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Aspirin-induced activation of the NF-B signaling pathway: a novel mechanism for aspirin-mediated apoptosis in colon cancer cells¹

Colon Cancer Genetics Group, University of Edinburgh Department of Oncology, Division of Clinical and Molecular Medicine and MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland

²Correspondence: Colon Cancer Genetics Group, MRC Human Genetics Unit, Western General Hospital, Crewe Rd., Edinburgh EH4 2XU, U.K. E-mail: Lesley.Stark{at}hgu.mrc.ac.uk

SPECIFIC AIM

The aim of this study was to determine whether aspirin mediates an anti-tumor effect by modulation of NF-B signaling.

PRINCIPAL FINDINGS

1. Aspirin induces cell death by an active apoptotic process
A dose-dependent reduction in cell viability was observed in SW480 colorectal cancer cells treated with aspirin in the dose range 0–10 mM for 24 h or 0–2 mM aspirin for 48 h. These doses are comparable to salicylate levels we measured in serum (0.05–1.13 mM) from human subjects given a short analgesic dose (600 mg qid) of aspirin. The reduction in cell viability was accompanied by an increase in cell death due to apoptosis, as determined by quantitation of cells showing phosphatidylserine externalization and cell morphology. Treatment in the presence of cycloheximide showed that aspirin-induced (10 mM) cell by death required de novo protein synthesis, confirming that death occurred through an active process, not passive necrosis.

2. Aspirin induces apoptosis in association with degradation of IB and nuclear translocation of NF-B
Next, we examined the involvement of the NF-B signaling pathway in the apoptotic response of SW480 cells to aspirin. Experiments were performed in the absence of tumor necrosis factor (TNF) or other stimulating cytokines, as it is under these experimental conditions that aspirin induced apoptosis. We found that prolonged treatment with aspirin (0.5–2 mM for 48 h or 3–20 mM for 24 h) induced a dose-dependent reduction in cytoplasmic IB levels that correlated with the reduction in the number of viable cells. Levels of control protein (Cu/ZnSOD) were unaffected by aspirin. Mutation of IB at the critical S32/36 phosphorylation sites (IB^S32/36-tag) and preincubation of cells with the MG132 proteasome inhibitor blocked aspirin-induced reduction in IB levels. These results indicate that aspirin mediates phosphorylation and subsequent proteosome-mediated degradation of IB and suggest that this degradation is associated with aspirin-induced cell death.

Electrophoretic mobility shift assays (EMSAs) revealed that aspirin-induced IB degradation was accompanied by a dose-dependent specific increase in nuclear NF-B (p50/p65) DNA binding complexes (Fig. 1A , B ). The findings from EMSAs were further corroborated by immunocytochemistry. Before aspirin treatment, p65 was localized mainly in the cytoplasm, but after 24 h treatment with 10 mM aspirin, there was extensive nuclear staining for the protein (Fig. 1D ). These results establish that 24 h exposure to aspirin activates the NF-B pathway colorectal cancer cells.

View larger version (58K): [in this window] [in a new window]   Figure 1. Aspirin induces nuclear translocation of NF-B. A,B) Electrophoretic mobility shift assays using NF-B consensus oligonucleotide, performed on nuclear extracts from SW480 cells treated with A) 0-20 mM aspirin for 24 h or B) 0-1 mM aspirin for 48 h. C) Competition electrophoretic mobility shift assays indicated the identity and specificity of NK-B-induced complexes. The band induced by 10 mM aspirin was supershifted when antibodies to p50 and p65 were included in the assay. Excess (100-fold) unlabeled wild-type (wt) NF-B oligonucleotide competed with binding of p50/p65 heterodimers to the labeled probe but not excess (100-fold) mutated (mt) NF-B sequences. D) Micrographs (63x) showing immunofluorescent analysis of SW480 cells either untreated (I) or treated with 10 mM aspirin for 24 h (II), then immunocytochemically stained with p65 antibodies. The signal was detected using FITC-conjugated secondary antibody.

3. Aspirin-induced IB degradation and NF-B nuclear translocation precede cell death
To investigate the possibility that NF-B nuclear translocation was a consequence of cell death, we studied the kinetics of the aspirin effects on NF-B signaling and apoptosis. Aspirin (10 mM) treatment induced complete degradation of IB after 2–5 h. Similarly, an increase in nuclear NF-B DNA binding was observed 2 h after treatment and persisted for > 16 h. In comparison, aspirin-induced apoptosis, determined by externalization of phosphatidylserine, was not detected until 16 h after treatment. These results show that apoptosis occurred after NF-B nuclear translocation, suggesting the possibility of causal relationship between aspirin-induced NF-B activation and subsequent cell death.

4. Inhibition of NF-B nuclear translocation inhibits aspirin-induced apoptosis
To definitively prove the relationship between NF-B nuclear translocation and aspirin-mediated growth inhibition, we generated stable transfectants of HRT18 and CT26 colon cancer cells that constitutively express the IB^S32/36-tag construct. Using Western blot analysis with the anti-tag antibody, two HRT18 (IB^S32/36h1, h28) and two CT26 (IB^S32/36ct3, ct4) clones were identified that expressed high levels of mutant protein. Expression of IB^S32/36 resulted in substantial inhibition of aspirin-induced nuclear translocation of NF-B in all clones compared with respective parental cells (Fig. 2A ).If nuclear translocation of NF-B were contributing to apoptosis, then inhibition should protect against aspirin-induced cell death. Indeed, the number of IB^S32/36 expressing viable cells actually increased in the presence of 1 mM aspirin compared with a 32.4% and 55.8% reduction in viable cell counts in parental HRT18 and CT26 cells, respectively (Fig 2B ). Similarly, 5 mM aspirin had significantly (P<0.05) less effect on the viability of mutant IB expressing clones than on the viability of parental lines (Fig. 2B ). These data indicate that aspirin-induced apoptosis of colorectal cancer cells requires phosphorylation and degradation of IB and subsequent nuclear translocation of NF-B complexes.

View larger version (35K): [in this window] [in a new window]   Figure 2. IBS32/36 inhibits aspirin mediated nuclear translocation of NF-B and apoptosis. HRT18 (h) and CT26 (ct) colorectal cancer cells were transfected with the IBs32/36-tag plasmid and stable clones generated. Anti-tag immunoblots identified 2 clones from each parental cell line that showed high levels of expression of the mutant protein (IBS32/36 h1, h28, ct3, and ct4) (not shown). A) EMSAs performed on nuclear extracts from HRT18 and CT26 parental cells and IBS32/36 h1, h28, ct3, and ct4 clones treated with aspirin for 24 h. Aspirin-induced p50/p65 complexes are not present in mutant IB-expressing cells. B) Hemocytometric counts of viable HRT18 (•) and CT26 () parental cells and IBs32/36 h1 (), h28 (), ct3 (), and ct4 () clones after 24 h of aspirin treatment. Data presented represent the mean of 4 independent experiments (± SE). At 1 and 5 mM aspirin, the difference between the number of viable treated and nontreated cells was significantly less in the clones expressing the mutant protein (P<0.05) compared with the parental cells.

5. Cell specificity of aspirin-induced IB degradation and apoptosis
Epidemiological studies indicate that nonsteroidal anti-inflammatory drug (NSAID) -mediated protection is relatively specific to colorectal tumors. Therefore, we wanted to determine whether cells that are not derived from colorectal tumors also responded to aspirin with similar effects on the NF-B pathway and cell viability. In all 4 (SW480, HRT-18, HCT116, CT26) colorectal cancer cell lines tested, 5 mM aspirin induced substantial IB degradation whereas 10 mM aspirin induced almost complete degradation. In contrast, 10 mM aspirin had no effect on IB in embryonic kidney (293) or in lung adenocarcinoma (A549) cells. Substantial growth inhibitory effects were observed in all colorectal cancer cell lines treated with 5 or 10 mM aspirin. Numbers of viable cells decreased by 2- to 6.7-fold after aspirin (5 mM) treatment. On the contrary, numbers of viable 293 cells increased in the presence of 5 mM aspirin while A549 cells only showed a 1.2-fold decrease in viability after treatment. These results suggest that aspirin-mediated IB degradation and apoptosis may be cell type specific.

6. Aspirin induces IB degradation in normal colonic mucosa and tumors from rectal cancer patients
To establish the potential clinical significance of our results, we investigated aspirin effects in clinical material. We treated biopsy samples of normal mucosa and tumor material from patients undergoing surgical resection for rectal cancer. IB degradation was observed in both the tumor and, to a lesser extent, in the normal mucosa, after 5 h treatment ex vivo with 10 and 20 mM aspirin. No change was observed in levels of control (Cu/ZnSOD) protein. These data confirm that clinical tumor biopsy material show the same IB response as observed in cell line experiments.

CONCLUSIONS

There is compelling evidence that NSAIDs have a protective effect against colorectal cancer. However, their detrimental side effects limit their potential use as chemopreventative agents. Therefore, there is a pressing need to understand the mechanisms by which NSAIDs exert their chemopreventative effects in order to allow development of safer alternatives.

We show here for the first time that aspirin mediates a reduction in cytoplasmic IB levels in colorectal cancer cells that is time and concentration dependent and due to phosphorylation and proteasome-mediated degradation of the protein. Using EMSAs and immunocytochemistry, we demonstrate that IB degradation results in nuclear translocation of p50/p65 NF-B complexes, confirming that aspirin stimulates the NF-B pathway. Although these data would appear to question studies showing that NSAIDs inhibit activation of NF-B through specific modulation of the IB kinase, previous studies examined only the very short-term effects of NSAIDs on activation of NF-B mediated by cytokines. We investigated the effects of aspirin alone on NF-B signaling, which is highly relevant to the anti-tumor activity of the agent.

The evidence of a link between NF-B translocation and apoptosis due to aspirin treatment, was initially implied by the correlation between IB degradation and reduced cell viability. Time course experiments indicated that the NF-B response preceded cell death, raising the possibility of a causal relationship. This was confirmed in cells we engineered to continuously express a dominant negative mutant IB (IB^S32/36). These cells showed inhibition of both aspirin-induced NF-B nuclear translocation and apoptosis vs. their parental counterparts. We also considered whether the lack of apoptotic response to aspirin in the mutant IB-expressing clones might be due to their slower rate of growth compared with parental cells. However, this is unlikely since these cells grew at a similar rate to other colorectal cancer cell lines (SW480) in which aspirin induced substantial apoptosis. Thus, we conclude that it is NF-B nuclear translocation that mediates aspirin-induced apoptosis of colorectal cancer cells.

These novel findings of a prolonged effect of aspirin on NF-B signaling provide new insight into the mechanism of action of aspirin against colorectal cancer and will inform chemoprevention strategies.

View larger version (27K): [in this window] [in a new window]   Figure 3. Summary diagram. Continuous treatment of colorectal cancer cells with aspirin (0.5–10 mM) results in degradation of IB, nuclear translocation of NF-B complexes, and apoptosis. IB degradation is observed 5 h after aspirin (10 mM) treatment and precedes apoptosis, which is evident 16 h after treatment. Expression of superrepressor IB, mutated at residues critical for phosphorylation and degradation, inhibits aspirin-induced NF-B nuclear translocation and apoptosis. These data indicate that activation of NF-B is required for the anti-tumor activity of aspirin.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0529fje ; to cite this article, use FASEB J. (March 20, 2001) 10.1096/fj.00-0529fje

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