Current Application of Selective COX-2 Inhibitors in Cancer Prevention and Treatment

ABSTRACT: The multistep process of carcinogenesis, which can take many years, provides many opportunities for intervention to inhibit disease progression. Effective chemoprevention agents may reduce the risk of cancer by inhibiting the initiation stage of carcinoma through induction of apoptosis or DNA repair in cells harboring mutations, or they may act to prevent promotion of tumor growth. Similarly, chemoprevention may entail blocking cancer progression to an invasive phenotype. Over the past decade, in vitro, preclinical, and clinical data have supported the hypothesis that cyclooxygenase (COX)-2 plays a central role in oncogenesis and that treatment with COX-2 inhibitors offers an effective chemoprevention strategy, as exemplified by the activity of celecoxib (Celebrex) in familial adenomatous polyposis. These COX-2 data have contributed to initiation of clinical trials testing COX-2 inhibitors for the chemoprevention of a wide variety of cancers that overexpress COX-2. [ONCOLOGY 16(Suppl 4):37-51, 2002]

Cyclooxygenases (COXs) are enzymes thatcatalyze the rate-limiting step in the conversion of arachidonic acid toprostaglandins (Figure 1).[1-3] Prostaglandins, along with other arachidonicacid products such as thromboxane and 15-hydroxy-eicosatetraenoic acids, belongto the eicosanoid family of fatty acid molecules, which are known to regulatemany physiologic processes including the inflammatory response and other immuneresponse modulators,[4-6] ovulation,[7,8] and mitogenesis.[9,10] Paradoxically,prostaglandins also have been shown to have anti-inflammatory andimmunosuppressive effects. Studies conducted by Gualde and colleaguesdemonstrated that prostaglandins inhibited T-cell proliferation in vitro.[11]Furthermore, prostaglandins can block production of cytokines by Tlymphocytes.[12]

Synthesis of prostaglandins can be regulated at severaldifferent points in the pathway (Figure 1). In the first step, membranephospholipid is converted to arachidonic acid via phospholipase A₂.Subsequently, arachidonic acid is converted to prostaglandin H₂ through atwo-step process that involves COX activity to convert arachidonic acid toprostaglandin G₂, followed by a peroxidase reaction that is also catalyzed byCOX to produce prostaglandin H₂.[13-15]

The COX enzyme family comprises two known isoforms, COX-1 andCOX-2. Cyclooxygenase-1 is a membrane-bound hemoglycoprotein that isconstitutively expressed in the endoplasmic reticulum of cells in most healthytissues and is responsible for local prostaglandin synthesis. In contrast, COX-2is primarily an inducible COX isoform, although low basal expression is apparentin some tissues, including brain and kidney.[16,17] There are a number ofstructural differences between the COX-1 and COX-2 genes, including differencesin the cis elements within the promoter regions and 3´-untranslated domains.

The structure of the COX-2 gene suggests that it is animmediate, early gene product that can be switched on rapidly during theinflammatory response.[18,19] Cyclooxygenase-2 synthesis is inducible by avariety of stimuli, including proinflammatory cytokines such as interleukin-1alpha and -1 beta,[20,21] growth factors such as platelet-derived growthfactor[22,23] and epidermal growth factor,[24,25] and lipopolysaccharide andendothelin.[26,27]

COX-2 Inhibitors and Cancer

Most nonsteroidal anti-inflammatory drugs (NSAIDs) that arecommonly administered to patients inhibit both COX-1 and COX-2. However,inhibition of the inducible isoform, COX-2, is the primary anti-inflammatorymechanism.[5,28,29] Adverse effects associated with long-term use of NSAIDs,including gastritis and gastrointestinal ulceration, in addition to reversibleliver and kidney dysfunction, are thought to be primarily due to inhibition ofthe constitutively expressed COX-1 isoform.[30-32] In recent years, COX-2-specificNSAIDs, including celecoxib (Celebrex) and rofecoxib (Vioxx), have becomeavailable. Selective COX-2 inhibitors are advantageous because they may inhibitpain and the inflammation process in arthritis and oncogenesis. However, they donot inhibit COX-1 enzymes, the products of the "housekeeping genes"required for the maintenance of the gastrointestinal tract and for normal renaland hepatic function.

Rigas and colleagues have demonstrated that colorectal adenomasand adenocarcinomas express elevated levels of prostaglandins.[33] Furthermore,accumulation of prostaglandins is associated with increased expression of COX-2,but not of COX-1.[34] It is also known that prostanoid levels increase duringthe progression from adenoma to adenocarcinoma in patients with familialadenomatous polyposis.[35] In addition, elevated prostanoid expression isassociated with tumor growth, metastatic potential,[36] disease stage,[37]recurrence,[38] and survival[39] in a broad spectrum of tumor types.Furthermore, overexpression of COX-2 in humans has been documented in manycancer types and neoplastic precursor lesions (Table1).[40-81]

These data indicate that selective inhibition of COX-2 may be aneffective strategy for preventing colorectal cancer and also may haveapplication in other cancers. Furthermore, because COX-2 overexpression has beenobserved in both preneoplastic lesions and cancers, chemoprevention interventionis possible at multiple stages of carcinogenesis.

Overexpression of COX-2 may affect a broad range of mechanismsimplicated in the process of carcinogenesis, including angiogenesis, apoptosis,and immune function. Cancer prevention offers more than one opportunity toinhibit disease growth. Effective chemopreventive agents may reduce the risk ofcancer by preventing the initiation stage of carcinoma by inducing apoptosis orDNA repair in cells harboring mutations, or they may act to prevent tumor growthduring the promotion and progression stages of carcinogenesis (Figure2).Ongoing clinical trials evaluating COX-nonspecific and COX-2-specificinhibitors as chemoprevention and therapeutic agents are shown in Table 2[82-84]and are discussed in the following sections.

Colorectal Cancer

Colorectal cancer is a major national health problem. It is thethird leading cause of cancer death in the United States, with 2002 estimates of148,300 new cases and 56,600 deaths.[85] Some (approximately 15%) individualswho develop colorectal carcinoma belong to clinically identifiable high-riskgroups due to familial adenomatous polyposis and hereditary nonpolyposissyndromes.[86] However, the majority of cases of colon carcinoma developsporadically in patients who have no known predisposition for the disease.[87]It is estimated that adherence to the current American Cancer Society andGastrointestinal Society colorectal cancer screening guidelines could lower theannual mortality rate by at least 50% over the next decade.[88]

COX-2: Expression and Preclinical Data

In humans, overexpression of COX-2 has been documented incolorectal adenomas and cancers, but not in normal-appearing mucosa.[89] Forexample, Figure 3 shows immunohistochemical staining for COX-2 in colon adenomatissue. Similar overexpression of COX-2 has been documented in a wide range ofcancers and their precursors (Table 1).[40-81] The chemopreventive effects ofCOX-2 inhibitors on the development of colorectal cancer are the subject ofintense study, and animal models have been useful in investigating colorectalcancer pathogenesis.

A mutation in the adenomatous polyposis coli (APC) gene resultsin spontaneous adenoma formation in the small intestine of APC delta716 knockoutmice. Using this rodent model, Oshima and colleagues demonstrated that there isa cause-effect relationship between COX-2 overexpression and gastrointestinaltumor incidence.[90] It was shown that suppression of one allele of the COX-2gene reduced the number of intestinal polyps by 66%, and suppression of bothalleles resulted in a reduction of 86%.[91] Furthermore, treatment of COX-2-expressingazoxymethane-treated rats with oral celecoxib suppressed formation of colorectaltumors by > 90%, compared with a suppression of 40% to 65% followingadministration of a nonselective COX inhibitor.[91,92]

Reduced Incidence of Colorectal Neoplasia

In prospective cohort studies, long-term administration ofaspirin and other NSAIDs has been associated with a reduction in the incidenceof colorectal adenomas, cancer, and cancer mortality by 40% to 50%.[93-95] Aninverse relationship also has been demonstrated between the use of NSAIDs andthe incidence of colorectal cancer in several case studies.[96,97] Furthermore,clinical trials showed that the administration of sulindac, a commonlyprescribed NSAID, prescribed to familial adenomatous polyposis patients wasassociated with a reduction in the number and size of adenomas.[97-99]

The US Food and Drug Administration recently granted approval ofcelecoxib for treatment of familial adenomatous polyposis. Celecoxib, which wasinitially approved for the relief of signs and symptoms of osteoarthritis andrheumatoid arthritis, is highly selective for COX-2[100] (375-fold greaterselectively compared with COX-1) and has a significantly reduced incidence ofcommon gastrointestinal toxicities, such as bleeding and upper gastrointestinalulcers, associated with NSAIDs.[101]

The pivotal trials of celecoxib for the treatment of familialadenomatous polyposis enrolled 77 patients who were randomized to receive eitherplacebo or celecoxib (100 or 400 mg twice daily) for 6 months.[102] The primaryefficacy end point was the percent change in the number of colorectal adenomas(> 2 mm in size) at 6 months. There was a 4.5% reduction in theplacebo-treated group, a 11.9% reduction in the 100-mg celecoxib-treated group,and a 28.0% reduction in the 400-mg celecoxib-treated group. The decrease inincidence between the 400-mg celecoxib twice-daily group and the placebo groupwas statistically significant (P = .003). The prevalence of adverse events was similar among the treatmentgroups and consisted primarily of diarrhea, dyspepsia, fatigue, upperrespiratory infection, and rash.

The results from the pivotal trial of celecoxib in familialadenomatous polyposis support further investigation of COX-2 inhibitors for anoverall chemoprevention strategy for colorectal tumors in other populations atrisk, including patients with sporadic adenomatous polyps. As shown in Table2,[82-84] there are several recently initiated clinical trials of celecoxib inthe prevention or recurrence of colorectal adenomas. Two are being conductedunder the sponsorship of the Division of Cancer Prevention at the NationalCancer Institute.

One clinical trial is being led by Monica Bertagnolli, MD,Brigham and Women’s Hospital, Dana-Farber Cancer Institute, Boston. This trialis investigating two dose levels of celecoxib compared with placebo with 1- and3-year colonoscopy end points. A second phase III clinical trial (using afactorial design) is studying celecoxib (400 mg/d) vs selenium (in the form ofbaker’s yeast) vs the combination of celecoxib/selenium vs a double placebo.This study is being conducted at the Arizona Cancer Center, Tucson, by Drs.David Alberts and Peter Lance.

An additional randomized, phase III trial is evaluating thepotential for celecoxib to reduce the incidence of sporadic adenomas. Adenomarecurrence rates will be evaluated at a 3-year colonoscopy end point. Theresults of these trials will not be available for several years, but couldestablish COX-2 inhibitors as important components of the management strategyfor colorectal adenomas and the prevention of colon cancer.

Nonmelanoma Skin Cancer

It is well recognized that chronic sun exposure is a majoretiologic agent for skin cancer, contributing to over 1 million new cases ofbasal cell carcinoma and squamous cell carcinoma each year in the United States.Although the majority of skin cancers are basal cell carcinomas, which arerelatively benign, squamous cell carcinomas account for approximately 2,000deaths annually.[103] Ultraviolet (UV)-B exposure is responsible for mostsquamous cell skin cancers.[104-106] It is considered to be the principalcarcinogen in skin cancers and is involved with all stages of carcinogenesis(initiation, promotion, and progression). Ultraviolet-A is also capable ofcausing oxidative stress and is also associated with UV-induced carcinogenesis,particularly melanoma.[106-108] Actinic keratosis is considered a precursorlesion of squamous cell carcinoma, with approximately 60% of squamous cellcarcinomas evolving from actinic keratosis.[109,110]

COX-2: In Vitro and Preclinical Data

Several mechanisms for UV-induced skin carcinogenesis have beendefined, including dysregulation of cell signal transduction pathways[111-113]and upregulation of COX-2 expression.[114,115] In the normal epidermis, thebalance between proliferation of cells in the basal layer, cell differentiationin the suprabasal spinous and granular layers, and apoptosis at the transitionalzone (where the stratum granulosum and stratum corneum meet) is tightlyregulated. Cells lose their proliferative capacity as they undergo terminaldifferentiation and leave the basal cell layer.

When homeostasis is maintained in the epidermis, COX-1 isconstitutively expressed by keratinocytes and COX-2 is abnormallyexpressed.[116] Studies conducted by Athar and colleagues, however, havedemonstrated that COX-2 is expressed in the epidermis in response to tumorpromoting agents,[117] and other colleagues demonstrated that the differentialexpression of COX-2 in mouse skin carcinoma is regulated by cis elements withinthe promoter region of the COX-2 gene.[117,118] Neufang and colleagues showedthat, in a transgenic mouse model, induced expression of COX-2 in the epidermisresulted in abnormal epidermal differentiation.[119] Furthermore, proliferationof epidermal cells was increased and, in some regions of the epidermis, therewas an increase in the number of viable cornified layers.

Prostaglandins are actively synthesized by the human epidermis:COX-2-mediated overexpression of prostaglandin E₂ has been shown to increaseepidermal cell proliferation in vitro.[114] Prostaglandins generated by thearachidonic acid cascade have been implicated in various models of skintumorigenesis. Elevated levels of protaglandin E₂ have been observed in basalcell carcinoma, squamous cell carcinoma, and actinic keratosis. Data suggestthat prostaglandin E₂ overexpression may correlate with the propensity formetastatic and invasive behavior.[120]

In the context of skin tumorigenesis, chemical carcinogenesismodels have implicated COX-2-regulated prostaglandin expression in mediatingtumor promotion.[121,122] Upregulation of COX-2-induced prostaglandinproduction causes an increase in cell growth and, as shown by Tsujii and DuBois,skin carcinoma cells overexpressing COX-2 are resistant to apoptosis.[60,123]

Cyclooxygenase-2 overexpression has also been demonstrated inUV-exposed human skin cells. Buckman and colleagues showed that exposure ofhuman keratinocytes in vitro to UV-B caused a significant increase in expressionof prostaglandin E₂.[124] In cultured human keratinocytes, UV-B-induced COX-2activity was blocked completely by indomethacin, a nonspecific COX inhibitor,and by SC58125, a COX-2-specific inhibitor.[125]

Clinical Data and Clinical Trials

Kagoura and colleagues used immunohistochemistry to investigateCOX-2 expression in basal cell carcinoma, Bowen’s disease, squamous cellcarcinoma, and metastatic tumors of the skin.[126] Four of 16 basal cellcarcinoma cases tested positive for COX-2. In Bowen’s disease, the stainingintensity for COX-2 was greater than that observed in basal cell carcinoma.

In addition, 11 of 15 squamous cell carcinoma patients testedpositive for COX-2. The pattern of staining was heterogeneous, with more intensestaining in the centers of the tumor loci. In metastatic tumors, the percentageof COX-2-positive tumor cells and the intensity of staining was low comparedwith Bowen’s disease and basal cell carcinoma, suggesting that COX-2 may playa greater role in the early stages of squamous cell carcinogenesis.

Currently, there are several ongoing clinical trials examiningthe effect of COX-2 inhibition on skin cancer (Table2).[82-84] At theUniversity of California, San Francisco, a study is being conducted to examinewhether celecoxib prevents the development of basal cell carcinoma in patientswith basal cell nevus syndrome. Celecoxib is also being tested in severalongoing, placebo-controlled clinical trials for preventing the development ofnew actinic keratosis lesions.

A phase IIB, double-blind, placebo-controlled trial is ongoingat the University of Alabama with sponsorship from the National CancerInstitute. The primary end point of this study will be inducing actinickeratosis regression. Secondary end points include the effect of celecoxib onpotential surrogate end point biomarkers in areas of actinic keratosis,sun-exposed skin, and non-sun-exposed skin, and the correlation of thesebiomarkers with clinical outcome.

Reports examining COX-2 and skin carcinogenesis illustrate theneed for evaluation of the efficacy of COX-2-selective inhibitors aschemoprevention agents to inhibit the course of UV-induced skin cancer. Futureresearch should combine in vitro techniques, and available transgenic knockoutor overexpression mouse models, in addition to human studies, to more clearlydefine the role of COX-2 in UV-B- and UV-A-induced carcinogenesis of theskin.

Lung Cancer

Lung cancer is the leading cause of cancer deaths in the UnitedStates. Furthermore, lung cancer has a high propensity to spread by hematologicand lymphatic routes. More than 86% of cases occur in current or formersmokers.[127,128] Approximately 75% of lung cancers are classified as non-small-celllung carcinoma. The disease is usually diagnosed at an advanced stage when it istherapeutically intractable.[129,130] Clearly, understanding the biology of thedisease and discovering specific molecular targets to facilitate early detectionand treatment would be valuable.

COX-2: In Vitro and Preclinical Data

Over 30% of non-small-cell lung cancer tumors and cell linesharbor an activating mutation in the K-ras oncogene,[131] which appearsassociated with expression of COX-2 in a panel of non-small-cell lung cancercell lines.[132] Several reports also have shown that NSAIDs block thetransformed growth of non-small-cell lung cancers that express the K-rasmutation.[133-135] It has been hypothesized that the generation of bioactivelipids derived from the arachidonic acid metabolic pathway modulates physiologicand pathologic responses involved in tumor growth and tumor progression in thelung. This theory has been based on data indicating that COX inhibitors inhibitthe growth of human lung cancer cell lines in vitro and in animalmodels,[136,137] and abnormalities in arachidonic acid metabolism are present innon-small-cell lung cancer.[138]

Masferrer and colleagues studied the effect of celecoxib in theLewis lung model.[139] Mice were injected with cancer cells into the foot pad,then fed either a control diet or a diet supplemented with celecoxib. Over aperiod of 30 days, celecoxib caused a dose-dependent decrease in tumor volumeand, at higher doses, celecoxib induced a decrease in the size and number oflung metastases. It was hypothesized that the decrease in tumor size andmetastatic invasion was due to celecoxib-induced inhibition of angiogenicactivity.[140]

Clinical Data and Clinical Trials

Epidemiologic studies have shown that NSAIDs, including aspirin,significantly reduce the risk of lung cancer.[41,42,141] Furthermore, COX-2expression appears elevated in non-small-cell lung cancer,[131,132] andincreased COX-2 expression is associated with poor prognosis.[43] Using in situhybridization with a COX-2 antisense probe, Khuri and colleagues demonstratedthat COX-2 expression in stage I disease was associated with decreased survivalrates.[61]

Cyclooxygenase-2 is diffusely overexpressed in atypicaladenomatous hyperplasia, a possible precursor lesion of adenocarcinoma of thelung.[42] Lau and colleagues demonstrated COX-2 overexpression in 19 of 20 lungcancer specimens using immunohistochemical analyses.[138] In another study,COX-2 overexpression was observed in both squamous cell carcinoma andadenocarcinomas of the lung.[40] Thus, COX-2 may provide a molecular target fortherapy and prevention of lung cancer in smokers and nonsmokers. A pilot studyof COX-2 inhibitors in high-risk tobacco smokers is ongoing at the JonssonCancer Center, University of California, Los Angeles. Other studies evaluatingthe role of celecoxib in heavy smokers are ongoing. In addition, celecoxib isbeing studied in neoadjuvant and therapeutic trials in combination withchemotherapy.

Prostate Cancer

Prostate carcinoma is the most commonly diagnosed cancer and thesecond-leading cause of cancer deaths in men in the United States. It isexpected that approximately 189,000 new cases will be diagnosed and more than30,200 deaths due to prostate cancer will occur in 2002.[85] Treatment foradvanced prostate cancer often involves androgen ablation therapy and eithersurgical removal of the prostate, external beam radiation, or implantation ofradioactive "seeds" into the prostate (brachytherapy). Unfortunately,as prostate carcinoma progresses, it tends to become androgen independent and,therefore, refractory to hormone therapy.[142,143] Patients diagnosed inadvanced stages of the disease have a poor prognosis.[144-146] Agents capable ofinhibiting cell growth and sensitizing prostate carcinoma cells to stimuli thatinduce apoptosis, such as radiation therapy, would enhance the efficacy oftreatment.

COX-2: In Vitro and Preclinical Data

Accumulating evidence suggests that overexpression of COX-2 isassociated with resistance to apoptosis, thereby increasing the tumorigenicpotential of a cancer.[123] Furthermore, selective inhibition of COX-2 has beendemonstrated to induce apoptosis in prostate carcinoma cells in vitro.[76,147]These observations suggest that COX-2 inhibitors could be effectivechemoprevention agents.

Lim and colleagues have examined the potential for COXinhibitors to be used to induce apoptosis in prostate cancer.[77] The COX-1 and-2 inhibitor sulindac was tested in vitro for proapoptotic activity in theprostate cancer cell lines PC3 and LNCaP, and in a normal prostate epithelialcell line PrEC. Apoptosis was quantified following treatment with eithersulindac or sulindac sulfone (exisulind, Aptosyn). Forty-eight hours followingtreatment with either sulindac or exisulind, 50% of PC3 cells and 40% of LNCaPcells underwent apoptosis. However, PrEC cells showed no indication of apoptosisat similar concentrations of drug.[77]

Studies evaluating the effect of COX-2-specific inhibitors onangiogenesis in prostate carcinoma cell lines have been performed. Cell linesLNCaP and PC3, and the control cell line PrEC, were treated with two COX-2-specificinhibitors, etodolac (Lodine) and NS398.[78] Both compounds decreased cellproliferation in the carcinoma cell lines, but not in the normal prostatestromal cell line. A DNA fragmentation assay revealed that both compounds alsoinduced apoptosis in the two carcinoma cell lines, but not in the normal stromalcell line.

Clinical Data and Clinical Trials

Using immunohistochemical analyses, Uotila and colleaguesexamined expression of COX-1 and -2 in prostate carcinoma tumors.[148] Nosignificant difference in COX-1 expression was observed between normal prostateand prostate cancer tissue. However, their data revealed stronger staining ofCOX-2 in the prostate cancer cells compared with normal glandular epithelium ofcontrol prostates. Cyclooxygenase-2 expression was also elevated in theprecursor lesion of prostate carcinoma, prostate intraepithelial neoplasia.[148]

Using similar techniques to quantify COX-2 expression,Kirschenbaum et al showed COX-2 overexpression in 86% of prostateintraepithelial neoplasia lesions and 87% of carcinomas collected duringprostatectomy.[79,149] Furthermore, treatment of prostate carcinoma cells with aselective COX-2 inhibitor induced apoptosis in vivo and in vitro. The in vivoresults revealed that the COX-2 inhibitor decreased microvessel density andangiogenesis. The investigators hypothesized that the decrease in angiogenesiswas caused by inhibition of COX-2-induced expression of vascular endothelialgrowth factor.[79,149]

As a result of the preclinical experimental results showingactivity for exisulind against prostate cancer, this phosphodiesterase/COX-1 and-2 inhibitor currently is being evaluated in a series of phase I/II clinicaltrials. These trials are examining prostate-specific antigen response andmeasurable disease response rate with exisulind as a single agent or incombination with docetaxel (Taxotere) (Table2).[82-84] Secondary objectivesinclude time to disease progression and duration of response in patients withprostate carcinoma.

Celecoxib is also undergoing clinical development forprevention/treatment of prostate cancer (Table2).[82-84] In one phase I/IIstudy, patients will be randomized to receive either celecoxib or placebo priorto radical prostatectomy.[82] The effect of celecoxib on COX-2 expression andangiogenic factors in the prostate will be examined.

These trials of COX-2 inhibitors will help determine theirfuture role in treatment and chemoprevention of prostate cancer. Clearly, themechanisms by which these drugs exert a proapoptotic effect in prostatecarcinoma cells warrant further investigation.

Breast Cancer

The incidence of breast carcinoma varies with age andnationality. In the United States, the incidence increases rapidly to about age45 years, then increases more slowly. At age 25 years, the incidence isapproximately 5 cases in 100,000 women; at age 50 years, the incidence increasesto 150 cases per 100,000 women. At age 75 years, the incidence rises to 200 per100,000 women.[150,151] The development of breast cancer appears to be relatedto ovarian function and hormone production. However, a young age at firstpregnancy, late menarche, and late menopause are factors with a protectiveeffect against breast cancer.[152-154] Furthermore, oophorectomy before age 35years reduces the risk of developing breast cancer by 70%.[155,156]

Therapy usually consists of a combination of surgery, radiation,and chemotherapy. Clearly, molecular target-specific chemoprevention agents withfew or no adverse effects would be valuable to reduce morbidity and mortalityrates from breast cancer.

COX-2: In Vitro and Preclinical Background

Prostaglandins have been implicated in breast carcinogenesis.Breast carcinoma cell lines[53,54] and rodent models[157] express elevatedlevels of COX-2 and prostaglandins compared with the normal tissues from whichthey arise. Inhibition of COX by NSAIDs reduces the development of chemicallyinduced and transplantable mammary cancers.[55,158,159] It is also interestingthat addition of hormones to human breast cancer cell lines[160] or rat mammarycarcinoma cell lines[161] induces expression of prostaglandins.

Studies conducted by Rozic and colleagues examined the role ofprostaglandins in the proliferation, survival, migratory, and invasive behavior,and angiogenic capacity in a highly invasive murine mammary tumor cell line.[55]Northern and Western blot analyses revealed a high expression level of COX-2mRNA and protein, respectively. Their results indicated a high level ofprostaglandin E₂ expression, which was completely abrogated by COX-2-specificinhibitors.

Migratory and invasive behavior was measured with an in vitrotranswell migration/invasion assay. Cyclooxygenase-2-specific antagonistsinhibited migration, while a COX-1-specific inhibitor had no effect ontranswell migration. The COX-2-specific inhibitors also blocked angiogenesisin an in vivo assay. These studies suggest that COX-2-specific inhibitors maybe effective for preventing breast tumor development and/or progression.[55]

Other studies also have shown that COX inhibitors blockformation of breast tumors in mouse models.[55,162,163] Some studies suggestthat COX-2 inhibitors may not only prevent mammary carcinogenesis, but may alsoprevent multidrug resistance in breast carcinoma.[164] The molecular mechanismsof prostaglandin-mediated progression of breast cancer are not characterized;however, one mechanism may involve the prostaglandin E₂ receptor. Fulton andcolleagues demonstrated that advanced and metastatic breast carcinomas oftenexpress a mutation in the prostaglandin E₂ receptor. This mutation may also beinvolved in multidrug resistance.[165]

The potential of celecoxib to prevent mammary cancer was studiedin the dimethylbenzanthracene (DMBA)-induced rat mammary tumor model by Harrisand associates.[166] In this study, animals were fed a control diet or a dietsupplemented with either ibuprofen (a COX-1 and -2 inhibitor) or celecoxib for 1week prior to a single dose of DMBA. Both ibuprofen and celecoxib groups showeda statistically significant reduction in tumor incidence. However, the reductionin tumor incidence observed in the celecoxib group was greater than thatobserved in the ibuprofen-treated group. Furthermore, Alshafie and colleaguesdemonstrated that celecoxib also had a therapeutic effect in the DMBA-inducedrat mammary model.[167]

Clinical Data and Clinical Trials

Hwang and colleagues demonstrated that COX-2 is overexpressed inbreast hyperplasia and atypical hyperplasias.[56] Similarly, these investigatorsshowed that COX-2 is overexpressed in > 56% of breast cancers. Interestingly,COX-2 overexpression was more apparent in ductal carcinomas in situ than inmetastatic breast cancers, suggesting that COX-2 may play an important role inthe early carcinogenesis of the breast.[40] The COX-2-specific inhibitors maybe ideal candidates for chemoprevention of breast cancer for several reasons.The toxicity profile appears to be acceptable for premenopausal women becauseCOX-2 inhibitors do not cause perimenopausal symptoms. However, long-termeffects of the use of celecoxib in these subjects is being evaluated in clinicalstudies. They may be effective in both estrogen-receptor-positive andestrogen-receptor-negative disease.

Several clinical trials with celecoxib are at different pointsof development. One such trial has been initiated examining efficacy ofcelecoxib and trastuzumab (Herceptin) in women with HER2/neu-overexpressingmetastatic breast cancer that is refractory to trastuzumab (Table2).[82-84]Multiple trials are ongoing to evaluate the effect of celecoxib in precancerous,in situ, and invasive breast carcinoma.

Gastric Cancer

The incidence of gastric cancer varies remarkably in differentparts of the world. The incidence is relatively high in Japan and Chile, withapproximately 58.4 cases per 100,000 men and 29.9 cases per 100,000 women.[168]The incidence of gastric cancer is lowest in the Dominican Republic andThailand, where the rate is approximately 5% of that reported in Japan.[168,169]The high rate of gastric carcinoma in Japan is thought to be caused in part bythe high consumption of cured meat and fish. These products contain a highconcentration of N-nitroso compounds that cause formation of potentiallycarcinogenic endogenous nitrosamines.[170,171] In the United States, 21,600cases of gastric cancer are expected in 2002.[85] However, the incidence in theUnited States and Japan has dropped in recent years, perhaps due to an increasedawareness of proper dietary habits.[172,173]

COX-2: Clinical Data

Some studies suggest that a Helicobacter pylori infectionpredisposes a patient to gastric cancer, perhaps owing to increased expressionof COX-2.[174-176] Leung and colleagues examined the association betweenexpression of COX-2 and the presence of a missense mutation in the tumorsuppressor gene p53.[177] Wild-type p53 normally binds to cis elements withinthe promoter region of the COX-2 gene and inhibits its transcription. Of 39patients with gastric cancer, 19 (49%) overexpressed COX-2.[66] Of these, thepatients with the strongest COX-2 expression also had a mutated p53 gene.

In addition, the cancer in these patients with higher COX-2expression and mutated p53 gene was more aggressive, with more lymphaticinvasion and metastases. These data suggest that mutation of the p53 gene mayhave a direct effect on expression of COX-2 in gastric carcinoma, and thatincreased COX-2 expression may be associated with poor prognosis.[66,177]

Other studies revealed a direct relationship between expressionof COX-2 mRNA and increased tumor invasion. Thus, COX-2 inhibitors may provideeffective prevention in patients with H pylori infection and in patients withthis risk factor for gastric cancer. Clinical trials should be developed to testthese agents in this patient population.

Bladder Cancer

Bladder cancer is the fourth leading cause of cancer in men andthe eighth in women. Furthermore, it is the ninth and fourteenth leading causeof US cancer deaths in men and women, respectively.[178] Many etiologic factorshave been identified including occupation (eg, close contact with chemicalcarcinogens),[179,180] diet,[181,182] chronic bladder infections,[183,184] andsmoking.[185,186]

The vast majority of bladder cancers arise from bladderpapilloma precursors, providing significant opportunities for the development ofchemoprevention strategies.

COX-2: In Vitro and Preclinical Background

Recent animal studies indicate that nonspecific COX inhibitorsand specific COX-2 inhibitors reduce the incidence of bladder carcinoma inducedby chemical carcinogens.[187,188] Khan and colleagues examined COX-1 and -2expression in normal dogs and in dogs with transitional cell carcinoma.[49]There was no difference in COX-1 expression between normal and malignant bladdertissue; however, COX-2 was only expressed in carcinoma and in new blood vesselsin the tumor tissue.

Using two rodent models of bladder cancer, Grubbs and colleaguesdemonstrated that the COX-2-specific inhibitor celecoxib was effective ininhibiting chemically induced bladder cancer.[189] Male mice treated withN-butyl-N-(4-hydroxy-butyl)nitrosamine (OH-BBN) compared with no treatmentdeveloped transitional and squamous cell urinary bladder cancer with highmorbidity. Mice pretreated with celecoxib 7 days before initiation of 12 weeklydoses of OH-BBN had a 75% decrease in the incidence of bladder cancer. There wasno decrease in development of preneoplastic lesions, suggesting that COX-2 mayplay a role during the progression stages of bladder carcinoma.

Similar results were observed in a rat model using the samechemical initiator and time course.[189] These data suggest that COX-2-specificinhibitors may not prevent initiation of carcinogenesis in the urinary bladder.However, they may be effective in preventing bladder carcinoma in individualsidentified as high risk and for treatment of early-stage bladder cancer.

Clinical Data and Clinical Trials

In humans, several studies have shown that COX-2 is expressed ininvasive transitional cell carcinoma, but not in normal bladder tissue.Expression of COX-2 is localized to the carcinoma cells, whereas there appearsto be no COX-2 expression in the stroma.[50-52] It is also interesting that instudies examining the molecular characteristics of bladder carcinoma in patientsin close proximity to Chernobyl following the nuclear accident in 1986,Romanenko and colleagues showed that overexpression of COX-2 was associated withmutations in the p53 gene.[190] These findings are similar to data indicating arelationship between mutant p53 and COX-2 expression in gastric cancer.[177]

Currently, a large clinical study examining inhibition of COX-2and recurrence of bladder cancer is ongoing at The University of Texas M. D.Anderson Cancer Center, Houston (Table 2).[82-84] This study is designed tocompare the time to recurrence following treatment with celecoxib or placebo inpatients with superficial transitional cell carcinoma of the bladder at highrisk for recurrence. This study will also correlate the modulation of one ormore biomarkers with recurrence of bladder cancer and evaluate the quality oflife of patients enrolled on each of the two regimens.

Esophageal Cancer

During the last decade, the incidence of esophagealadenocarcinoma has increased at a disproportionately rapid rate.[191] Themajority of esophageal cancers arise in patients with the premalignant conditionBarrett’s esophagus, which manifests by the replacement of normal esophagealepithelium with a columnar type.[192-195] Barrett’s esophagus is frequentlyassociated with high-grade dysplasia and aneuploidy in esophagealepithelium.[196-199] The condition develops from chronic, severegastroesophageal reflux. Patients afflicted with Barrett’s esophagus have a30- to 40-fold increased risk for development of esophagealadenocarcinoma.[200,201] Postoperative morbidity is high for esophageal cancer,and survival is not favorable (13% to 30% at 5 years).[202-205] Clearly,chemoprevention strategies are needed.

COX-2: In Vitro and Preclinical Background

Research conducted by Li and colleagues demonstrated thataspirin retarded cell growth in an esophageal adenocarcinoma cell line.[206]Growth inhibition was shown to be dose and time dependent.[63] Subsequently,they tested the effect of the COX-2-specific inhibitor NS398 on apoptosis andexpression of genes that regulate apoptosis. In vitro, the COX-2-specificinhibitor induced apoptosis in several esophageal adenocarcinoma cell linesthrough a cytochrome C-dependent pathway. There was a direct relationshipbetween apoptosis and the level of COX-2 expression. Caspase-9 and caspase-3were activated by NS398, and addition of a caspase inhibitor reversed theapoptotic effect of the COX-2 inhibitor.[206] These data suggest that COX-2specific inhibitors may be promising for chemoprevention and treatment ofesophageal adenocarcinoma.

Clinical Data

Kandil and colleagues examined esophageal pinch biopsy specimensfrom patients with Barrett’s esophagus from normal and abnormal tissue.[207]Western analyses for COX-2 protein showed an increase in COX-2 expression in 41%of the Barrett’s esophagus tissue biopsies. No COX-2 expression was observedin adjacent normal tissue. It is of interest that COX-2 was found in Barrett’sesophagus tissue with or without dysplasia, suggesting that COX-2 may play arole in the early stages of development of adenocarcinoma.[45] Additionally,COX-2 is overexpressed in adenocarcinoma arising from Barrett’s esophagus atall stages.[46,208,209]

Currently, a clinical study is ongoing coordinated by the JohnsHopkins Comprehensive Cancer Center, Baltimore, to evaluate the efficacy andsafety of celecoxib in patients with Barrett’s esophagus (Table

Pancreatic Cancer

Pancreatic cancer is a highly invasive, aggressive disease andis the fifth leading cause of cancer deaths in the United States.[210] Althoughthe DNA synthesis inhibitor gemcitabine (Gemzar) can have a modest effect onsurvival, the majority of patients succumb to their disease within 6 monthsfollowing diagnosis.[211,212]

COX-2: In Vitro and Preclinical Data

Studies were conducted to measure the effects of COX-nonspecificand COX-2-specific inhibitors on cell growth and apoptosis in four pancreaticcancer cell lines expressing COX-2.[213] Cell growth, measured by[³H]-thymidineincorporation, showed a dose-dependent decrease in cell proliferation with boththe nonspecific COX inhibitors and the COX-2-specific inhibitor NS298.

Yip-Schneider and colleagues examined the effects of COXinhibitors in combination with gemcitabine in vitro.[74,214] The COX-nonspecificand COX-2-specific inhibitors caused cell cycle arrest primarily in the G1/G0phase through decreased expression of cyclins, whereas gemcitabine caused arrestin the S phase due to its incorporation into DNA. It is of great interest thatCOX inhibitors in combination with gemcitabine had an additive effect oninhibition of cell growth. No significant effect on apoptosis was observed inany of the test groups, suggesting that the drugs alone and in combination mayinduce cell senescence.[74,214]

Clinical Data

Tucker and colleagues examined expression of COX-2 mRNA intissue isolated from pancreatic cancer patients using quantitative reversetranscriptase polymerase chain reaction.[75] These studies revealed a 60-foldincrease in expression of COX-2 mRNA in tissue isolated from 9 of 10 pancreaticadenocarcinomas compared with adjacent normal pancreatic tissue.Immunohistochemical staining indicated that COX-2 expression was localized tothe malignant epithelium. It was also demonstrated that COX-2 was expressed inhuman pancreatic carcinoma cell lines.[75]

Clearly, the effectiveness of COX inhibitors in combination withgemcitabine may have potential for treatment of pancreatic cancer, but continuedclinical development is required. Several studies evaluating the effect ofcelecoxib in combination with chemotherapy for pancreatic cancer have beeninitiated, including a phase II clinical trial of celecoxib and gemcitabine atthe Arizona Cancer Center.

Conclusions

Cyclooxygenase-2 is known to be a mediator of inflammation andother immune processes. It is therefore of interest that several of themalignancies in which COX-2 is overexpressed are associated with a chronicinflammatory condition. For example, esophageal carcinoma is associated withBarrett’s esophagus, and nonmelanoma skin cancers are associated with UVdamage. In addition, gastric carcinoma is associated with an ulcerativecondition caused by infection with H pylori. The data suggest that inhibition ofCOX-2 may inhibit carcinogenesis at a very early stage and may completely blocktumor formation in tumors that arise from nonmalignant inflammatory precursors.As discussed in this review, preclinical and clinical data support thehypothesis that COX-2 plays a role in oncogenesis, and that COX-2 inhibitors mayoffer effective chemoprevention strategies (summarized in Figure4).

Epidemiologic studies revealing an inverse correlation betweenthe incidence of colon cancer and regular use of NSAIDs provided initial cluessuggesting that COX inhibition may be an effective intervention approach topreventing cancer.[215-217] Because NSAIDs are known to block prostaglandinsynthesis by inhibition of the COX enzymes, it was hypothesized that aberrantprostaglandin synthesis may contribute to colorectal neoplasia. Furtherinvestigations documented that COX-2 is barely detectable in normal colonmucosa, but is significantly upregulated in colon carcinoma.[34,47]Cyclooxygenase-2 also is overexpressed in intestinal adenomas in rodent modelsof intestinal tumorigenesis and in a high percentage of colorectaladenomas.[218,219]

More recently, in familial adenomatous polyposis patients, theCOX-2-specific inhibitor celecoxib caused a 28% reduction in colorectaladenomas after 6 months of treatment at a dose of 400 mg twice-daily, whereascontrols given placebo experienced no significant reductions in adenomanumbers.[102] This finding has established celecoxib as a potentially valuablechemoprevention agent in patients at high risk of sporadic colorectal adenomarecurrence.[48,220-222]

Currently, there are three phase III trials under way to testdifferent doses of celecoxib in the prevention of sporadic colorectal polyprecurrence. Clearly, further examination of COX-2 inhibitors in prevention andtreatment of cancer is warranted. Because of the favorable safety profile inhealthy individuals, COX-2 inhibitors may provide an effective and safe optionfor cancer chemoprevention.

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