Aspirin and other nonsteroidal anti-inflammatory
drugs inhibit prostaglandin synthesis and tumor growth in many experimental systems, but it is unclear which
of these tumor models are relevant to humans. We have reported reduced risk of fatal colon cancer among persons
who used aspirin in a large prospective study. This analysis examines other fatal cancers in relation
to aspirin among 635,031 adults in that study who provided information in 1982 on the frequency and duration of
their aspirin use and did not report cancer. Death rates were measured through 1988. Death rates decreased
with more frequent aspirin use for cancers of the esophagus, stomach, colon, and rectum but not generally for other
cancers. For each digestive tract cancer, death rates were approximately 40% lower among persons who
used aspirin 16 times/month or more for at least 1 year compared to those who used no aspirin. The trend of decreasing
risk with more frequent aspirin use was strongest among persons who had used aspirin for 10 years or
more; it remained statistically significant, except for esophageal cancer, in multivariate analyses that adjusted
for other known risk factors. Biases such as early detection or aspirin avoidance among cases do not
appear to explain the results. Our data suggest that regular, prolonged use of aspirin may reduce the risk of fatal
cancer of the esophagus, stomach, colon, and rectum. Future epidemiological and basic research should
examine all digestive tract cancers in considering the chemopreventive or therapeutic potential of nonsteroidal
antiinflammatory drugs.
Aspirin Use and the Risk for Colorectal
Cancer and Adenoma in Male Health Professionals
Edward Giovannucci; Eric B. Rimm; Meir J. Stampfer;
Graham A. Colditz; Alberto Ascherio; and Walter C. Willett
15 August 1994 | Volume 121 Issue 4 | Pages 241-246
Objective: To determine whether regular use of aspirin decreases
the risk for colorectal cancer.
Design: Prospective cohort study.
Setting: Male health professionals throughout the United States.
Patients: 47 900 male respondents to a mailed questionnaire
in 1986, who were 40 to 75 years of age.
Measurements: Questionnaires in 1986, 1988, and 1990 about use
of aspirin and other variables including occurrence of cancer.
Results: 251 new patients were diagnosed with colorectal cancer during the study period. Regular
users of aspirin ( 2 times per week) in 1986 had a lower risk for total colorectal cancer (relative risk
[RR] = 0.68; 95% CI, 0.52 to 0.92) and advanced (metastatic and fatal) colorectal cancer (RR = 0.51; CI, 0.32
to 0.84) after controlling for age; history of polyp; previous endoscopy; parental history of colorectal
cancer; smoking; body mass; physical activity; and intakes of red meat, vitamin E, and alcohol. The
incremental association was greater among men who reported regular use of aspirin consistently on subsequent
questionnaires. The total number of colorectal adenomas discovered among aspirin users was lower with or
without overt or occult fecal blood. Thus, earlier diagnosis and treatment of adenomas did not account
for the inverse association between aspirin and cancer.
Conclusions: These
results support laboratory, clinical, and other epidemiologic evidence that regular use of aspirin is associated
with a decreased risk for colorectal cancer.
Comment in:
· Science. 1995 Dec 22;270(5244):2017-9.
Inhibition of NF-kappa B by sodium salicylate and aspirin.
Kopp E, Ghosh S.
Department of Cell Biology, Yale
University School of Medicine, New
Haven, CT 06536.
The transcription factor
nuclear factor-kappa B (NF-kappa B) is critical for the inducible expression of multiple cellular and viral genes involved
in inflammation and infection including interleukin-1 (IL-1), IL-6, and adhesion molecules. The anti-inflammatory drugs sodium
salicylate and aspirin inhibited the activation of NF-kappa B, which further explains the mechanism of action of these drugs.
This inhibition prevented the degradation of the NF-kappa B inhibitor, I kappa B, and therefore NF-kappa B was retained in
the cytosol. Sodium salicylate and aspirin also inhibited NF-kappa B-dependent transcription from the Ig kappa enhancer and
the human immunodeficiency virus (HIV) long terminal repeat (LTR) in transfected T cells.
· Epidemiology. 1994 Mar;5(2):133-5.
Aspirin use and lung, colon, and breast cancer incidence in a prospective study.
Schreinemachers DM, Everson RB.
Health Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle
Park, NC 27711.
A large body of experimental
data and several recent epidemiologic studies indicate that aspirin use may decrease cancer risk. The experimental studies
found effects at many anatomic sites, whereas the epidemiologic studies saw the greatest effect on mortality from digestive
cancers. To provide further human data, we examined the association between aspirin use and cancer risk using data from the
National Health and Nutrition Examination Survey I (NHANES I) and the NHANES I Epidemiologic Follow-up Studies (NHEFS). Characterization
of aspirin use was based on questions in the baseline interview asking whether subjects used aspirin during the previous 30
days. Data were available from 12,668 subjects age 25-74, at time of initial examination for NHANES I, who were followed for
an average of 12.4 years. Among these subjects, 1,257 were diagnosed with cancer more than 2 years after their NHANES I examination.
Incidence of several cancers was lower among persons who reported aspirin use: the incidence rate ratios (and 95% confidence
intervals) for all sites combined were 0.83 (0.74-0.93), lung cancer 0.68 (0.49-0.94), breast cancer in women 0.70 (0.50-0.96),
and colorectal cancer in younger men 0.35 (0.17-0.73). These findings were not readily explained by potentially confounding
factors. The data suggest an association between aspirin consumption and decreased cancer incidence at several cancer sites.
Risk of aspirin-associated major upper-gastrointestinal bleeding with enteric-coated or buffered product.
Kelly JP, Kaufman DW, Jurgelon JM, Sheehan J, Koff RS, Shapiro S.
Slone Epidemiology Unit, School
of Public Health, Boston University
School of Medicine, Brookline, Massachusetts
02146, USA.
BACKGROUND: Aspirin
products are known to cause irritation and injury to the gastric mucosa. The belief that enteric-coated and buffered varieties
are less likely to occasion major upper-gastrointestinal bleeding (UGIB) than plain aspirin was tested in data from a multicentre
case-control study. METHODS: 550 incident cases of UGIB admitted to hospital with melaena or haematemesis and confirmed by
endoscopy, and 1202 controls identified from population census lists, were interviewed about use of aspirin and other non-steroidal
anti-inflammatory drugs (NSAIDs) during the 7 days before the onset of bleeding (cases) or interview (controls). Relative
risks of UGIB for each type of aspirin used regularly (at least every other day) were calculated overall, and according to
dose, by multiple logistic regression, with control for age, sex, marital status, date, education, cigarette smoking, alcohol
use, and use of NSAIDs. FINDINGS: The relative risks of UGIB for plain, enteric-coated, and buffered aspirin at average daily
doses of 325 mg or less were 2.6, 2.7, and 3.1, respectively. At doses greater than 325 mg, the relative risk was 5.8 for
plain and 7.0 for buffered aspirin; there were insufficient data to evaluate enteric-coated aspirin at this dose level. There
were no important differences in risk attributable to the three aspirin forms according to bleeding site (gastric vs duodenal),
or when users of NSAIDs were excluded. INTERPRETATION: Use of low doses of enteric-coated or buffered aspirin carries a three-fold increase in the risk of major UGIB. The
assumption that these formulations are less harmful than plain aspirin may be mistaken.
ASPIRIN INDUCES CELL DEATH OF COLON CANCER CELLS
Aspirin-induced activation of the NF- B signaling pathway: a novel mechanism for aspirin-mediated apoptosis in colon
cancer cells1
LESLEY A. STARK2,
FARHAT V. N. DIN, RALF M. ZWACKA and MALCOLM G. DUNLOP
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@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 I B 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
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
cell death.
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.
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 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.
