant binding affinity for PG-binding sites (3), which led us to postulate that RBA of G17 for PG-binding sites is dictated by the presence or absence of CCK2R (45). The results of the binding studies (Fig. 1) provide further evidence for our above hypothesis. Several years ago, we had identified a 33 to 36 kDa protein as a high-affinity binding protein for PG/gastrin peptides, which was pharmacologically different from CCK1R and CCK2R (46). Recently, we discovered that ANX-II represented the novel p36 receptor protein (11). Unlike CCK2R antibodies, ANX-II antibodies completely attenuated growth effects of PG on AR42J cells (Fig. 1D), confirming our previous findings with IEC and colon cancer cells (11).

Besides PG, other gastrin-like peptides also activate NF-{kappa}B via either CCK1R (47) or CCK2R (48). CCK2R antagonist and a PKC inhibitor completely attenuated gastrin-induced activation of NF-{kappa}B (48); surprisingly, inhibition of p38 MAPK had no effect on NF-{kappa}B activation (48). However, our studies suggest that p38 MAPK plays a critical role in mediating activation of NF-{kappa}B in response to PG in AR42J cells. These findings once again show that, although many ligands, ligand receptors, and kinases activate NF-{kappa}B, the specific kinases that signal to NF-{kappa}B activation are stimulus and/or cell specific. The findings thus far suggest that binding of PG/G17-like peptides to their cognate receptors (such as ANX-II, CCK1R, and CCK2R) can potentially result in activation of NF-{kappa}B via many different pathways, resulting in differential effects on different cell types.

Binding of various ligands with ANX-II has been reported to result in the activation of several signaling pathways, including NF-{kappa}B, JAK/STAT, p38 MAPK, and MEKK4, as recently reviewed (49). It is possible that PG binding to ANX-II may provide a similar platform for multimeric complex formation of various kinases resulting in the observed activation of several signaling molecules in response to PG, including Src, PI3K/Akt, JAK2, STAT5/3, ERKs, p38 MAPK, and NF-{kappa}B (current studies; refs. 12, 50), as diagrammatically presented in our recent review article (49).


   Acknowledgments
 
Grant support: NIH grants CA97959 and CA114264 (P. Singh) and CA099121 (S. Umar).

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

We thank Dr. Sanjeev Choudhary (Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX) for his help with some of the methods and Cheryl Simmons and Lyn Schilling for the secretarial help.


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

3 S. Umar, S. Cowey, S. Sarkar, P. Singh, unpublished data. Back

Received 4/ 5/07. Revised 5/10/07. Accepted 5/30/07.


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 Introduction
 Materials and Methods
 Results
 Discussion
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