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Biomedicine & Aging Pathology 2 (2012) 67–72

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OX-2 as a molecular target of colon cancer chemoprevention: Promise andeality

asmeet Kaura, Vivek Vaishb, Sankar Nath Sanyalb,∗

Mercer University School of Medicine and Memorial University Medical Center, Hoskins Research Building, 4700, Waters Avenue, Savannah, GA 31404, USADepartment of Biophysics, Panjab University, Chandigarh 160 014, India

r t i c l e i n f o

rticle history:eceived 17 April 2012ccepted 9 July 2012vailable online 28 August 2012

eywords:hemopreventionolon cancerOX-2oxibsSAIDs

a b s t r a c t

The decisive epidemiological observation that non-steroidal anti-inflammatory drugs (NSAIDs) preventcolon and possibly other cancers as well has spurred novel approaches to cancer chemoprevention.The known inhibitory effect of NSAIDs on the prostaglandin production prompted studies focusing oncyclooxygenase (COX) and its products. The increased prostaglandin E2 levels and the over-expression ofCOX-2 in colon and many other cancers provided the rationale for clinical trials with COX-2 inhibitors forcancer chemoprevention. However, their modest efficacy in the prevention of sporadic colon and othercancers, and the recent withdrawal of Rofecoxib, a COX-2 specific inhibitor (coxib), because of its sideeffects are causes of concern about this class. There is evidence to suggest that COX-2 may not be theonly or ideal target in the eicosanoid pathway for cancer chemoprevention. Various observations like therelatively late induction of COX-2 in carcinogenesis, the modest clinical efficacy of coxibs in compari-son to traditional NSAIDs (tNSAIDs) in cancer prevention trials, the finding that NSAIDs may not require

inhibition of COX-2 for their effect, that currently available coxibs have multiple non-COX-2 effects, thepossibility that concurrent inhibition of COX-2 in non-malignant cells may be harmful and the possibilitythat COX-2 inhibition may modulate alternative eicosanoid pathways in a way that promote carcinogen-esis support this concept. Given the limitations of COX-2-specific inhibitors and the biological evidencefor the existence of other potential targets, we suggest that targets other than COX-2 should be exploredas unconventional or paired approaches to cancer prevention.

© 2012 Elsevier Masson SAS. All rights reserved.

. Introduction

‘Cancer prevention is better than treatment’ has emerged as aiable option for reducing mortality from this disease. However,n contrast to other diseases, like cardiovascular and infectiousnes, whose prevention contributed significantly towards theireduced morbidity and mortality, gain in cancer prevention haseen limited. Therefore, there is need for the identification of poten-ial targets for chemoprevention for the development of safe andffective agents. This review describes the background for theevelopment of one such target, cyclooxygenase-2 (COX-2) andaking a step further, we suggest a search for other alternativeargets of drug development.

∗ Corresponding author. Tel.: +911722534122.E-mail address: sanyalpu@gmail.com (S.N. Sanyal).

210-5220/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved.ttp://dx.doi.org/10.1016/j.biomag.2012.07.007

2. Background

The first epidemiological evidence that non-steroidal anti-inflammatory drugs (NSAIDs) prevent human colon cancerrecognized the possibility of cancer chemoprevention [1]. Based onthe two sets of data, one from epidemiological studies which doc-ument an association between NSAID use and reduced cancer riskand second from interventional clinical trials that demonstrate thatthe administration of NSAIDs can actually prevent cancer; it is nowdecisively established that the long-term use of NSAIDs preventsan array of cancers.

The epidemiological studies reported by Thun et al. collectivelydescribing results on more than one million subjects, have pointedout the protective effect of NSAIDs against colon [2]. Two random-ized interventional studies using polyp recurrence as a generalendpoint demonstrated the preventive effect of aspirin on coloncancer [3,4]. The overall significance of these landmark studies

proves the general concept of chemoprevention by NSAIDs.

NSAIDs were discovered to act by inhibition of the enzyme COX,which catalyzes the synthesis of prostaglandins (PGs) from arachi-donic acid (AA) [5]. The COX gene was cloned by three separate

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esearch groups in 1988 [6–8] and two isoforms of COX have sinceeen identified: COX-1 and COX-2 [9]. These isoforms possess anpproximate 60% amino acid homology, similar tertiary structuresnd similar, but not identical, active sites [10].

The expression of COX-1 is found to be constitutive throughouthe body [11] and regulates homeostatic processes such as plateletggregation, gastrointestinal (GI) protection and renal function. Itsxpression is only slightly upregulated (two- to four-fold) in someells in response to hormones or growth factors [12]. In contrast,OX-2 is expressed predominantly in response to inflammation andther stressors and its expression is markedly upregulated (10- to0-fold) by a variety of mediators [13]. It is involved in the synthesisf PGs mediating pathologic processes such as pain, inflammation,ever and carcinogenesis [14]. Expression of COX-2 has been knowno facilitate several oncogenic processes, including tumor invasion,ngiogenesis and metastasis [15].

These distinct expression patterns have led to the theoryhat COX-1-derived prostaglandins are largely responsible forhysiologic (housekeeping) functions [16] and COX-2-derivedrostaglandins mediate pathophysiological and inflammatory pro-esses. Traditional NSAIDs (tNSAIDs) which inhibit both COX-1 andOX-2 [17] are known to cause GI toxicity due to mucosal erosion.herefore it was hypothesized that selective COX-2 inhibitors (cox-bs) would have the advantages of conventional NSAIDs but wouldot interfere with GI protection or homeostasis [18].

. COX-2 as a target for colon cancer chemoprevention

In the normal GI tract, very little COX-2 or undetectable levelf COX-2 is expressed [19]. COX-2 expression has been found toncrease in approximately 80% of human colorectal adenocarcino-

as collected surgically or by endoscopic removal by 2- to 50-fold20]. These findings decisively indicate that COX-2 plays an impor-ant role in the development of colon cancer. Chronic use of aspirinnd other traditional NSAIDs, in more than 50 population-basedtudies, was found to be associated with a 40%–50% reduction in theisk for developing colon cancer [21]. Biological activity of sulindac,

non-specific COX-2 inhibitor in the treatment of familial adeno-atous polyposis (FAP), was demonstrated in a double blinded,

andomized, placebo-controlled trial [22], which showed a signifi-ant reduction in the number and size of adenomas compared withhe patients treated with placebo over a 9-month period.

The effect of treatment with COX-2 inhibitors on tumor growthas first investigated in animal studies. Human colonic adeno-

arcinoma cells that had very high levels of COX-2 expressionere grafted into immunocompromised mice [23]. After these cellseveloped into tumors, a COX-2-specific inhibitor was added to theice’s diets, resulting in a significant inhibition of tumor growth.

imilarly, rats treated with celecoxib, a specific COX-2 inhibitorhowed a significant reduction in the number of adenomas and,f non-invasive and invasive adenocarcinomas [24]. Evidence thatOX-2-specific inhibitors reduce tumor growth was also shown

n a study involving the multiple intestinal neoplasia (Min) mice, genetic model for FAP [25]. In humans, 77 patients with FAPere treated with celecoxib 100 mg BID, celecoxib 400 mg BID, orlacebo [26]. At 6 months, patients receiving celecoxib 400 mg BIDemonstrated a significant reduction in polyp burden comparedith placebo (30.7% vs 4.9%). It leads to culminate in celecoxib

eceiving US FDA approval for cancer prevention in patients withAP.

Thus, COX-2 inhibitors possess outstanding potential as chemo-

reventive agents and support the notion that cancer prevention

s a viable clinical option. COX-2 inhibitors are effective in therevention of colon cancer in several animal models and cane administered for long periods with fewer toxic effects than

Pathology 2 (2012) 67–72

nonselective NSAIDs. They are prescribed mainly for the treatmentof arthritis and pain, so there is a large clinical experience with theiruse in the general population. Since COX-2 appears to be expressedat high levels in many different types of human tumors, but not insurrounding normal tissue, it seems likely that the paradigm devel-oped for colon cancer can be expanded to other organ sites. Thus,there is a strong base from which to design human trials.

4. Challenging the central role of COX-2 in carcinogenesisand its chemoprevention

The intellectual beauty and utter simplicity of the concept thatCOX-2 induction is central to the pathogenesis of colon and othercancers, and its inhibition would prevent and regress them, hasbeen challenged by various key observations in recent times. Wediscuss these observations briefly below.

5. COX-2 expression during various stages of carcinogenesis

The first challenge is the relatively late induction of COX-2 dur-ing carcinogenesis which does not entirely fit to the model thatCOX-2 is central to carcinogenesis. Nobuoka et al. have shown thatthere is no COX-2 expression in human aberrant crypt foci, theearliest recognizable premalignant lesion in the colon [27]. COX-2 expression in aberrant crypt foci would be highly advantageous,if not a requirement, for COX-2 to be the ideal chemopreventiontarget, because chemoprevention would be easiest at this stage oflower complexity. Moreover, the expression of COX-2 commencesonly at the adenoma stage when only 45% of the adenomas arepositive; of the carcinomas, 85% are positive [20]. This pattern ofCOX-2 over-expression evokes an alternative interpretation thatCOX-2 expression is the result of and not a dominant contributorto carcinogenesis.

The strongest argument against this interpretation, however,are the animal studies demonstrating that disruption of eitherthe COX-1 or COX-2 gene reduces the development of tumorsin mice [28]. Findings from our lab after six weeks of 1, 2-dimethylhydrazine (DMH, colon-specific carcinogen) treatment inmale Sprague-Dawley (SD) rats have shown the occurrence ofmultiple plaque lesions (MPLs), aberrant crypt foci (ACF) as wellas increased COX-2 expression in the colonic mucosa comparedto the control rats [29,30]. This was significantly prevented afterco-treatment with COX-non-specific and COX-2 selective or pref-erential NSAIDs [31–40].

Takeda et al. advanced an interesting idea and demonstratedthat most COX-2-expressing cells in the polyps of Apc�716 miceare stromal fibroblasts in which COX-1, COX-2 and mPGES (humanmicrosomal prostaglandin E synthase) co-localize [41]. COX-2 wasinduced only in polyps more than 1 mm in diameter, but COX-1was found in polyps of any size. Based on these data, they proposedthat COX-1 expression in the stromal cells secures the basal level ofPGE2 that can support polyp growth to ∼1 mm and that simultane-ous inductions of COX-2 and mPGES support the polyp expansionbeyond ∼1 mm by boosting the stromal PGE2 production.

6. Concern about efficacy and safety of coxibs

Recent observations about limited clinical efficacy of coxibs incancer prevention and, increased risk of myocardial infarctions andstroke in clinical trials have raised concerns about this class ofNSAIDs. It may be emphasized that celecoxib is the only coxib so far

approved by the FDA to be used for the treatment in FAP patients.However, the data shows that the suppression of the neoplasticprocess was modest [26]. The recently reported failure of celecoxibcombined with trastuzumab to have an effect on patients with

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uman epidermal growth factor receptor-2/neu-over-expressing,rastuzumab-refractory metastatic breast cancer is another dis-ppointing result [42]. A strong link between over-expression ofuman epidermal growth factor receptor-2/neu and COX-2 activityas however, been demonstrated in preclinical studies. Rofecoxib,nother COX-2 specific inhibitor too had a statistically significantut marginal effect on the number of polyps in FAP patients (6.8%eduction from baseline values) [43]. Also, in a phase II study ofetastatic colon cancer, rofecoxib in combination with chemother-

py showed increased toxicity and no efficacy [44].Two theories could explain these results: either COX-2 is not

entral to the carcinogenic process or COX-2 specific inhibitor likeelecoxib is not a sufficiently strong COX-2 inhibitor. Against theatter, all available clinical and preclinical evidence suggests thatelecoxib is a strong COX-2 inhibitor [45]. The point of interestemains however, that the dose required inhibiting COX-2 activityignificantly i.e. by 80%; has also been shown to cause a 60% inhibi-ion in the COX-1 activity [46]. Conversely sulindac, a tNSAID, has

pronounced effect on colorectal polyps in FAP patients. A statis-ically significant decrease occurred in the mean number of polypsnd their mean diameter in patients treated with sulindac (150 mgrally twice a day) when compared with those given placebo [47].nother pilot study by Winde et al. using rectally applied sulin-ac in colectomized FAP patients showed a complete reversion ofdenoma after 12 months at 53 and 67 mg of sulindac per day peratient on average, respectively, compared with the control group48]. Also tissue PGE2 levels were greatly reduced. Although notirectly comparable, given the different duration of drug admin-

stration it seems that sulindac may have a stronger effect thanhe coxibs. These findings suggest that inhibition of targets otherhan or in addition to COX-2 may contribute to sulindac’s strongerhemopreventive effect in the colon.

In view of safety, the Celecoxib Long-Term Arthritis Safety StudyCLASS) trial showed this agent to have fewer GI side effects ando increase in cardiovascular risk at 6 months, but the 12-monthata suggest that celecoxib did not differ from the tNSAIDs in

ts gastrointestinal effects. Importantly, a retrospective analysis ofhe data suggests signs of increased cardiovascular risk [49]. Thedenomatous Polyp Prevention on Vioxx study to evaluate the effi-acy of rofecoxib in patients with colorectal adenoma showed that.5% of rofecoxib recipients and 1.9% of placebo recipients sufferedyocardial infarctions or strokes during the trial. This prompted

he termination of this and all related trials and subsequently theermanent withdrawal of rofecoxib. This has been a setback to these of COX-2 inhibitors to prevent colon and perhaps other cancerss well. This has also provoked the concerns for a “class (side) effect”f the coxibs [50]. It has been suggested that since COX-2 is the prin-ipal enzyme involved in the production of PGI2, its inhibition byOX-2 inhibitors could increase cardiovascular risk by tipping thealance toward platelet aggregation and vasoconstriction [50].

. COX-2 expression in normal cells

The expression of COX-2 is not restricted to tumor cells andhus concurrent inhibition of COX-2 in non-neoplastic cells may bearmful. COX-2 is induced in a limited repertoire of cells, notably

n monocytes, macrophages, neutrophils and endothelial cells [51]y bacterial lipopolysaccharides, growth factors, cytokines andhorbol esters. COX-2, although detectable at only low levels inascular endothelium, may be expressed in response to normallood flow [52]. Activated and proliferating vascular tissues, such

s angiogenic microvessels and atherosclerotic tissues also expressigh levels of COX-2. Eicosanoids often form “opposing pairs”hose balance determines the final result. A classic pair consists

f thromboxane A2 and prostacyclin, which have opposite effects

Pathology 2 (2012) 67–72 69

on platelets and vascular tone [53]. Shifting the balance of such apair could have either beneficial (e.g., prevention of cardiovascularevents by low-dose aspirin) or catastrophic effects. The latter mayaccount to the cardiovascular side effects of coxibs as COX-2 is amajor source of systemic prostacyclin (PGI2) biosynthesis [54].

8. Stimulation of other pro-carcinogenic pathways whileCOX-2 is suppressed

Other than COX, phospholipases (PLA2), cytochrome P450,lipoxygenases (LOXs) and the so-called “terminal enzymes”, i.e.,those converting endoperoxides to the end products, generate anarray of biologically active eicosanoids from polyunsaturated fattyacids, arachidonic and linoleic acids. Inhibition of COX may shiftits substrate fatty acid to a non-COX pathway and generate a pro-carcinogenic end product. An increased pulmonary production ofPGI2 by lung-specific over-expression of prostacyclin synthase,which operates downstream of COX, decreases lung tumor inci-dence and multiplicity in both chemically induced murine lungcancer models and in a tobacco smoke exposure model [54,55],suggesting a distinctive role of terminal enzymes in cancer.

Recent observations show that besides products of COX iso-forms, LOX products may also be important in carcinogenesis. SomeLOX products have pro-tumorigenic activities, whereas others areanti-tumorigenic [56]. Inhibition of COX-2 could shift arachidonicacid to the LOX pathway, thereby suppressing apoptosis - not adesirable effect in cancer prevention. A recent human study sug-gested that oral celecoxib increased leukotriene B4 production inthe lung microenvironment under physiological conditions [57].Such findings suggest that COX inhibition may not be always desir-able for cancer control.

9. COX-independent effects of NSAIDs

Against the concept that COX-2 has a central role in carcinogene-sis, NSAIDs do not require the presence of COX-2 to prevent cancer[58]. This was based on the finding that in vitro NSAIDs displayeffects compatible with cancer prevention, such as inhibition of cellproliferation, induction of apoptosis, inhibition of angiogenesis andmany others, in the absence of COX-1 or -2. This initial observationis now firmly established by many studies that point to an arrayof molecular targets affected by NSAIDs [59]. In this regard, NO-donating aspirin also inhibited cell proliferation, induced apoptosis,and inhibited nuclear factor-�B activation and Wnt signaling whileinducing COX-2 expression without affecting COX-1 expression inhuman colon cancer cells [60].

However, it has been proposed that these non-COX effects ofNSAIDs occur only at “industrial strength” concentrations of NSAIDs[61], which is not entirely accurate or necessarily relevant. Forexample, some tNSAIDs have IC50 values for the inhibition of coloncancer cell growth in the micromolar range, with probably sulin-dac sulfide, the active metabolite of sulindac, having the lowest(approximately 175 �M) and aspirin the highest (2.5 mM); the IC50for sulindac approaches 1 mM [62]. The corresponding IC50 forsome COX-2 inhibitors seems to be around 30 �M [63] indicating aclear discrepancy between cell culture results and pharmacologicalefficacy. As discussed above, sulindac is more effective in the pre-vention of colon cancer in patients with FAP than coxibs, a completereversal of the conclusions that might have been reached from thecell culture data. It should also be kept in mind that cell culturesystems are by their very nature time-sensitive. Most studies using

cell culture systems have to be terminated by 72 to 96 hours; thus,high concentrations of an agent are at times necessary to detect aneffect. These observations prompt a reminder of the limitations ofthe cell culture system, whose findings should be viewed for what

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hey are: a mere indication of what may happen in the complexityf the human organism.

In contrast, in in vivo chemoprevention studies, compara-ively lower doses of an agent are administered for much longereriods of time, and the effect of the agent may thus becomepparent. The “low dose–long duration” or “high dose–short dura-ion” balance needs to be considered so that potentially usefulgents are not unjustifiably abandoned. Supporting this, our in vivoesults demonstrate the contribution of various COX-independentechanisms of NSAID towards colon cancer chemoprevention

ike inhibition of cell proliferation [29], induction of apoptosis29,30,37,38,64], inhibition of oncogenic phoshatidylinositol (PI)3-inase/Akt, Wnt [29,39,65] and NF-�B [33,37,40,66] pathways,nduction of PPAR� [33,67] and ROS production [38,67].

0. COX-2-independent effects of coxibs

Extensive data establish that COX-2 independent activities ofoxibs may account at least for part of their cancer-preventiveroperties. These are inhibition of cell proliferation [68], inductionf apoptosis [69], induction of cell cycle block [70], expression ofurvivin [71], release of cytochrome c [72], inhibition of carbonicnhydrase [73] and induction of NSAID-activated gene-1 (NAG-1)74].

Celecoxib also inhibits the growth of various cancer cellines [63], including hematopoietic cell lines [75] that areOX-2-deficient. Moreover, celecoxib inhibited the growth of COX--deficient colon cancer xenografts in nude mice, providing atrong evidence for its COX-2-independent antitumor effect [63].ecently, a selective COX-2 inhibitor reduced tumor growth andngiogenesis in COX-2-positive pancreatic cancer, whereas thepposite effect was observed in COX-2-negative pancreatic cancer.e. it actually increased angiogenesis and tumor growth [76].

Thus considering the limited efficacy of COX-2 inhibitors in FAPnd the in vitro evidence that COX-2 inhibitors act also beyondOX-2 inhibition suggest that COX-2 inhibition alone is not suffi-ient to arrest carcinogenesis.

1. Future insight

That tNSAIDs and COX-2-specific inhibitors alike modulateargets other than COX-2 is now beyond doubt and should be con-idered when analyzing their effects on cancer prevention. Aboveata make it clear that inhibition of COX-2 alone could not pre-ent cancer and even COX-2-specific inhibitors may require theontribution of their non-COX-2 effects for their chemopreven-ive actions. Combined with the recent withdrawal of one COX-2nhibitor, these data make a strong case for an evaluation of drugevelopment targets beyond COX-2.

Thus there is a strong need to actively pursue the idea that mod-lating targets other than/alongwith COX-2 can prevent cancer. Asancer represents a process rather than an abrupt transition fromormalcy to malignancy, it is crucial to assess the mechanistic rele-ance of each pathway modulated by tNSAIDs or coxibs. Developing

rational approach to cancer prevention would likely require atrategy capitalizing on two facts: firstly, NSAIDs do prevent can-er in humans, and secondly, NSAIDs act on multiple molecularargets, of which COX-2 is but one. Thus it is clear that the study ofhe non-COX-2 targets is likely to point to opportunities for cancerrevention thus far unappreciated. This represents the promise ofmolecular targets beyond COX-2”.

The foregoing analysis provides us three clear conclusions. First,NSAIDs prevent colon and other cancers most likely by modulat-ng several molecular targets besides COX-2. However, their safetyimitations preclude their application to humans for this purpose.

[

Pathology 2 (2012) 67–72

Second, focusing exclusively on COX-2 inhibition may not be aneffective strategy for cancer prevention. Third, exploring targetsbeyond COX-2 is likely to yield a productive approach to cancerprevention. The most pressing need in cancer chemoprevention isto identify agents or combinations of agents that combine high effi-cacy with minimal toxicity. The future approach should rest on thesolid fact that tNSAIDs prevent several cancers in humans and ifthe molecular targets mediating their effect are identified, it willbe feasible to enhance this effect with new agents that will meetthe general criteria of a successful chemopreventive agent eithersingly or in a paired approach.

Disclosure of interest

The authors declare that they have no conflicts of interest con-cerning this article.

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