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Carcinogenesis Advance Access originally published online on November 28, 2006
Carcinogenesis 2007 28(5):968-976; doi:10.1093/carcin/bgl220
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Aspirin activates the NF-{kappa}B signalling pathway and induces apoptosis in intestinal neoplasia in two in vivo models of human colorectal cancer

Lesley A. Stark*, Kirsten Reid1, Owen J. Sansom2, Farhat V. Din1, Sylvie Guichard3, Iain Mayer3, Duncan I. Jodrell3, Alan R. Clarke1 and Malcolm G. Dunlop

Division of Oncology, School of Clinical and Molecular Medicine, University of Edinburgh, Colon Cancer Genetics Group, MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
1 Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3US, UK
2 Beatson Institute for Cancer Research, Garscube Estate, Glasgow, UK G61 1BD
3 Pharmacology and Drug Development Group, Cancer Research UK Centre, University of Edinburgh, Edinburgh EH4 2XR, UK

* To whom correspondence should be addressed. Tel: +44 131 467 8440; Fax: +44 131 343 2620; Email: lesley.stark{at}hgu.mrc.ac.uk


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Substantial evidence indicates that aspirin has antitumour activity against large bowel cancer and modulation of the NF-kappaB (NF-{kappa}B) signalling pathway has been identified as a key mechanism in this effect. However, studies examining how aspirin affects the NF-{kappa}B pathway to promote apoptosis have been restricted to in vitro analysis in tissue culture systems and have produced contrasting results. Here, we employed two animal models of human colorectal cancer to determine aspirin effects on the NF-{kappa}B pathway in colorectal neoplasia in vivo, and the relationship of such effects to the induction of apoptosis. We demonstrate that aspirin induces phosphorylation and degradation of cytoplasmic I{kappa}B{alpha} in xenografted HT-29 tumours and in adenomas from APCMin+/– mice. Furthermore, we show that this response occurs in a time-dependent manner and is paralleled by nuclear translocation of p65 and caspase activation. Using high performance liquid chromatography analysis, we demonstrate that >0.5 mM salicylate levels are achievable in xenografted tumours after low-dose aspirin (40 mg/kg) treatment and that these levels, which are pharmacologically relevant to humans, are sufficient to stimulate an NF-{kappa}B and apoptotic response. We demonstrate that activation of the NF-{kappa}B pathway is associated with increased apoptosis in neoplastic epithelial cells, but found that aspirin has a minimal effect on nuclear p65 and apoptosis in normal intestinal mucosa from APCMin+/– mice. These in vivo findings further establish that aspirin induces activation of the NF-{kappa}B pathway in neoplastic epithelial cells and provide further support that this effect is important for the antitumour activity of the agent. These data have considerable relevance to cancer prevention and therapy.

Abbreviations: NF-{kappa}B, NF-kappaB; NSAIDs, non-steroidal anti-inflammatory drugs; I{kappa}B, I-kappaB; WT, wild-type; SR, super-repressor


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Strategies focussed on cancer prevention are now considered the optimal approach for combating the significant morbidity and mortality associated with colorectal cancer (http://info.cancerresearchuk.org/). Substantial evidence from epidemiological, clinical, animal and laboratory studies indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have antitumour activity against colorectal cancer and the potential for primary prevention of this disease (1–3). However, the potential of both aspirin-like and COX-2-specific NSAIDs is limited by the toxicity associated with their long-term use (4,5). The predominant antitumour activity of NSAIDs is recognized to be the selective induction of apoptosis in neoplastic cells (6–11). Understanding the fundamental molecular mechanisms underlying this proapoptotic activity will allow the rational design of safer, more effective chemopreventative agents.

We, and others, have shown that modulation of the NF-kappaB (NF-{kappa}B) signal transduction pathway is a key mechanism for the proapoptotic activity of aspirin and related NSAIDs (12–16). However, studies examining how NSAIDs affect the NF-{kappa}B pathway to promote apoptosis have all been carried out using tissue culture systems in vitro, and have produced contrasting results dependent upon experimental design. The NF-{kappa}B transcription factor generally exists as a heterodimer of the p50 and p65 polypeptides, bound in the cytoplasm by the inhibitor protein I-kappaB (I{kappa}B) (17). Following cellular stimulation by specific inducers, I{kappa}B{alpha} is phosphorylated at serines 32 and 36 by the I{kappa}B kinase complex, then degraded by the 26S proteosome. Subsequently, NF-{kappa}B translocates to the nucleus where it regulates transcription of its target genes, which include many that control cell growth and apoptotic cell death (18).

Data from this laboratory have shown that aspirin activates the NF-{kappa}B pathway to induce apoptosis of colorectal cancer cells. We demonstrated that prolonged treatment of colorectal cancer cells with pharmacologically relevant doses of aspirin, in the absence of additional cytokines or other NF-{kappa}B activating agents, induces phosphorylation and proteosomal-mediated degradation of I{kappa}B{alpha} and nuclear translocation of NF-{kappa}B (16,19). This NF-{kappa}B response to aspirin occurred in a time- and dose-dependent manner, consistent with aspirin-induced apoptosis. Furthermore, using colon cancer cells we generated to express a mutant form of I{kappa}B, that is resistant to aspirin-induced phosphorylation/degradation (I{kappa}B-SR), we demonstrated that inhibiting nuclear translocation of NF-{kappa}B blocks the apoptotic response to aspirin. In recent studies, we used p65 null mouse embryo fibroblasts, reconstituted with wild-type (WT) p65, to confirm that aspirin induces nuclear translocation of the p65 component of NF-{kappa}B and that this response is essential for aspirin-mediated apoptosis (16). In addition to aspirin, the NSAID diclofenac (20), and the COX-2-specific inhibitors, celecoxib and NS-398 (21,22), have also been shown to induce degradation of I{kappa}B and nuclear translocation of NF-{kappa}B in the absence of additional NF-{kappa}B stimulators. In contrast to these data, we and others have demonstrated that in short-term in vitro experiments, aspirin inhibits inflammatory cytokine-induced activation of NF-{kappa}B in colorectal cancer cells (12,23,24).

Here, we employed the HT-29 xenograft and APCMin+/– mouse models of colon carcinogenesis to determine the effect that aspirin has on the NF-{kappa}B pathway in colorectal neoplasia in vivo. We also investigated the relationship between such effects on NF-{kappa}B and the proapoptotic activity of the agent. Use of these two animal models allowed study of aspirin effects on the NF-{kappa}B pathway and apoptosis in vivo in both premalignant neoplastic epithelial cells and also in malignant colon cancer cells. Using the HT-29 xenograft model, we examined the pharmacokinetics of low- and high-dose aspirin in plasma and tumours and related salicylate levels to effects on the NF-{kappa}B pathway. Our data demonstrate that aspirin activates the NF-{kappa}B pathway in intestinal neoplasia in vivo and that this effect is paralleled by the induction of apoptosis. These studies identify an intrinsic activity of aspirin in vivo that is highly relevant to colon cancer chemoprevention in humans.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Tissue culture
HT-29 cells were obtained from the American Type Culture Collection and maintained in supplemented dulbecco's modified eagle's medium. Cultured cells were exposed to aspirin as described previously (25).

Western blot analysis and electrophoretic mobility shift assays
To extract protein from normal mucosa and tumour material, 0.5 cm3 of tissue was suspended in three volumes of lysis buffer (19) then subjected to three cycles of snap freeze/thaw and homogenization with five strokes of an eppendorf pestle (Eppendorf, Hamburg, Germany). Debris was removed (1000 r.p.m., 2 min) then cytoplasmic and nuclear fractions were prepared from supernatant as described previously (19). Western blot analysis was performed as described previously (19) using the following antibodies: sheep polyclonal anti-I{kappa}B{alpha} [gift from Prof. R.Hay (Dundee)], sheep polyclonal anti-Cu/ZnSOD (The Binding Site Birmingham, UK), rabbit polyclonal anti-p65 (Santa Cruz, USA), goat polyclonal anti-procaspase-3 (uncleaved) (R&D Systems, Abingdon, UK), mouse monoclonal actin Merck Biosciences Ltd, (Nottingham, UK), rabbit antiphosphorylated S32 I{kappa}B{alpha} (Cell Signalling Technologies, Danvers, USA) and mouse anti-pk-tag [a kind gift from R.Hay (Dundee)]. Densitometry was performed on scanned western blots using Bio-Rad QuantityOne software. The results presented are the mean p65 band intensity, relative to control (actin) band intensity, for three western blots (from three separate mice) per time point (±SE). Electrophoretic mobility shift assays (EMSAs) were carried out as described previously (19).

Transfections
The I{kappa}B{alpha}-WT and I{kappa}B{alpha}-super-repressor (SR) plasmids were a gift from R.Hay (Dundee) and consist of either WT or SR (mutated at residues S32/36) I{kappa}B{alpha} cloned into pcDNA3 with a C-terminal pk-tag (19). Cells were transiently transfected using Lipofectamine [as described by manufacturers (Gibco BRL, Paisley, UK)], grown for 24 h in low serum (0.5% fetal calf serum) medium then treated with aspirin (0–5 mM) continuously for a further 24 h. Immunocytochemistry was performed on transfected cells as described below and western blot analysis on cytoplasmic extracts as described above.

Aspirin administration to mice bearing HT-29 xenograft tumours
Xenografts were established by s.c. implantation of either a 0.1 cm3 piece of third to sixth generation source HT-29 tumours (100 mg/kg aspirin experiments) or 107 HT-29 cells in serum-free media (40 versus 400 mg/kg experiment), on each flank of female Nu/Nu mice. All animal experiments were carried out according to UK Co-ordinating Committee on Cancer Research guidelines. Aspirin was administered by i.p. injection (100 mg/kg experiments) or by oral gavage (40 versus 400 mg/kg experiment) when tumours reached 50–100 mm3 (3–4 weeks later). A minimum of three mice were analysed per time point. Tumours and plasma were collected at necropsy at the time points specified.

Aspirin administration to APCMin+/– mice
C57BL/6 mice heterozygous for APCMin, aged until they developed obvious signs of intestinal neoplasia (usually bleeding from the anus or anaemia scored through whitening of the feet), were given a single i.p. injection of aspirin (100 mg/kg). The entire intestine was removed at necropsy, flushed with phosphate-buffered saline (PBS) then mounted en face. Large adenomas and normal appearing mucosa were removed macroscopically, then intestines fixed in methacarn (4:2:1 methanol:chloroform:glacial acetic acid), wound into a ‘gut roll’ and paraffin embedded for histological analysis and immunohistochemistry.

Caspase assays
Caspase 3/7 activity was measured in cytoplasmic extracts from tumour/normal mucosa using the Apo-ONE® Homogeneous Caspase-3/7 Assay kit (Promega, Southampton, UK). Protein content was quantified by performing Bradford assays (in triplicate) then 25 mg of protein, made up to a volume of 50 ml with lysis buffer, mixed with 50 ml assay buffer containing caspase 3/7 substrate in a 96-well plate as per manufacturers' instructions. Samples were incubated at room temperature for 48 h then caspase activity was determined by fluorometric analysis as per manufacturers' instructions. Results presented are the mean of three replicas of samples from three independent mice per time point (±SE).

High performance liquid chromatography analysis of plasma and tumour aspirin/salicylate levels
Plasma and tumour aspirin and salicylic acid concentrations were measured using a validated high performance liquid chromatography (HPLC) assay with UV detection. For plasma, the sample was precipitated with a 3-fold excess of acetonitrile and the supernatant analysed by RP-HPLC using a Polymer Labs PLRP-S-100A column with a water/acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA) and UV detection at 236 nm. For tumour, the sample was extracted by homogenization with a 3-fold excess of 0.1% phosphoric acid in acetonitrile. The extract was dried and resuspended in water prior to analysis by RP-HPLC using a Phenomenex Gemini C18-110A column with a water/acetonitrile gradient containing 0.1% TFA and UV detection at 236 nm. The linear range validated for both compounds is 0.35–50 µg/ml in plasma and 0.25–25 µg/ml in tumour tissue. Inter- and intra-assay variability were <15%. Validation details are available in an internal report, No. EOU002.

Immunocytochemistry and histological analysis
Paraffin-embedded tissues were sectioned at 10 mm. Fluorescent immunohistochemistry was carried out as described previously (19), with some modifications for paraffin-embedded sections. Briefly, sections were deparaffinized, boiled three times in antigen unmasking solution (Vector Laboratories), then washed 5x in PBS prior to commencing the immunocytochemistry protocol. Antibodies used were a rabbit antiactive caspase 3 (R&D Systems), a rabbit polyclonal anti-p65 (Santa Cruz) and a fluorescein isothiocyanate-labelled antirabbit secondary antibody (Jackson laboratories, Maine, USA). Stained sections were mounted in Vectasheild (Vector Laboratories, Peterborough, UK) containing 20 mg/ml 4'6-diamidino-2-phenylindole·2HCl. Fluorescent microscopy and image capture were performed as described (16). Analysis of the intensity of nuclear p65 in aspirin-treated adenomas and normal mucosa is described in Figure 4.

To determine levels of apoptosis, gut rolls were stained with haematoxylin and eosin and then the number of apoptotic bodies was scored using a Highly Optimized Microscope Environment microscope. Apoptosis in normal crypts were scored per 25 half crypts. For each adenoma, the number of apoptotic bodies per 500 cells was scored and for each mouse at least five adenomas were scored producing an average per mouse. At least three mice were used for each time point.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Aspirin activates the NF-{kappa}B pathway to induce apoptosis in HT-29 cells in vitro
To examine aspirin effects on NF-{kappa}B signalling and apoptosis in colorectal neoplasia in vivo, we initially used the HT-29 xenograft model of human colorectal cancer. Firstly, we wished to confirm that aspirin activates the NF-{kappa}B signalling pathway in the HT-29 colorectal cancer cell line and that this is causally involved in the apoptotic effects of the agent. Using western blot analysis, we observed a dose-dependent reduction in the levels of cytoplasmic I{kappa}B{alpha} in HT-29 cells in response to aspirin (0–10 mM) (Figure 1A). This reduction in cytoplasmic I{kappa}B{alpha} was paralleled by an increase in nuclear NF-{kappa}B complexes, as demonstrated by EMSAs (Figure 1B), and by the induction of apoptosis, as determined by cleavage of procaspase-3 (Figure 1C) and annexinV apoptosis assays (data not shown (25)). To determine whether activation of the NF-{kappa}B pathway was required for these apoptotic effects, we utilized SR I{kappa}B that we have shown previously to be resistant to aspirin-induced degradation and to inhibit aspirin-induced stimulation of the NF-{kappa}B pathway and apoptosis of SW480, HRT18 and CT26 colorectal cancer cells (16,19) We found that aspirin mediated a significant increase in apoptosis in HT-29 cultures expressing WT I{kappa}B{alpha}, as determined by active caspase-3 immunocytochemistry (Figure 1D and E). However, this response was abrogated in cultures expressing SR-I{kappa}B{alpha} (Figure 1D and E). Western blot analysis, using an antibody directed against the C-terminal pk-tag of the recombinant WT and mutant protein, confirmed that aspirin induced the degradation of WT I{kappa}B{alpha} in HT-29 cells, but had no effect on levels of the SR mutant (Figure 1F). These data confirm that stimulation of the NF-{kappa}B pathway is required for aspirin-induced apoptosis of HT-29 cells.


Figure 1
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  		Fig. 1. Aspirin effects on the NF-{kappa}B pathway and caspase-3 in HT-29 colorectal cancer cell lines. (A–C): HT-29 colorectal cancer cells were treated in vitro with aspirin (0–10 mM). (A) Western blot analysis showing cytoplasmic levels of I{kappa}B{alpha}. (B) EMSA demonstrating increased nuclear NF-{kappa}B DNA-binding complexes in response to aspirin (5 mM). The upper band consists of p50/p65 heterodimers and the lower band is non-specific, as previously determined by supershift analysis and competition with a non-labelled NF-{kappa}B probe, respectively (16,19). (C) Western blot analysis demonstrating a decrease in levels of procaspase-3 in response to aspirin, suggestive of cleavage and the induction of apoptosis. D–F: HT-29 cells were transiently transfected with pCDNA3.1 expressing WT or SR, pk-tagged, I{kappa}B{alpha} prior to aspirin [0 (–) or 5 mM, 16 h (+)] stimulation. (D) Immunomicrograph demonstrating active caspase-3 (bright cells) activity in transfected cultures. Nuclei are stained with DAPI. (E) The number of cells showing active caspase-3 (from D above) was quantified in at least 200 cells. Data are the mean of three independent experiments ±SE. The increase in apoptosis in response to aspirin observed upon expression of WT protein is blocked by expression of I{kappa}B{alpha}-SR. (F) Anti-pk-tag western blot analysis performed on cytoplasmic extracts. Aspirin induces degradation of WT I{kappa}B{alpha} but has no effect on levels of SR mutant protein.

 
Aspirin effects on the NF-{kappa}B signalling pathway and apoptosis in HT-29 xenograft tumours
Next, we determined whether aspirin stimulated the NF-{kappa}B pathway in association with apoptosis in vivo, in the context of a whole tumour environment. In two independent protocols, athymic nude mice (3 mice per time point per protocol), bearing established bilateral flank HT-29 tumour xenografts, were administered a single i.p. injection of aspirin (100 mg/kg). Animals were killed and tissues harvested at either 0 and 6 h (protocol 1) or 0, 6 and 24 h (protocol 2). Western blot analysis, performed on cytoplasmic protein extracts from tumour material, demonstrated a marked reduction in cytoplasmic levels of I{kappa}B{alpha} 6 h after aspirin treatment (Figure 2A and B). This reduction in I{kappa}B{alpha} corresponded with a reduction in cytoplasmic p65 and an increase in nuclear p65, indicating activation of the NF-{kappa}B pathway (Figure 2A and B). Furthermore, in both experiments, aspirin-induced activation of the NF-{kappa}B pathway at 6 h was paralleled by a decrease in levels of procaspase-3, suggestive of cleavage of the protein and caspase activation (Figure 2A and B). Densitometric analysis of western blots confirmed a consistent increase in nuclear p65 band intensity (relative to actin controls) 6 h post-aspirin treatment and caspase 3/7 activity assays (Promega Apo-ONE® Homogeneous Caspase-3/7 Assay), performed on tumour protein extracts, confirmed that this nuclear translocation of p65 preceded a significant increase in caspase activity at 24 h posttreatment, indicative of the induction of apoptosis (Figure 2C and D).


Figure 2
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  		Fig. 2. Aspirin effects on the NF-{kappa}B pathway and apoptosis in human adenocarcinoma cells in the HT-29 xenograft model of colorectal cancer. In two independent protocols, Nu/Nu mice bearing established HT-29 tumour xenografts were given a single i.p. injection of aspirin (100 mg/kg). Tissues were harvested at either (A) 0 and 6 h (protocol 1) or (B) 0, 6 and 24 h (protocol 2). Western blot analysis shows cytoplasmic levels of I{kappa}B{alpha} and p65, nuclear levels of p65 and cellular levels of caspase-3. Cu/Zn SOD and actin were used as controls for protein loading. Three animals were analysed per time point per protocol. The data shown are representative. (C) Densitometry was performed on nuclear p65/actin western blots using Bio-Rad QuantityOne software. The data presented are the mean p65 intensity relative to actin intensity (±SE) of three western blots (from three mice) per time point. (D) Caspase 3/7 activity was determined in cytoplasmic extracts from xenografted tumours using a fluorometric assay (Apo-ONE® Homogeneous Caspase-3/7 Assay, Promega). Data presented are the mean of three replicas of samples from three independent mice per time point (±SE). FU = fluorescent units.

 
Time and dose dependency of aspirin effects on the NF-{kappa}B signalling pathway and apoptosis in HT-29 xenografted tumours
We wished to further explore aspirin effects on the NF-{kappa}B pathway in vivo, and in particular, determine whether the dose of aspirin administered affected this response. Hence, athymic nude mice bearing established bilateral tumour xenografts were administered with a single dose of either 40 or 400 mg/kg aspirin by oral gavage. Tumours and plasma were extracted 0–24 h later, at time points specified in Figure 3.


Figure 3
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  		Fig. 3. Time and dose dependency of aspirin effects on the NF-{kappa}B pathway and caspase-3 cleavage/activity in HT-29 xenograft tumours. (A, B): HPLC analysis of (A) plasma and (B) tumour salicylate concentrations 0–18 h after a single oral administration of 40 or 400 mg/kg aspirin to Nu/Nu mice bearing established tumours. (C, D) Western blot analysis demonstrating cytoplasmic levels of phosphorylated (I{kappa}Bp) and total cytoplasmic I{kappa}B{alpha}, nuclear p65 and caspase-3 (casp-3) in corresponding tumours. Actin is used as a control for protein loading. (E, F) Densitometry was performed on nuclear p65/actin western blots using Bio-Rad QuantityOne software. Three mice were analysed per dose per time point. The data presented are the mean relative (to actin) p65 intensity (±SE). (G, H) Caspase 3/7 activity was determined in cytoplasmic extracts from xenografted tumours as for Figure 2. Results presented are the mean of three replicas of samples from three independent mice per time point (±SE).

 
Firstly, we used HPLC analysis to examine tumour and plasma aspirin/salicylate levels after treatment (25). We detected >0.5 mM of salicylate, the active form of aspirin, in both plasma and tumour only a few minutes after administration (Figure 3A and B), indicating that the agent is rapidly absorbed and deacetylated. Peak plasma salicylate levels were higher than those achieved in the tumour and, as expected, plasma salicylate concentrations were dependent upon the dose of aspirin administered (Figure 3A). However, peak tumour salicylate concentrations were similar for both aspirin doses, suggesting saturable uptake into the tumour (Figure 3B). ‘Exposure’ to salicylate, as represented by the area under the concentration time curve, was higher in the 400 mg/kg dosed animals compared with the 40 mg/kg animals for both the plasma (27.3 versus 3.9 mM h) and the tumour (9.9 versus 2.6 mM h). Taken together, these data demonstrate that appreciable salicylate levels can be achieved in tumours, even after low-dose aspirin treatment (40 mg/kg). A dose of 40 mg/kg equates to 120 mg/m2 in humans, equivalent to a clinical dose of 208 mg for average body surface area, or approximately one aspirin tablet taken for analgesic purposes.

In parallel with the above studies, tumours were analysed for effects on NF-{kappa}B signalling. Western blot analysis, using an I{kappa}B/S32 phospho-specific antibody, revealed a time-dependent increase in phosphorylated I{kappa}B{alpha} in xenografted tumours in response to aspirin (Figure 3C and D). For both aspirin doses, this increase in phosphorylated I{kappa}B was apparent 1 h post administration, peaked at 8 h, and corresponded with a time-dependent decrease in total cytoplasmic levels of the protein (conducive with proteosome-mediated degradation subsequent to phosphorylation). The aspirin effects on I{kappa}B also corresponded with a time-dependent increase in nuclear p65 indicating activation of the NF-{kappa}B pathway (Figure 3C–F). Furthermore, these aspirin effects on the NF-{kappa}B pathway preceded the induction of apoptosis, as evidenced by cleavage of procaspase-3 (data not shown) and an increase in caspase 3/7 activity 18 h after treatment with both 40 and 400 mg/kg aspirin (Figure 3G and H). These data provide further evidence that aspirin activates the NF-{kappa}B pathway in colorectal tumours in vivo in association with apoptosis. Our finding that the kinetics of aspirin-mediated activation of NF-{kappa}B and apoptosis are similar after 40 and 400 mg/kg indicates that the lower dose is sufficient to overcome the effect threshold.

Aspirin effects on the NF-{kappa}B signalling pathway and apoptosis in intestinal adenomas from APCMin+/– mice
To determine whether aspirin has a similar effect on the NF-{kappa}B pathway and apoptosis in precancerous intestinal lesions, we utilized the C57BL/6JMin+/– (APCMin+/–) mouse. This mouse carries a heterozygous inactivating mutation of the adenomatous polyposis coli (APC) gene and develops numerous spontaneous intestinal adenomas.

APCMin+/– mice with an established load of intestinal neoplasia were given a single i.p. injection of aspirin (100 mg/kg), animals were killed 0–48 h later and tissues harvested. Figure 4A demonstrates an increase in phosphorylated cytoplasmic I{kappa}B{alpha} in intestinal adenomas within 6 h of aspirin treatment. This increase in phosphorylated I{kappa}B{alpha} was again associated with a time-dependent decrease in total cytoplasmic levels of the protein (Figure 4A). To examine nuclear translocation of p65 in neoplastic adenoma cells in response to aspirin, we used anti-p65 fluorescent immunohistochemistry, performed on fixed sections of intestinal gut rolls. Adenomas were identified by low power microscopy, then cellular localization of p65 in neoplastic epithelial cells analysed at high magnification (Figure 4B). We found that prior to aspirin treatment, p65 was mainly present in the cytoplasm, with minimal nuclear staining. However, in response to aspirin, there was a marked increase in nuclear staining for p65. Quantitative measurements by computer-assisted cell image analysis (see figure 4B) demonstrated that levels of nuclear p65 were significantly greater 6 h after aspirin treatment (Figure 4C). These data provide further compelling evidence that aspirin activates the NF-{kappa}B pathway in intestinal adenomas. Western blot analysis and assays for caspase activity demonstrated that this aspirin-induced activation of the NF-{kappa}B pathway was paralleled by the induction of apoptosis, as determined by cleavage of procaspase-3 (Figure 4D) and increased caspase 3/7 activity (Figure 4E). Furthermore, an increase in apoptosis in response to aspirin was also demonstrated by analysis of haematoxylin and eosin-stained sections for morphological changes indicative of apoptosis (apoptotic bodies) (Figure 4F).


Figure 4
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  		Fig. 4. Aspirin effects on the NF-{kappa}B signalling pathway and apoptosis in intestinal adenomas from APCMin+/– mice. APCMin+/– mice with established intestinal neoplasia were given a single administration of aspirin (100 mg/kg) then tumours harvested macroscopically at necropsy 0–48 h later. (A) Western blot analysis showing cytoplasmic levels of phosphorylated (I{kappa}Bp) and total cytoplasmic I{kappa}B{alpha}. CuZn SOD represents a control for protein loading. (B, C) Anti-p65 immunocytochemistry performed on sections of gut rolls. DAPI staining depicts nuclei. Adenomas were identified at low power (20x) then cellular localization of p65 examined at high power. To analyse the intensity of nuclear p65 staining, 50 random nuclei from two separate adenomas per intestine were outlined in the DAPI channel using IPLab 3.6 software. Outlines were transferred to the FITC (p65) channel [to delineate the nuclei in this channel (shown in (B))] then the average pixel intensity within individual nuclei determined using IPLab software. Data presented show average p65 nuclear intensity [with background (FITC intensity in area with no cells) subtracted] for one representative experiment that is two adenomas from one intestine per time point. (D) Western blot analysis showing levels of procaspase-3 in adenomas in response to aspirin. (E) Caspase 3/7 activity was determined in cytoplasmic extracts from adenoma extracts using a commercially available kit (see Materials and methods). Results presented are the mean of three replicas of samples from three independent mice per time point (±SE). FU = fluorescent units. (F) Heamatoxylin and eosin-stained sections of gut rolls were analysed for morphological changes indicative of apoptosis in adenomas using the Highly Optimized Microscope Environment system. For each adenoma, the number of apoptotic bodies per 500 cells was scored and for each mouse at least five adenomas were scored producing an average per mouse. At least three mice were used for each time point.

 
Aspirin effects on the NF-{kappa}B signalling pathway and apoptosis in normal intestinal mucosa
Next we wished to determine whether the NF-{kappa}B and apoptotic response to aspirin were restricted to neoplastic tissue, or whether aspirin has a similar effect on normal intestinal mucosa. In parallel with tumour analysis (described above), aspirin effects on normal intestinal mucosa from APCMin+/– mice were examined. Using western blot analysis, we found that basal levels of I{kappa}B{alpha} were increased in intestinal adenomas from the APCMin+/– mouse (Figure 4A), compared with adjacent normal mucosa (Figure 5A). This difference in basal levels of I{kappa}B appeared to reflect the NF-{kappa}B response to aspirin. In normal small intestine, there was an increase in cytoplasmic I{kappa}B{alpha} 6 h after aspirin treatment, with levels returning to basal within 24 h (Figure 5A). This is compared with the reduction in I{kappa}B{alpha} observed in adenomatous tissue (Figure 4A). Furthermore, there was no change in the levels of nuclear p65 (as determined by quantitative immnuhistochemistry as previously) (Figure 5B and C), an increase in procaspase-3 (Figure 5D) and no significant change in caspase 3/7 activity (Figure 5E) or the number of apoptotic cells (Figure 5F) in normal intestinal mucosa in response to aspirin. These data demonstrate that the NF-{kappa}B-related apoptotic response to aspirin is enhanced in neoplastic epithelial cells.


Figure 5
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  		Fig. 5. Aspirin effects on the NF-{kappa}B signalling pathway and apoptosis in normal mucosa from APCMin+/– mice. In parallel with tumour analysis described above, aspirin effects on normal mucosa from APCMin+/– were analysed. (A) Western blot analysis showing cytoplasmic levels of I{kappa}B{alpha}. CuZn SOD is used as a control for protein loading. (B) Anti-p65 immunocytochemistry showing the cellular localization of p65 before and 6 h after aspirin treatment. DAPI is used to identify nuclei. (C) The nuclear intensity of p65 before and after aspirin treatment was determined as described in Figure 4. (D). Western blot analysis showing levels of procaspase-3 in response to aspirin. Actin demonstrates equal protein loading. (E) Caspase 3/7 activity [presented in fluorescent units (FU)] in normal mucosa in response to aspirin. Results presented are the mean of three replicas of samples from three independent mice per time point (±SE). (F) The percentage of apoptotic cells in normal intestine was scored as in Figure 4.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We previously reported that activation of the NF-{kappa}B pathway by aspirin is critical for the proapoptotic activity of the agent against colorectal cancer cells in vitro. The present study was undertaken to determine the effect that aspirin has on the NF-{kappa}B pathway and apoptosis in vivo, in the context of a whole tumour environment. The work presented provides two important new insights into the antitumour activity of the agent. Firstly, we show that aspirin activates NF-{kappa}B signalling associated with apoptosis in vivo, at doses relevant to human treatment protocols. Secondly, that NF-{kappa}B and apoptosis are induced by aspirin in benign neoplasms as well as in malignant cells.

We demonstrated that aspirin mediates a time-dependent increase in phosphorylated I{kappa}B in HT-29 xenograft tumours and in adenomas from APCMin+/– mice. In both models of intestinal carcinogenesis, this increase in phosphorylated I{kappa}B was paralleled by a decrease in total cytoplasmic levels of the protein, indicative of proteosome-mediated degradation consequent upon phosphorylation. Furthermore, aspirin-induced degradation of I{kappa}B corresponded with a decrease in cytoplasmic p65 and a time-dependent increase in nuclear levels of the protein. Therefore, we conclude that in the cellular context of premalignant and malignant intestinal neoplasia, aspirin activates the NF-{kappa}B pathway.

We demonstrate that aspirin-induced degradation of I{kappa}B/nuclear translocation of p65/NF-{kappa}B is required for apoptosis of HT-29 cells and precedes the induction of apoptosis in HT-29 tumour xenografts and in intestinal adenomas from APCMin+/– mice, as determined by cleavage of procaspase-3, activation of caspases and an increase in cells showing morphological signs of apoptosis (apoptotic bodies). These data are consistent with previous in vitro data from this laboratory showing that nuclear translocation of p65/NF-{kappa}B is required for aspirin-induced apoptosis (16,19), but, would appear to go against the conventional view that nuclear NF-{kappa}B activity has an antiapoptotic role in the colon (26–29). However, we have shown previously that aspirin-mediated nuclear translocation of p65/NF-{kappa}B causes a decrease in basal levels of NF-{kappa}B-driven transcription in colorectal cancer cells (16). Furthermore, we demonstrated that this decrease in NF-{kappa}B activity was causally involved in the apoptotic response to aspirin. Campbell et al. (30) have also demonstrated that stimulation of the NF-{kappa}B pathway by specific agents causes nuclear translocation of p65/NF-{kappa}B complexes that repress transcription of NF-{kappa}B-regulated antiapoptotic genes. Therefore, we propose that in vivo, the apoptotic response to aspirin is also caused by nuclear translocation of repressive p65/NF-{kappa}B complexes and decreased expression of NF-{kappa}B-driven antiapoptotic genes.

Interestingly, Cho et al. (21) recently demonstrated that the NSAID diclofenac downregulates beta-catenin/T cell factor-driven transcription in colorectal cancer cells through activation of the NF-{kappa}B pathway. Since downregulation of beta-catenin signalling is associated with the induction of apoptosis in colorectal cancer cells (31,32) and with the chemopreventative effects of NSAIDs (33–36), it may be that aspirin-induced nuclear translocation of p65/NF-{kappa}B causes apoptosis of neoplastic intestinal epithelial cells through inhibition of beta-catenin activity. We are currently exploring this possibility.

These data showing activation of the NF-{kappa}B pathway in intestinal neoplasia are in contrast to previous in vivo studies demonstrating that aspirin inhibits NF-{kappa}B activity in the aorta, kidney and heart in animal models of inflammation/organ damage (37–39). However, we show here that aspirin effects on the NF-{kappa}B pathway in normal intestinal mucosa differ from those in intestinal tumours. Therefore, we propose that modulation of the NF-{kappa}B pathway by aspirin is dependent upon the cell type and tissue environment. Indeed, previous data from this laboratory indicate cell type specificity for aspirin-mediated activation of NF-{kappa}B (25).

Despite the identification of a number of molecular targets for NSAIDs in vitro (10,40–43), data on the mechanisms underlying their antitumour activity in vivo are limited (35,44–47). The data presented here provide further understanding of the mechanisms by which aspirin mediates apoptosis of colorectal cancer cells in vivo, and will inform the design of safer chemopreventative agents.


   Acknowledgments
 
We gratefully acknowledge the gift from Ron T.Hay (University of Dundee) of the I{kappa}B{alpha} and PK-tag antibodies and the WT and I{kappa}B SR plasmids. P.Perry wrote the scripts for image capture. This work was supported by grants from the Scottish Executive Chief Scientist's Office (K/MRS/50/C2719 and CZB/4/41), the Association of International Cancer Research (02-330) and the Cancer Research UK (CRUK) (C20658/A6656). L.A.S. is a Caledonian Research Fellow. M.G.D. is supported by CRUK Programme Grant funding (C348/A3758).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 16, 2006; revised October 6, 2006; accepted November 3, 2006.


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