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@hgu.mrc.ac.uk
The aim of this study was to determine
whether aspirin mediates an anti-tumor effect by modulation of NF-
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 I B and nuclear translocation of NF- B
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
I B levels that correlated with the reduction in the number of viable cells.
Levels of control protein (Cu/ZnSOD) were unaffected by aspirin. Mutation of I B at the critical S32/36 phosphorylation sites (I BS32/36-tag) and preincubation of cells with the MG132 proteasome
inhibitor blocked aspirin-induced reduction in I B levels. These results indicate that aspirin mediates phosphorylation and
subsequent proteosome-mediated degradation of I B and suggest that this degradation is associated with aspirin-induced
Electrophoretic mobility shift assays (EMSAs)
revealed that aspirin-induced I B 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.
3. Aspirin-induced I B 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 I B 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
I BS32/36-tag construct. Using Western blot analysis with
the anti-tag antibody, two HRT18 (I BS32/36h1, h28) and two CT26 (I BS32/36ct3, ct4) clones were identified that expressed high
levels of mutant protein. Expression of I B 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 I BS32/36 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 I B 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 I B and subsequent nuclear translocation of NF- B complexes.
5. Cell specificity of aspirin-induced
I B 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 I B degradation whereas 10 mM aspirin induced almost complete degradation.
In contrast, 10 mM aspirin had no effect on I B 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 I B degradation and apoptosis may be cell type specific.
6. Aspirin induces I B 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. I B 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 I B response as observed in cell line experiments.
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 I B 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 I B 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 I B 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 I B 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
I B (I B 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 I B -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.