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Mechanisms of the antitumoural effects of aspirin
in the gastrointestinal tract
q
Annalisa Bruno, PharmD, PhD Student
a,c, Melania Dovizio, PhD, Post-Doc
b,c,
Stefania Tacconelli, PhD, Researcher
b,c,
Paola Patrignani, PhD, Professor of Pharmacology
b,c,*aDepartment of Medicine and Aging,“G. d’Annunzio” University, School of Medicine, Italy bDepartment of Neuroscience and Imaging,“G. d’Annunzio” University, School of Medicine, Italy cCenter of Excellence on Aging (CeSI), Via dei Vestini, 31, 66100 Chieti, Italy
Keywords: Aspirin Platelets Cyclooxygenase Tumourigenesis
a b s t r a c t
A recent clinical study showed that after five years of taking aspirin, at doses of at least 75 mg once daily, death rates were 54% less for gastrointestinal (GI) cancers. Thefinding of aspirin benefit at low-doses used for cardioprevention, locates the antiplatelet effect of aspirin at the centre of its antitumour efficacy. At low-doses, aspirin acts mainly by an irreversible inactivation of platelet cyclooxygenase (COX)-1 activity. We propose that platelet activation is involved in the early stages of colorectal carcinogen-esis in man through the induction of a COX-2-mediated paracrine signalling between stromal cells and epithelial cells within adenomas. In this scenario, aspirin causes a chemopreventive effect by countering platelet activation which seems to play a role in early event in GI tumourigenesis.
Ó 2012 Elsevier Ltd. All rights reserved.
Introduction
A large body of clinical evidence suggests that long-term use of aspirin (acetyl salicylic acid) reduces the incidence and mortality due to cancer in the gastrointestinal (GI) tract, in particular oesophageal and colorectal (CRC)[1]. Indirect comparisons of the chemopreventive effect against CRC by different aspirin doses showed that a maximal benefit is detected at 75 mg daily[2], i.e. a low-dose of the drug
q NSAIDs and Aspirin; Benefits and Harms for the Gut.
* Corresponding author. Department of Neuroscience and Imaging, “G. d’Annunzio” University, School of Medicine, Italy. Tel.: þ39 0871 541473; fax: þ39 0871 3556718.
E-mail addresses:ppatrignani@unich.it,p.patrignani@alice.it(P. Patrignani).
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which acts by inhibiting almost exclusively platelet cyclooxygenase (COX)-1 activity and thus affecting thromboxane (TX)A2-dependent platelet activation[3]. Similarly, aspirin is causing a restraining action
on atherothrombosis without an apparent dose-dependent effect with a maximal benefit detected at low-doses (75–150 mg daily). In contrast, at higher doses it was shown a trend towards a reduced efficacy[4].
Altogether thesefindings pave the way for novel views on the mechanism of CRC development and suggest that it may share some physiopathological features with atherothrombosis. We propose that arterial occlusion and adenoma formation may represent different phenotypes of the same abnormal repair process mediated by platelet activation at distinct sites of injury. In this setting, endothelial products, such as COX-2-dependent prostacyclin (PGI2) may play a protective role by restraining
platelet function [5]and offsetting the release of growth and angiogenesis factors from platelet
a
-granules and from other cells[6].In this review, we will utilize the knowledge of clinical pharmacology of aspirin to support our hypothesis that the antiplatelet effect of aspirin plays a central role in the long-term benefit of aspirin as antitumour agent in the alimentary tract, in particular in the colorectum.
Clinical results on the chemopreventive effect of aspirin against GI tumourigenesis
Information on the chemopreventive effect of aspirin against CRC was obtainedfirst in epidemio-logical studies. In fact, both case-control and cohort studies have found that regular, continued use of aspirin is associated with approximately 50% reduction in the incidence and mortality of CRC, both in men and women[2,7]. However, this benefit may not be evident until after at least a decade of regular aspirin consumption[8].
Several randomized clinical trials (RCTs) with different aspirin doses (81–600 mg daily) were performed in different clinical conditions, i.e. in healthy individual [9,10], in average/high-risk (i.e. patients with a history of colorectal adenomas)[11–15]or high-risk population [i.e. hereditary non-polyposis colorectal cancer (lynch syndrome) and the adenomatous non-polyposis coli gene (APC)-asso-ciated polyposis condition, i.e. familial adenomatous polyposis (FAP)] (Table 1)[16,17].
In the two RCTs performed in healthy individuals [The Physicians’s Health (PHS) and Women’s Health (WHS) Studies], aspirin (325 and 100 mg, respectively) did not significantly reduce the risk of CRC (Table 1)[9,10]. Aspirin failure found in these RCTs might be explained by some limitations in the design of the trials, i.e. inadequate treatment duration or follow-up (in PHS) and/or administration schedule (in WHS the drug was given every other day).
In contrast, in prospective, placebo-controlled, RCTs performed in average/high-risk population, aspirin at low–medium doses (81–325 mg daily) significantly reduces the risk of colorectal adenoma reoccurrence[11–13,15](Table 1).
A RCT was performed to explore the effect of aspirin at a medium–high dose (600 mg) on the risk of colorectal cancer, in carriers of hereditary colorectal cancer (CAPP2 trial) and it was found that drug administration was associated with reduced colorectal cancer incidence after 55.7 months (Table 1)
[17]. Despite these findings are very interesting it is unknown whether aspirin may have the same effect at lower doses.
Limited information is available on the chemopreventive effect of aspirin in FAP. In the study performed by Burn et al[16], an international, multicenter, randomized, placebo-controlled trial of aspirin (600 mg/day) and/or resistant starch (RS) (30 g/day) (CAPP1) performed in young FAP patients (from ten to 21 years of age), the daily administration of aspirin 600 mg from one to 12 years did not realize the primary end-point consisting in the reduction of polyp number in the rectum and sigmoid colon. In contrast, the drug treatment significantly reduced the size of polyps (secondary-end-point). These data are promising even if they are not conclusive due to several limitations, such as a lack of standardization of the extent of endoscopic examination and surveillance done by multiple endo-scopists at 12 different treatment centres[16,18].
Recently, Rothwell and colleagues[1,19]analysed individual patient data from cardiovascular (CV) RCTs, in which patients took low-doses of aspirin once daily, as recommended for the prevention against heart disease[3]and found that those taking aspirin had a lower incidence of colorectal and other solid cancers than controls. The chemopreventive effect of aspirin was evidenced after at least
T able 1 Randomized clinical trials which ha v e compar ed the effect of aspirin in the pr ev ention of color ectal adenoma or cancer v ersus compar at or . Author Treatment Patients Primary end-point Results (95% CI) Primary prevention Gann et al, 1993 (PHS) [9] Placebo or aspirin 325 mg every each day for fi ve years Male physicians aged between 40 and 84 years Incidence of total cancer RR: 1.15 (0.80 – 1.65) for colorectal cancer Cook et al, 2005 (WHS) [10] Placebo or aspirin 100 mg every other day for an average of 10.1 years Healthy women ag ed at least 45 years Con fi rmed newly diagnosed invasive cancer at any site RR: 1.01(0.94 – 1.08) for total cancer; RR: 0.97(0.77 – 1.24) for colorectal cancer Secondary prevention Baron et al, 2003 [11] Placebo, aspirin 81 mg or 325 mg daily for 2.8 years Patients with a recent history of histologically documented (removed) adenomas Proportion of patients in whom one or mo re colorectal adenomas were detected Any ade noma; RR: 0.81 (0.69 – 0.96) for ASA 81 mg, P ¼ 0.04; RR: 0.96 (0.81 – 1.13) for ASA 325 mg Advanced lesion; RR: 0.59 (0.38 – 0.92) for ASA 81 mg; RR: 0.83 (0.55 – 1.23) for ASA 325 mg Sandler et al, 2003 [12] Placebo or enteric coated-aspirin 325 mg daily for 2.6 years Patients who had histologically documented colon or rectal cancer with a low risk of recurrent disease. Detection of adenomas in the large bowel by either colonoscopy or sigmoidoscopy after randomization RR: 0.65(0.46 – 0.91) P ¼ 0 .004 Benamouzig et al, 2003 [13] Placebo or soluble aspirin (160 or 300 mg daily) for one year Patients with a history of colorectal adenomas Adenoma recurrence afte r one year RR: 0.73 (0.52 – 1.04) P ¼ 0.04 for both doses Benamouzig et al, 2011 [14] Placebo or soluble aspirin (160 or 300 mg daily) for four years Patients with a history of colorectal adenomas Adenoma recurrence afte r four years RR: 0.96(0.75 – 1.22) for both doses Logan et al, 2008 [15] Aspirin (300 mg daily) versus folate supplements (0.5 mg/day) for about 2.6 years Patients with an adenoma removed in the six months before recruitment A colorectal adenoma diagnosed after baseline RR: 0.79(0.63 – 0.99) P ¼ 0 .04 Burn et al, 2011 (CAPP1) [16] Aspirin (600 mg/day) and/or resistant starch (30 g/day) for 17 years FAP young patients Polyp number in the rectum and sigmoid colon RR: 0.77 (0.54 – 1.10) Burn et al, 2012 (CAPP2) [17] Aspirin (600 mg/day) and/or resistant starch (30 g/day) for up to four years Lynch syndrome (hereditary non-polyposis colon cancer or HNPCC) Development of color ectal canc er HR: 0.63 (0.35 – 1.13) for the entire postrandomization period HR: 0.41(0.19 – 0.86) P ¼ 0.02 for 2 years of treatment RR: Relative risk; HR: Hazard Ratio; PHS: Physicians Health Stu dy; WHS: Women ’s Health Study; CAPP: Colorectal Adenoma/Carcinoma Prevention Programm e.
five years of treatment. Rothwell et al[1]showed that 20-year risk of cancer death was reduced by approximately 20% [Hazard Ratio (HR): 0.80, 0.72–0.88] for all solid cancers and by 35% (HR: 0.65, 0.53– 0.78) for GI cancers in patients treated with aspirin forfive years or longer. This benefit increased with duration of aspirin treatment; in fact, the 20-year risk of cancer death was reduced by approximately 30% (HR: 0.69, 0.54–0.88) for all solid cancers and by approximately 60% (HR: 0.41, 0.26–0.66) for GI cancers in RCTs in which the patients took aspirin for more than 7.5 years[1]. It is noteworthy that, in one of the CV RCTs (Thrombosis Prevention Trial)[20]analysed by Rothwell and colleagues, the use of a controlled-release formulation of aspirin (75 mg) with negligible systemic bioavailability [21], leading to a selective inhibition of platelet COX-1 pathway [22], was associated with the chemo-preventive effect of aspirin on the long-term follow-up[1]. For individual cancers of GI tract, the risk of cancer death was reduced by about 40% for bowel cancer and 60% for oesophageal cancer, while that for stomach cancers was difficult to quantify because of smaller numbers of deaths. The reduction in deaths due to oesophageal cancer was confined to adenocarcinoma (HR 0.36, 0.21–0.63, P ¼ 0.0001), although the number of squamous-cell cancers was small (9/6258 in the aspirin groups versus 2/4244 in the control groups)[1].
It is quite interesting that across all cancers, aspirin only reduced deaths due to either histologically proven adenocarcinomas or primary cancers in which adenocarcinoma predominates (stomach, small bowel, pancreas, bile duct, colon, rectum, breast, uterus, ovary, and prostate).
Altogether the results of clinical studies suggest that:
(1) Aspirin causes a chemopreventive effect against tumourigenesis with an apparent maximal effect at low-doses;
(2) This effect is associated with the administration of the drug once daily; (3) This chemopreventive effect requires a long-term treatment with aspirin;
(4) The drug seems to be more efficacious in the prevention of sporadic colorectal adenomas than FAP; despite both pathological conditions are characterized by altered functions of adenomatous pol-yposis coli (APC) gene, in FAP this is driven by germline mutations.
(5) The drug showed to be very effective also in the chemoprevention of esophageal cancer which is associated with hypermethylated APC DNA that may cause inactivation of tumour suppressor genes such as APC[23,24].
Pharmacodynamic and pharmacokinetic of aspirin
Aspirin and other nonaspirin NSAIDs are analgesic and anti-inflammatory agents which act by inhibiting the generation of prostanoids [25]. Prostanoids are a family of biologically active lipid mediators which comprise prostaglandin (PG)E2, PGF2a, PGD2, prostacyclin and TXA2; they play
important roles in many cellular responses and pathophysiologic processes, such as modulation of the inflammatory reaction and its resolution, erosion of cartilage and juxtaarticular bone, GI cytoprotection and ulceration, angiogenesis and cancer, haemostasis and thrombosis, renal haemodynamics and progression of kidney disease, atheroprotection and progression of atherosclerosis[26,27]. Prostanoids are generated intracellularly from arachidonic acid (AA) mainly through, but not exclusively, the activity of phospholipase A2(PLA2)[28](Fig. 1). Once released, intracellular free AA is transformed to
PGH2by the activity of prostaglandin H synthases [named COX-1 and COX-2]; then, PGH2is
metab-olized to the prostanoids by different synthases expressed in a tissue-specific fashion[28]. Both COX-1 and COX-2 share the same catalytic activities[29]: the cyclooxygenase activity which oxidizes AA to PGG2and the peroxidase activity which reduces PGG2to the unstable endoperoxide PGH2. COX-1 gene
is considered a‘housekeeping gene’ and it is highly expressed in platelets and gastric epithelial cells where it plays a role in causing platelet activation, via the generation of TXA2, and gastric
cytopro-tection, via the generation mainly of PGE2 [26,27,30]. COX-1 plays a role in several pathological
conditions, such as thrombosis, atherosclerosis and tumourigenesis[31–33].
Differently from COX-1 gene, COX-2 is a primary response gene with many regulatory sites [34]. However, COX-2 is constitutively expressed in some cells in physiologic conditions, such as endothelial cells [35], where COX-2-dependent-PGI2 induces an antithrombotic and vasoprotective signalling
[5,36]. A constitutive overexpression of COX-2 has been detected in cancer cells where the major product is PGE2[37]. PGE2modulates processes fundamental to tumour cell survival such as enhanced
proliferation and resistance to apoptosis[37–41].
Aspirin and nonaspirin NSAIDs reduce prostanoid generation by inhibiting the cyclooxygenase activity of COX-1 and COX-2 [25,30]. However, aspirin, but not nonaspirin NSAIDs, causes an irre-versible inactivation of COX-isozymes[3].
COX-isozymes are homodimers ofw72 kDa and each monomer is a heme-containing glycoprotein
[29]. Recent evidences have displayed a functional cross-talk between the two monomers of each COX enzyme and, in particular, both isoforms exhibit half of sites COX activity[42,43]. Both monomers bind the substrate AA but a monomer acts as an allosteric subunit (regulatory) which transforms the partner monomer into the catalytic one transforming AA into PGG2; then PGG2is transformed to PGH2by the
peroxidise activity of COX-1 and COX-2. Aspirin binds to one monomer of COX-1 and COX-2 by the interaction with Arg120 residue and modifies covalently COX isozymes by the acetylation of Ser529 and Ser516 on COX-1 and COX-2, respectively; the acetylated monomer becomes the allosteric subunit, and the partner monomer becomes the catalytic monomer. Acetylation of the allosteric subunit of COX-1 and COX-2 by aspirin causes an irreversible inactivation of the COX activity[44]. The acetylated COX-2 has a significantly compromised ability to form PGG2 but produces an alternative product,
15R-hydroxyeicosapentaenoic acid (15R-HETE) from AA [45]. Studies performed by Smith’s group [46]
showed that aspirin acetylation of the regulatory monomer of COX-2 is associated with an irrevers-ible inhibition of the catalytic monomer to form PGG2. In contrast, the acetylated monomer forms
primarily 15R-HETE from AA. Thus, the effect of aspirin on COX-2 is an incomplete allosteric inhibitory effect compared with that seen with COX-1[46].
Several studies in vitro have shown that 15R-HETE is then metabolized to the epi-lipoxins (LXs) in monocytes and leukocytes through the action of 5-lipoxygenase (5-LO)[47,48](Fig. 1), the enzyme also responsible for initiation of leukotriene synthesis. Epi-LXs seem to be potent inhibitors of cell prolif-eration and angiogenesis. However, convincing evidence that these lipid mediators triggered by aspirin are generated in vivo in humans are lacking, in particular because the analytical assays (mainly immunoassays) used to measure their levels in urinary collections[49]were not rigorously validated by comparison with mass-spectrometry analysis.
The plasma concentration of aspirin decays with a half-life of 15–20 min[3,44]. Despite the rapid clearance of aspirin from the circulation, the inhibitory effect of COX-1 and COX-2 is long-lasting because of the irreversible inactivation of the COX-isozymes. Thus, in a nucleated cell treated with aspirin, the biosynthesis of prostanoids recovers as a result of de novo protein synthesis of COXs which
Fig. 1. Effects of aspirin on arachidonic acid metabolism. (A) Aspirin acetylates COX-1 enzyme, thus blocking the biosynthesis of prostanoids COX-1-derived. Differently, acetylated COX-2, conserves the catalytic ability to transform free AA to HETE. (B) 15R-HETE can be metabolized by cells expressing the enzyme 5-lipooxygenase (5-LO) (such as leukocytes), to 15-epi-LXA4.
requires roughly 3–4 h. In contrast, in platelets which have limited capacity of protein synthesis[50], the irreversible inhibition of COX-1 (the only COX-isozyme expressed in platelets in physiological conditions) by aspirin lasts for the life span of the platelet[51]. This explains the use of aspirin once daily in the antithrombotic therapy and the necessity to administer multiple doses to obtain anti-inflammatory and analgesic effects[3,44].
Clinical pharmacology of aspirin
In vitro experiments show that aspirin is 60-fold more potent to inhibit platelet COX-1 than monocyte COX-2[52]. When administered in vivo to healthy subjects once daily, aspirin causes a dose-dependent inhibition of platelet COX-1 activity ex vivo, as assessed by the measurement of the generation of TXB2[53]in whole blood allowed to clot for 1 h at 37C (serum TXB2is a capacity index of
platelet COX-1 activity). However, as shown inFig. 2, the maximal inhibition of platelet COX-1 activity is obtained at a low-dose of 75–100 mg. At these doses, aspirin inhibits platelet COX-1 activity >95%, at 1 h after dosing, and this effect persists up-to 24 h[51]. This profound inhibitory effect of platelet COX-1 is not explained by systemic circulating levels of aspirin[21], but it has been shown to be caused by higher levels of the drug reached in the presystemic circulation. In fact, oral aspirin is mainly absorbed by the stomach and is subjected tofirst-pass metabolism both in the gastrointestinal tract and in the liver[54,55]. Presystemic deacetylation of aspirin after oral administration is responsible for reduced systemic levels of aspirin and increased levels of salicylate which is roughly 100-times less potent to inhibit platelet COX-1 than aspirin[52]. The almost complete inhibition of platelet capacity to generate TXA2by low-dose aspirin is associated with a profound inhibition of TXA2-dependent platelet function
which persists throughout dosing interval (i.e. 24 h)[53].
The almost complete and persistent inhibition throughout dosing interval of platelet COX-1 represents a fundamental requisite to obtain an antithrombotic effect [3,44]. In fact, even tiny concentrations of TXA2can activate platelets and they can synergize with low-concentrations of other
agonists to cause a complete platelet aggregation[56]. This effect can be obtained by aspirin which is an irreversible inhibitor of platelet COX-1 while nonaspirin NSAIDs, which affect platelet COX-1 reversibly, cause an intermittent inhibition of platelet TXA2generation in the interval between doses that is not
adequate to translate into an antithrombotic effect[57,58]. As shown in Fig. 2, an almost complete inhibitory effect of platelet COX-1 activity is obtained ex vivo after dosing with 75–100 mg/day of aspirin and this is coincident with a maximal antithrombotic effect detected in RCTs[4].
Fig. 2. Differential dose-response relationships to inhibit platelet COX-1 ex vivo and systemic PGI2in vivo by aspirin: correlation
The oral administration of low-doses of aspirin once daily is associated with a maximal systemic drug concentration (approximately 7
m
M) [21] which may affect only marginally COX-2 activity expressed in blood cells and/or vascular or epithelial cells. In addition, de novo synthesis of the acetylated COX-2 in a nucleated cell may cause a rapid recovery of prostanoid biosynthesis. Thus, the administration of low-dose aspirin did not significantly affect whole blood COX-2 activity ex vivo[59,60]. Moreover, systemic biosynthesis of vascular PGI2 (as assessed by the measurement of
a major enzymatic urinary metabolite, 2,3-dino-6-keto-PGF1a, PGI-M), mainly derived from the
activity of COX-2 [5], was only partially affected by low-doses of aspirin (Fig. 2)[59]. However, at higher doses of aspirin a profound inhibitory effect on the biosynthesis in vivo of PGI2was found
[61], which might contribute to the apparent reduced antithrombotic benefit detected at high doses of the drug.
The administration of low-dose aspirin doubles the relative risks (RR) of upper gastrointestinal bleeding (UGIB) in comparison to aspirin nonusers [62–64]. Since after oral dosing with low-dose aspirin once daily, the levels of the drug in the systemic circulation [21] are insufficient to cause a substantial inhibition of the biosynthesis of cytoprotective prostanoids in the GI tract, it is plau-sible that the anti-platelet effect of low-dose aspirin contributes to enhanced incidence of UGIB[26]. However, at high aspirin doses (600 mg daily), the inhibitory effect of COXs of the GI tract contribute to the almost 4-fold increase in the risk of UGIB compared with nonaspirin users [63]
(Fig. 2).
The role of platelets in GI tumourigenesis
Thefinding of RCTs showing that low-dose aspirin given once daily causes a chemopreventive effect against atherothrombosis[3]and CRC[1,19]suggests that enhanced platelet activation is involved in the development of the two pathological conditions. In fact, these aspirin doses and dosing intervals are consistent with a selective inhibitory effect of aspirin on platelet COX-1 activity and on TXA2
-dependent platelet function.
Platelets represent an important linkage between tissue damage/dysfunction and the inflammatory response initially acting to repair the damage, but then, uncontrolled platelet activation may translate into pathological conditions, such as atherothrombosis and cancer.
Activated platelets may play a role in tumour progression and metastasis by the release of: (i) several factors that regulate the angiogenic process and cell growth[65], (ii) microparticles (MPs) and exosomes[66](Fig. 3). After activation, various angiogenic-regulating factors, such as vascular endo-thelial growth factor (VEGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), endostatin, thrombospondin-1, tissue inhibitor of metalloproteinases (TIMP) are released from different sets of
a
-granules, in which these products are stored [67]. In addition, platelet-derived interleukin (IL)-1b
, generated by the interaction of platelets with monocytes, has been shown to contribute to monocytic COX-2 induction through a post-transcriptional mecha-nism which stabilizes COX-2 mRNA[68].MPs released from activated platelets are approximately 0.1–1.0
m
m in diameter in humans and express P-selectin (CD62P) and GP IIb–IIIa. They adhere to a variety of cells, can activate endothelial cells, leukocytes and other platelets, and deliver signals through chemokines[69]. Exosomes, range in size from 0.04 to 0.1m
m, arise from the internal membrane vesicles of multivesicular bodies and plateleta
-granules. Unlike MPs, exosomes do not show a similar surface phenotype of activated platelets. However, both MPs and exosomes are able to carry and deliver cellular signals, suggesting a potential role in platelet-derived signalling. Exosomes can be also released from cancer cells and can be detected in the systemic circulation[70]. Interestingly, it has been proposed that exosomes may be used as surrogate diagnostic markers for biopsy profiling[71], due the fact that their protein and RNA repertoire resembles that of the origin tumour, thus suggesting their potential application for the early tumour detection in asymptomatic patients.The generation of TXA2, a major product of platelet COX-1, which promotes platelet aggregation and
vasoconstriction [5] represents another important mechanism by which platelets can affect tumourigenesis. It has been shown that enhanced TXA2 generation, by the introduction of the
tumour angiogenesis, tumour growth in vivo[72]and promotes the interaction between metastasizing tumour cells and the host haemostatic system [72], thus suggesting a role of TXA2 in promoting
angiogenesis and the development of tumour metastasis[73].
Enhanced TXA2 biosynthesis in vivo (by assessing a urinary enzymatic metabolite of TXA2,
11-dehydro-TXB2, TX-M, which is an index of TXA2generation in vivo) [74]has been recently detected
in patients with FAP[75]. Studies with low-dose aspirin in humans have convincingly shown that TX-M is mainly from platelets[76]and this biomarker has been used in many clinical studies as an index of platelet activation in vivo. Thefinding that the administration of the selective COX-2 inhibitor celecoxib did not affect enhanced urinary levels of TX-M in FAP strongly supports a COX-1 origin[75]. We have also shown that enhanced TX-M is detected in patients with colorectal cancer and that a very low-dose of aspirin (50 mg daily) caused a cumulative inhibition of TX-M suggesting a platelet origin [77]. Enhanced systemic biosynthesis of TXA2was also detected[75]in an experimental model of FAP in
mice [ApcMin/þmice][78]. To support a platelet origin of enhanced TXA2generation detected in vivo in
intestinal tumourigenesis, we performed experiments in vitro of coculture of platelets and colon cancer cells, and we showed that TXA2is enhanced and that its origin is from platelets[75]. In this
scenario, vascular COX-2-dependent PGI2 may play a protective role by restraining the release of
growth and angiogenesis factors from platelets but also from different cell types, such as vascular cells. In fact, Dovizio et al[75]found that circulating levels of angiogenin, an inducer of angiogenesis present in platelets and released in response to agonist stimulation[79], were inversely related to urinary PGI-M in FAP patients treated with celecoxib. There was also an inverse Spearman’s correlation between urinary excretion of PGI-M and plasma levels of two mediators of angiogenesis and tumour progres-sion, i.e., FGF-2[80]and HGF[81].
The possible antitumourigenic role of PGI2 might explain why high doses of aspirin are not
associated with enhanced chemopreventive effects against CRC. Systemic circulating levels of aspirin on the one side may have an anti-proliferative effect by inhibiting COX-2-dependent-PGE2 in the
tumour and on the other side they may remove the antiangiogenic effects of vascular COX-2-dependent-PGI2.
Fig. 3. Platelet-mediated mechanisms in gastrointestinal tumourigenesis Platelets may play an important role in promoting the early events of tumourigenesis through the release of: (i) several factors that regulate the angiogenic process and cell growth, (ii) microparticles (MPs) and exosomes, which can activate stromal cells (macrophages andfibroblasts), the earliest cells which over-express COX-2 in CRC. The stromal COX-2 over-expression may result in the release of high levels of prostanoids and growth factors. This step may contribute to the overexpression of COX-2 in epithelial cells. During tumour progression there will be the induction of COX-2 also in endothelial cells (EC) and this will contribute to a proangiogenic response. In this scheme of intestinal tumourigenesis, we postulate a role played by both COX-1 and COX-2 pathways and that the two COX pathways operate sequentially. Abbreviations: ADP (Adenosine diphosphate).
We propose that enhanced platelet activation may act in early phase of tumourigenesis by stimu-lating stromal cells to express COX-2 and release enhanced levels of PGE2and other inflammatory
mediators, growth factors and proangiogenic factors. Altogether these events may participate in epithelial cell transformation in part as a consequence of COX-2 overexpression.
The role of COX-2 in intestinal tumourigenesis is supported by several clinical studies, in which the administration of selective COX-2 inhibitors (such as celecoxib) was associated to a reduced the risk of colorectal adenoma recurrence [82,83]. However, the use of selective COX-2 inhibitors as chemo-preventive agents is limited by their ability to interference with CV homeostasis due to the coincident inhibition of vascular COX-2-dependent PGI2[5].
Thus, we propose that in intestinal tumourigenesis, both COX-1 and COX-2 pathways are involved and they operate sequentially (Fig. 3). This is strongly sustained by experimental animal studies in which the loss of either COX-1 or COX-2 genes blocks intestinal polyposis in mouse models of FAP by about 90%[32,84].
Evidences for COX-independent mechanisms of the antitumoural effects of aspirin
It is generally believed that pharmacological effects of NSAIDs arise from their inhibitory action on COXs[85–87]. However, it has been shown that some NSAIDs, including aspirin, are able to inhibit the proliferation and to induce apoptosis of colon cancer cells in vitro even in the absence of any apparent involvement of either COX-1 or COX-2[85–87].
Thus, alternative mechanisms for their activity on tumour cell growth have been proposed [85–
87](Fig. 4).
Hopefully, the elucidation of extra-COX molecular targets and the downstream signalling pathways involved in cell proliferation and/or induce apoptosis affected by NSAIDs and aspirin will facilitate the development of more effective approaches for cancer prevention and treatment. However, it is
Fig. 4. COX-independent mechanisms of the antitumoural effects of aspirin NSAIDs, including aspirin, are able to inhibit the proliferation and to induce apoptosis of colon cancer cells in the absence of any apparent involvement of either COX-1 or COX-2. Alternative mechanisms for their activity on tumour cell growth include: (a) inhibition of the most essential oncogenic pathway in CRC, the Wnt/b-catenin pathway, which plays a role in the expression of genes involved in tumourigenesis; (b) the inhibition of the binding of c-Raf with Ras, leading to the inhibition of extracellular signal-regulated protein kinase (ERK) pathway, which is activated in a variety of cell types by different extracellular stimuli; its activation leads to the phosphorylation of various down-stream substrates involved in a multitude of cellular responses such as cell proliferation, cell differentiation, cell survival, and cell motility; (c) inhibition of I-kappa kinase (IKK)b, thereby preventing the activation by NF-kB and its capacity to regulate the expression of several genes that cause suppression of the apoptotic response in cancer cells. Abbreviations: Tcf (T-cell factor).
important to underline that probably these COX-independent pathways are not clinically relevant in aspirin chemopreventive action. In fact, their involvement in the antiproliferative and proapoptotic effects of aspirin has been detected in vitro at very high concentrations of aspirin, often in the millimor range, which is not possible to obtain in vivo, even with high anti-inflammatory doses of aspirin. Summary
Aspirin is the only NSAID which causes an irreversible inactivation of COX-1 and COX-2 [3,44]. Several lines of evidence support the hypothesis that the inhibitory effect of platelet COX-1 by aspirin is at the centre of its antitumour efficacy. The first important evidence comes from several observational studies and from the long term analyses of CV RCTs and RCTs of adenoma recurrence[1,2,19], which showed the apparent saturability of the chemopreventive effect of low-doses aspirin, given once daily (as recommended for the prevention against heart disease). A remarkably similar saturability of the cardioprotective effect of low-dose aspirin is explained by: (i) the irreversible nature of COX-1 inactivation in platelets [3,44] and (ii) the limited capacity of human platelets of de novo protein synthesis[50].
Moreover, several factors contribute to exclude the possibility that the chemopreventive effect of low-dose aspirin may occur through a direct inhibition of COX in nucleated cells throughout the 24 h dosing interval: (i) the short half-life of aspirin in human circulation (approximately 20 min)[3,44]; (ii) the capacity of nucleated cells to resynthesize the acetylated COXs and (iii) circulating levels of aspirin, which are below the IC50values for inhibition of COX-2[21,52].
Platelets may play an early role in tumourigenesis (through the release of products and/or exo-somes) by causing the activation of stromal cells and over-expression of COX-2 which results in the release of higher levels of growth factors. They may induce intestinal epithelial cell transformation, in part as a consequence of COX-2 expression. Later in tumour progression there will be the induction of COX-2 also in endothelial cells and this will contribute to a proangiogenic response. In this scheme of intestinal tumourigenesis, we postulate a role of COX-1 and COX-2 and that the two COX pathways operate sequentially.
It has been proposed that some NSAIDs, including aspirin might act as chemopreventive agents by modulating COX-independent biological responses[85–87]. The majority of experimental evidences suggesting the involvement of COX-independent mechanisms in the anti-proliferative and pro-apoptotic actions of aspirin derives from in vitro studies, which assess the effects of aspirin at high concentrations on colon cancer cell lines. Since it is not possible to obtain the same concentrations in vivo (even with high anti-inflammatory doses of aspirin), it seems unlike that these COX-independent pathways may contribute to the chemopreventive action of aspirin in a clinically rele-vant fashion.
Practice points
The finding that aspirin at doses of at least 75 mg daily, used for cardioprevention, reduced long-term incidence and mortality due to colorectal cancer locates the antiplatelet effect of aspirin at the centre of its antitumour efficacy.
Given the short half-life of aspirin in human circulation (approximately 20 min) and the capacity of nucleated cells to resynthesize the acetylated COX-isozyme(s), it seems unlikely that a nucleated target could be suppressed through the 24-h dosing interval
Activated platelets may play an important role in promoting the early events of intestinal tumourigenesis by the induction of a COX-2-mediated paracrine signalling between stromal cells and epithelial cells within adenomas.
COX-independent mechanisms of aspirin, such as the inhibition of NF-kB signalling and Wnt/
b
-catenin signalling, have been suggested to play a role in its chemopreventive effects. However, their relevance remains to be demonstrated in vivo at therapeutic doses.Conflict of interest None.
Acknowledgements
This study was supported by grants [2005–2007 and IG–12111] by the Associazione Italiana per la Ricerca sul Cancro (P.P.). We wish to thank Dr Carlo Patrono (Catholic University, Rome, Italy) and Dr Luis A Garcia Rodriguez (Ceife, Madrid, Spain) for fruitful discussions and suggestions.
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