Annals o f Clinical & Laboratory Science, vol. 30, no. 1, 2000 3

Molecular Pathology of Cyclooxygenase-2 in Neoplasia

Egil Fosslien

Department of Pathology, University of Illinois College of Medicine, Chicago, Illinois

Abstract. Cyclooxygenase (COX)-2 levels are elevated in several types of human cancer tissues. Nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit both the COX-1 and COX-2 protein, the two enzymes that convert arachidonic acids to prostaglandins. Regular use of such NSAIDs significantly reduces the risk and spread of some cancers. The objective of this study was to elucidate the molecular pathology of neoplasms that overexpress COX-2. Epidemiological data and clinical studies were analyzed and compared with results of studies of human tumor tissues, animal models, and cultured tumor cells. COX-2, but not COX-1, is highly expressed in human colon carcinoma, squamous cell carcinoma of the esophagus, and skin cancer. COX-2 is inducible by oncogenes ras and scr, interleukin-1, hypoxia, benzo[a]pyrene, ultraviolet light, epidermal growth factor, transforming growth factor beta, and tumor necrosis factor alpha. Dexamethasone, antioxidants, and tumor-suppressor protein p53 suppress COX-2 expression. COX-2 synthesizes prostaglandin E2 (PGE2) which stimulates bcl-2 and inhibits apoptosis, and induces interleukin-6 (IL-6) which enhances haptoglobin synthesis. PGE2 is associated with tumor metastases, IL-6 with cancer cell invasion, and haptoglobin with implantation and angiogenesis. Drastic reduction in polyp number results from COX-2 gene knockout as well as from selective COX-2 inhibition in a mouse model of human familial adenomatous polyposis. Nonselective NSAIDs, for instance aspirin, and selective COX-2 inhibitors such as celecoxib (SC-58635) and NS-398 suppress azoxymethane-induced colon carcinogenesis in rats. Aspirin, indomethacin, and ibuprofen decrease cultured lung cancer cell proliferation. Selective inhibition of COX-2 is preferable to nonselective inhibition. It reduces cancer cell proliferation, induces cancer cell apoptosis, and spares COX-1—induced cytoprotection of the gastrointestinal tract.

Keywords: cyclooxygenase-2, carcinogenesis, cancer invasion, metastasis, apoptosis, antioxidants, angiogenesis

Introduction

Chemoprevention is the use of pharmacological or natural agents to prevent, suppress, interrupt, or reverse the process of carcinogenesis [1]. Results of epidemiological studies argue that nonsteroidal anti­inflammatory drugs (NSAIDs) may be used for chemoprevention [2]. Many, but not all studies show that long-term use of aspirin and other non-selective NSAIDs reduces the risk o f cancer (Fig. 1). Nonselective NSAIDs inhibit cyclooxygenases (COX-1 and COX-2), the two enzymes that convert

Address correspondence to Egil Fosslien, M.D., Department of Pathology, University of Illinois College of Medicine, Chicago, IL 60612. Tel: 312 996 7323; Fax: 312 996 7586; E-mail: efosslie@uic.edu

arachidonic acids to prostaglandins [3]. COX-2 is highly expressed in a number of human neoplastic proliferations where it stimulates tumor cell division and angiogenesis and inhibits programmed cell death (apoptosis).

Significant anticancer effects of NSAIDs derive from their inhibition of COX-2. For instance, NSAIDs help restore apoptosis and reduce tumor mitogenesis and angiogenesis. COX-2 is highly inducible by proinflam m atory cytokines. Proinflammatory prostaglandins produced by COX-2 play a pivotal role in inflammation [4]. Through inhibition o f COX-2, nonselective NSAIDs are effective analgesic and antiphlogistic agents. In contrast, COX-1 is constitutively expressed. It synthesizes cytoprotective prostaglandins in the gastrointestinal tract. Inhibition of COX-1 can

0091-7370/00/0100-003 $4.50; © 2000 by the Association o f Clinical Scientists, Inc,

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therefore lead to serious gastrointestinal ulceration and bleeding [4], Newer NSAIDs that selectively inhibit COX-2 have recently passed FDA approval for the treatment of rheumatoid arthritis. They are presently being intensely investigated for their potential as anticancer drugs.

Selective inhibition of COX-2 appears more desirable than nonselective inhibition in suppressing proliferation and in blocking the inhibition of apoptosis of cancer cell lines that express COX-2. However, is COX-2 inhibition the best logical approach to the treatment of cancers that overexpress COX-2? Is COX-2 overexpression in the tumors the principal carcinogenic event or is it rather secondary to some other alteration? How do some selective COX- 2 inhibitors suppress in vitro proliferation of cancer cell lines that do not express COX-2 (Fig. 2)? This paper discusses the pathophysiology of COX-2

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Fig. 1. Epidemiology: Several, but not all, studies show that regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer. Illustrated here are the results of studies on the effect of NSAIDs on the risk of cancer of the ovary, stomach, esophagus, breast, and colorectum. Only data from references listed on the diagram are illustrated. For results of other studies, see text.

overexpression in neoplasia in order to answer these questions.

Epidemiological and clinical studies

There is extensive evidence that NSAIDs can lower the risk of developing cancer and inhibit carcinogenesis (Fig. 1). As an example, ingestion of NSAIDs in 341 women with invasive carcinoma of the breast was inversely associated with the size of the primary tumor and the status and number of involved axillary lymph nodes [5]. Regular NSAID use can significantly reduce the risk of developing breast cancer. A case-control study of 511 breast cancer patients and 1,534 population control subjects produced odds ratios of0.69 and 0.57 for the NSAIDs aspirin and ibuprofen respectively [6]. The highest level of use was daily intake of aspirin over 5 years and was associated with the greatest risk reduction. A 5-year prospective cohort study of the association between NSAIDs and breast cancer in 32,505 women in Ohio revealed an incidence o f323 breast cancers among non-users and 183 breast cancers among heavy ibuprofen or heavy aspirin users [7]. Regular use of ibuprofen or aspirin decreased breast cancer rates by about 50% and 40% respectively.

Similarly, another population-based case-control study showed that aspirin and other NSAIDs significantly reduce the risk of adenocarcinoma and squamous cell carcinoma of the esophagus and gastric noncardia adenocarcinoma [8]. However, use of NSAIDs did not alter the risk of gastric cardia adenocarcinoma. Aspirin [9] and other NSAIDs [10] can significantly prevent the development of colon cancer. Next to lung cancer, this malignancy is the most common cause of cancer death in the United States [11], accounting for approximately 57,000 deaths per year [1]. Regular use of non-aspirin NSAIDs by 104,217 members enrolled in the Tennessee Medicaid Program significantly reduced the risk of colon cancer [10]. All members of the program were over 65 years old and had been enrolled for at least 5 years. Members who used non-aspirin NSAIDs regularly for at least 48 months had a relative risk of0.49 for colon cancer compared with members who did not use NSAIDs. Surprisingly, low doses of NSAIDs appeared to be at least as effective as high doses. Furthermore, no specific NSAID provided a

ESOPHAGUS

CO LO N & RECTUM

Cyclooxygenase-2 in neoplasia 5

particular protective effect. Patients with rheumatoid arthritis treated with NSAIDs for several years have a low rate of colon cancer [12]. Follow-up for up to 35 years of 862 rheumatoid arthritis patients who used non-aspirin NSAIDs for at least 48 months detected colorectal cancer in only half the expected number.

However, interpretation of epidemiological studies is complex [13]. There may be inherent biases in observational studies, such as lifestyle factors, and sparse information about dose and duration of NSAID use [14]. For example, many NSAIDs are available as over-the-counter analgesics and estimation of their use from questionnaires is imprecise. Bleeding induced by aspirin may lead to earlier diagnosis and treatment than usual. And while several epidemiological studies of NSAID users have shown a significant reduction in colorectal cancer risk, two studies revealed a slight augmentation in risk. One of the studies, the United States prospective Physicians Health Study, involved 22,071 male physicians who were 40 to 84 years of age [15]. It failed to detect any protective effect of ingestion of 325 mg aspirin every other day on the incidence of colorectal cancer after five years.

It appears that NSAID use significantly protects against some but not all types of human cancer. As examples, one study showed a significant inverse association between paracetamol use and ovarian cancer [16] whereas aspirin use resulted only in a modest reduction of the ovarian cancer rate. Furthermore, regular use of ibuprofen did not lower the risk of ovarian cancer. A hospital-based case-control study investigating a possible association between cancer and a prior history of coronary heart disease revealed that aspirin use was protective for colorectal but not for prostate cancer [17].

Aberrant crypt foci of the colon may be precursors of adenoma and cancer of the colon. A clinical study using magnifying endoscopy established that therapy with sulindac, a nonselective NSAID, significantly reduces the number of aberrant foci [18]. Patients with familial adenomatous polyposis coli (APC) who are treated with sulindac experience a regression of intestinal adenomas [19]. These patients harbor germ- line mutations in the APC gene leading to enhanced expression of COX-2 [20]. Left untreated, they develop hundreds to thousands of precancerous aden­omatous colorectal polyps. Remarkably, treatment of

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Fig. 2. In vitro inhibitory effects (IC50) of indomethacin, a nonselective, nonsteroidal anti-inflam m atory drug (NSAID), and NS-398, a COX-2 selective inhibitor, on Colo320 and THRC colon carcinoma cell lines. Both agents inhibit cancer cell proliferation and enhance apoptosis. NS- 398 inhibits cancer cell growth at lower doses than does indomethacin. It increases apoptosis ofTHRC and Colo320 cells about 7- and 9-fold, respectively, compared to non­treated cells. The NS-398-induced apoptosis is about 3- fold compared with that of indomethacin. Remarkably, the colon carcinoma S/SK cell line, which expresses no detectable COX-2, is also inhibited by NS-398, raising the question of a different (COX-3?) form of cyclooxygenase. Alternatively, NS-398 may also inhibit cancer cell growth through pathways not yet known. Data from Hara et al. [118] and Elder et al. [119].

such patients with NSAIDs causes regression of adenomas that were already present before initiation of therapy [21,22,19].

However, NSAIDs vary in their inhibition of the COX-1 and COX-2 enzymes [23]. As an example, in human blood, flurbiprofen and ketoprofen are COX-1 selective, ibuprofen and naproxen nonselective, and diclofenac and mefenamic acid are COX-2 selective [24]. The COX-1 selectivity in the blood correlated well with the COX-1 selectivity in gastric biopsies from the same volunteers. However, this was not the case for COX-2 selectivity. Some NSAIDs presumably selective for COX-2 still had sufficient COX-1 activity to potently inhibit gastric cytoprotective prostaglandin synthesis.

Cyclooxygenase expression in human neoplasia

Significant overexpression of COX-2 mRNA and protein in several types of cancer tissues strongly

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suggests that COX-2 expression is important in the process o f carcinogenesis. Enhanced COX-2 expression is related to the grade or differentiation of the tumor. For instance, well-differentiated carcinoma of the lung [25] and well-differentiated primary hepatocellular carcinoma (HCC) [26,27] show augmented COX-2 expression compared with the less differentiated forms. However, COX-2 expression may vary in different types of tumor of the same organ.

Immunostaining of biopsies from squamous cell carcinoma of the skin shows strongly increased staining for COX-2 protein compared to non-sun-exposed skin [28]. COX-2, but not COX-1, expression is elevated in human colon [29,30] and esophageal carcinomas [31]. COX-2 is upregulated by 2- to 50-fold in 85- 90% of colorectal adenocarcinomas [9]. Larger colon carcinomas and tumors with deep invasion produce significantly more COX-2 than smaller tumors, but expression levels do not correlate with whether patients have metastases or not [32].

Nevertheless, some im m unohistochem ical findings question a relationship between COX-2 overexpression, cell proliferation, and degree of malignancy [33]. This doubt originated from an assessment of the immunostaining intensity and distribution of COX-2 protein, Ki-67, as an indicator of cell proliferation, and the tumor-suppressor protein p53 as a marker of malignancy in colorectal cancer tissues from 21 patients [33]. Nine tumors were differentiated well, 11 moderately well, and 1 poorly. COX-2 and p53 stained positive in 38% and Ki-67 in 48% of the cases. The distribution of the analytes in this study failed to indicate any relationship between COX-2 expression, cell proliferation, or the grade of malignancy. Moreover, in another study, chromatography-mass spectrometry was used to determine the levels of prostaglandin (PG) D2, E2, F2a, 6-ketO 'Fla, and thromboxane B2 in biopsy specimens from 4 familial adenomatous polyposis (FAP) patients treated with sulindac [34], The measured levels varied widely, ranging from an increase of 19% to a decrease of 89%. The results were interpreted as pointing to an uncoupling of tissue prostaglandin levels and the process of carcinogenesis.

These conflicting results of COX-2 expression in some cancers may appear confusing. However, in te rp re ta tion o f these data requires careful

consideration of three obvious variables. First, tumor tissue levels might not accurately reflect the tumor cell levels. Second, it is implausible that all the cells in the biopsy m aterial were tum or cells. T hird , the distribution of COX-2 overexpression in human colon carcinoma cells is heterogeneous [35].

A high percentage of pre-invasive tumors over­express matrilysin, an important matrix metallo- proteinase (MMP) involved in cancer invasion and metastases [36]. An immunohistochemical study of hum an colon carcinomas designed to look for coexpression of MMP and COX-2 revealed that 80% of the specimens overexpressed both COX-2 and matrilysin in the neoplastic epithelium [37]. COX-2 immunostaining was strongest in well-differentiated tum or regions, while matrilysin expression was strongest in the more dysplastic and invasive areas of the tumors. Moreover, the regional distribution of COX-2 and matrilysin differed. These results provide compelling evidence that involvement of COX-2 and matrilysin in carcinogenesis are not closely linked.

In contrast, COX-2 and transforming growth factor beta (TGF-P) expressions in cancer tissues are related. Deregulation ofTGF-(3l expression is an early event in colorectal carcinogenesis [38]. Similar distribution of expression of COX-2 and TGF—(3 in colon adenocarcinomas suggests that they cooperate in the process of carcinogenesis [39]. The enzyme and the growth factor are abundant in malignant cells [39], and moreso in primary than in metastatic tumors. T G F -p l was low or undetectable in the normal mucosa. O n the o ther hand, in an earlier immunostaining study, 8 of 10 adenomas and 46 of 48 carcinomas expressed TGF-P 1 in the epithelial cells[40]. However, 52 of 58 samples also showed TGF— p l protein in epithelial cells of a normal colon. Interestingly, the upper parts of the crypts showed more immunostaining compared with the proliferative compartment [40], suggesting a growth inhibitory function of TGF—P in the upper parts of the crypts.

Supplementary immunohistochemical and cell culture (vide infra) findings indicate that TGF—(3 is involved in the growth and spread of colon cancer. As an example, primary colon carcinomas expressing elevated TG F—p i levels have an 18-fold rate of recurrence as distant metastases compared to primary

Cyclooxygenase-2 in neoplasia 7

colon cancers with low TGF—p i levels [41]. In addition, results from a series of samples from 47 colorectal tumors confirm the notion that TGF—P expression in the tum or is im portant in colon carcinogenesis [38] and that the measuring of TG F- P l expression has prognostic value. TGF—P i was detected in 9 of 13 adenomas and 30 or 34 ad­enocarcinomas [38], T G F -P l levels were low in adenomas w ithout dysplasia, higher in tubular adenomas with dysplasia, and highest in carcinomas. Both epithelial and stromal cells were positive forTGF- P l in dysplasia and cancer. In contrast, epithelial cells of normal mucosa were T G F -P l negative. However, normal tissue endothelial cells occasionally stained positive for T G F -P 1.

In addition to COX-2 overexpression, tumors of the colon in humans and azoxymethane (AOM)- induced colon tumors in rats overexpress inducible nitric oxide synthase (iNOS) [42], The importance of iNOS on the induction of COX-2 was demonstrated in a study of aberrant crypt formation in AOM-treated F344 rats. The specific iNOS inhibitor S,S’-1,4- phenylene-bis(l ,2-ethanediyl)bis-isothiourea (PBIT) reduced the AOM-induced formation of aberrant crypts by 58% compared to rats not receiving PBIT.

Interestingly, elevation of COX-1 has been detected in the stroma next to malignant cells suggesting that stromal cells react to the presence of tumor cells [43]. COX-1 is often referred to as a constitutively expressed enzyme; however, COX-1 was elevated in tissues of 30 of 44 breast cancer patients as compared with normal breast tissues from 14 patients. Remarkably, immunohistochemistry revealed that COX-1 was not localized in the tumor cells, but in the adjacent stromal cells [43]. These findings suggest that cancer cells that overexpress COX-2 release substances, for instance hormones, that might induce COX-1 expression. As an example, COX-1 expression can be modulated by estrogen [44,45] and progesterone [45].

Cyclooxygenase gene structure and regulation

The cyclooxygenase proteins show structural and enzymatic similarities, both having a molecular weight of approximately 70 kDa. The COX-2 protein consists of 604 amino acids, just 5 more than the COX-1 enzyme. Exhibiting only 61% homology [46], the

COX-1 and COX-2 genes map to chromosome regions 9q32-q33.3 and Iq25.2-q25.3 respectively (Fig. 3) [47].1 The human COX-1 gene was cloned in 1988. It is also known as prostaglandin (PG) H synthase (EC1.14.99.1) and the gene is abbreviated PGHS-1. Three years later, a differentially regulated form of cyclo­oxygenase was detected. First, a Rous sarcoma virus (scr)-inducible form of cyclooxygenase was found in chicken embryo fibroblasts [48]. The majority of the scr-inducible mRNA present in such non-proliferating cells contained an unspliced intron that separated the signal peptide from the remainder of the protein. Mitogenic stimulation removed the intron and a fully spliced mRNA was formed, which translated into a functional protein. A short time later, another research group detected a highly cell type—restricted, phorbol ester-inducible2 mRNA, encoding a 604 amino acid long protein coded by the PGHS-2 gene [49]. The protein showed sequence similarity with murine, sheep, and human cyclooxygenase-1. Finally, a 4 kb COX-2 mRNA with sequence similarity to the 2.8-kb PGHS-1 cDNA was discovered [50]. The larger mRNA was transcrip tionally upregulated by serum and downregulated by glucocorticoids in C l27 mouse fibroblasts. The mRNA encoded a protein that was specifically immunoprecipitated by anticyclooxygenase antibody.

Sequences of the 3'-UTR transcripts of COX-1 and COX-2 are highly divergent, resulting in distinct divergence in their regulation of expression at the post- transcriptional and translational levels [51]. The 5 - flanking region of the COX-2 gene contains important regulatory elements such as a TATA box, CCAAT/ enhancer-binding protein (C/EBP), and cyclic AMP- responsive element (CRE) motifs. Moreover, there are transcription regulatory sequences for the activator binding protein-2 (AP-2), the ubiquitous DNA binding transcription factor SP1, and NF-kappa B (NF-kB) [52]. Human adenovirus E4 promoter binding protein (E4BP4) type elements, inducible by dexamethasone, are located in the promoter region of CO X-2, explaining a possible m echanism for glucocorticoid repression of the gene [53]. For example, dexamethasone inhibits induction of COX-2 by interleukin-1 alpha (IL-la) and by tumor necrosis

1 http://www.ncbi.nlm.gov2 12-0-tetradecanoylphorbol-13 acetate

8 Annals o f Clinical & Laboratory Science

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Antioxidants

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Fig. 3. COX genes. The main difference between COX-1 and COX-2 is that the latter contains a larger regulatory region. Inducers of COX-2 are: nitric oxide produced by inducible nitric oxide synthase (iNOS), high fat corn oil (HFCO), oncogenes ras and scr, interleukin-1 alpha (IL-lalpha), epidermal growth factor (EGF), transforming growth factor beta (TGF-p), tumor necrosis factor-alpha, ultraviolet light B (UVB), benzo[a]pyrene (B[a]P), and androgen. Inhibitors of COX-2 are tumor suppressor protein p53, high fat fish oil, estrogen, and several antioxidants (PDTC, Trolox, U75006). In some cells, inhibition by PDTC is post-transcriptional. COX-1 is inducible by estrogen and iNOS. Cytosolic phospholipase-2 (cPLA2), which maps to the same chromosome region as COX-2, may be coregulated with COX-2.

factor alpha (TNFa) [54]. E4BP4 motifs are also present in the genes for inducible nitric oxide synthase (iNOS) and cytosolic phospholipase A2 (cPLA2) [54],

In contrast to COX-1, COX-2 is highly inducible by proinflammatory cytokines such as IL-1 [55,56], and by growth factors, for instance epidermal growth factor (EGF), and transforming growth factor-beta (TGF-p) (Fig. 3). Androgen, the Rogs sarcoma virus, iNOS, and benzo[a]pyrene (B[a]P) induce COX-2 genes. Mesenchym-derived inflammatory cells exhibit high levels of COX-2 transcripts [46]. In cell models, IL-1 [57] and hypoxia [58] induce COX-2 expression via NF-kB, p65 binding to the matching sites in the COX-2 promoter region. Most importantly, EGF, TG F-p, and their receptors emerge as the more important COX-2 inducers in cancer development.

EGF induces both COX-2 messenger ribonucleic acid (mRNA) and COX-2 protein (Fig. 3). It has no effect on expression of COX-1 [59]. In a series of 97 fresh and 74 formalin-fixed bladder cancer tissues, EGF receptor (EGFR) expression was strongly associated with 5-bromodeoxyuridine (BrdU) labeling index, grade, and stage [60], In hepatocellular carcinoma, the ability to produce metastases directly correlates with elevated levels of EGF receptor mRNA and protein [61]. Interestingly, the gene for EGFR maps to chromosome region 7pl2 (Fig. 4) [62], and EGFR expression in cancer cells is associated with an increased copy number of chromosome 7 [61], In the A431 breast cancer cell line the EGFR gene is amplified 20- to 30-fold [63]. A prior study demonstrated that 4 of7 squamous cell carcinoma cell lines carried EGFR

Cyclooxygenase-2 in neoplasia 9

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Fig. 4. Schematic illustration of evidence-based and hypothetical regulation of COX-2 expression in neoplasia and its effect on neoplastic cell proliferation. COX-2 increases prostaglandin E2 (PGE2) synthesis, which induces cell proliferation. PGE2 can temporarily induce COX-2 expression, as shown by exogenous supplementation with dimethylprostaglandin E2 (dmPGE2) to cancer cells in vitro. Numbers in small boxes in the figure are indicated in legend by [ ]. Nonselective and selective COX-2 inhibitors, e.g. NS-398 [1], reduce the formation of PGE2 and reduce proliferation. Ultraviolet light disrupts (*) tumor suppressor protein p53 that normally inhibits COX-2 formation, leading to COX-2 overexpression in skin cancer. In vitro transforming growth factor-beta (TGF-P) induces the malignant phenotype by inducing COX-2. Epidermal growth factor (EGF) is a strong inducer of COX-2. The carcinogen azoxymethane (AOM) induces inducible nitric oxide synthase (iNOS) that induces COX-2 expression; the induction can be blocked by S,S’-l,4-phenylene-bis(l,2- ethanediyl)bis-isothiourea (PBIT), a specific iNOS inhibitor [2].

genes in amounts more than 20 times that of control cells [64],

Transient EGF transfection of human oral squamous carcinoma cells doubles the activity of the COX-2 promoter [65]. Retinoids suppress the COX- 2 induction. EGFR expression remains unaffected. Antisense EGFR RNA downregulates EGFR expres­sion and attenuates malignant behavior of Moser human colon cancer cells. The antisense molecules block the ability of exogenous EGF to stimulate malignant cell behavior [66].

The Epstein-Barr virus (EBV) induces the EGF receptor (Fig. 4). The virus is associated with smooth muscle cell tumors in various organs of children [67,68,69] or adults [70,71] with AIDS or adult immune-suppressed patients [72], EBV codes for two

latent membrane proteins (LMP-1, LMP-2A, and LMP-2B) in the virus-infected tumor cells [73,74]. The 54 kDa and 40 kDa proteins are encoded by different viral genes [75]. Expression of LMP-1 induces EGFR and protects cell death through induction of the anti- apoptotic zinc-finger protein A20 in C33A human epithelial cells [76] and A20 and anti-apoptotic protein bcl-2 in EBV-immortalized B-cell lines [77]. LMP-1 activates the NF-kB transcription factor that regulates expression of A20 [77].

Addition of transforming growth factor beta 1 (TGF—pi) to cultured rat intestinal epithelial (RIE) cells rapidly induces both COX-2 mRNA and protein (Fig. 4) [78], During the first 7 days of treatment, TG F-P 1 inhibits growth, but thereafter the cells become resistant to growth inhibition by TG F-pi.

10 Annals o f Clinical & Laboratory Science

After another 50 days of chronic TGF—pi exposure, these resistant cells lose contact inhibition, form foci in culture, grow in soft agar, and exhibit invasive potential. In addition, the cells become resistant to Matrigel and sodium butyrate-induced apoptosis. Their Type II TG F-P receptor (TGF-pRII) level decreases by 95%, and their COX-2 protein level increases 40-fold. Most importantly, cessation of exogenous TGF-P 1 exposure reverses the malignant behavior [39]. However, TGF—pi regulation of COX- 2 transcription is cell specific. For instance, in human lung fibroblasts, TGF-p 1 alone does not induce COX- 2 transcription, but potentiates the effect of IL-1 P and T N F -a —induced transcrip tion o f COX-2 by stabilization of the resulting transcript for TGF—pi[79].

Disruption o f the TG F—(3 Type II receptor attenuates in vitro invasiveness (Fig. 4). Human nonpolyposis colorectal cancer (HNPCC) cells which express a nonfunctional Type II receptor are non- invasive in vitro. Correcting the receptor defect by re­expressing wild-type Type II receptor restores their invasive potential [80]. The Type II receptor is somatically altered in hereditary H N PCC patients[81]. In addition, there is an association between TypeII receptor gene alteration and adenoma-to-carcinoma progression in HNPCC. An analysis of the occurrence of Type II receptor gene alterations in such tumors revealed that it progressed from 8 of 14 adenomas to11 of 13 human cancers [81].

Ultraviolet B (UVB) light is a risk factor in skin cancer. Irradiation of human keratinocytes by 30 mj/ cm2 UVB light results in a 6-fold increase in COX-2 levels and raises the levels of PGE2 compared to non­exposed cells [28]. Besides, biopsies of sun-protected skin of human volunteers irradiated with four times the minimal erythema dosage showed induction of COX-2 mRNA which peaked 12 hr after exposure [28]. Overexpression of COX-2 in mouse skin- carcinoma cells is associated with a change in CCAAT/ enhancer-binding protein (C/EBP) expression levels[82]. In addition, tumor-suppressor protein p53 normally inhibits COX-2 expression (Fig. 4) [83], and UVB irradiation of mouse skin induces mutations of the TP53 tumor suppressor gene that might reduce p53 inhibition of COX-2 expression [84].

COX-2 is overexpressed in Barretts esophageal

cancer [85]. In this disease, the most frequent of loss of heterozygosity of chromosome 17 is region 17pl3.1 where the TP53 gene is located [86]. Thus, loss of inhibition of expression of COX-2 by p53 might contribute to the elevation of COX-2. In addition, iNOS is overexpressed [85], which might lead to stimulation of COX-2 expression.

Antioxidants reduce COX-2 expression. The regulation is cell specific. For instance, in rat mesangial cells, the oxidant-scavenger pyrrolidine dithio- carbamate (PDTC) inhibits IL-l(3-induced COX-2 expression at the post-transcriptional level [87]. In comparison, addition of PDTC to cultured colorectal cancer cells decreases COX-2 expression at the transcriptional level [88]. Similarly, other antioxidants such as U74006, N-acetylcysteine, and 6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) lower COX-2 transcription in the colon cancer cells (Fig. 3).

Fifty percent o f colon carcinomas harbor mutations of the m-oncogene [89]. Activated ras oncogene transforms rat fibroblasts, induces COX-2, and stimulates proliferation (Fig. 4) [90]. The selective COX-2 inhibitor l-[(4-methylsulfonyl)phenyl]-3- trifluoro-methyl-5-[(4-fluoro)phenyl]pyrazole (SC- 58125) [88] inhibits the rcw-induced proliferation [89] and corroborates that COX-2 is directly involved in the growth control.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon in tobacco smoke, upregulates COX-2 transcription and PGE2 synthesis in cultured oral epithelial cells [91], suggesting a role for COX-2 in smoking-related carcinogenesis (Fig. 4).

Reports on the effect of sex hormones on COX- 2 expression vary, possibly because the stimulation apparently is indirect through EGF and its receptor (Fig. 4). Estrogen induces EGF [92] and progestins induce EGFR [93]. In bovine chondrocytes, COX-2 mRNA expression is inhibited by 17(3-estradiol [54], Estrogen-dependent MCF-7 breast cancer cells show a relatively high expression of COX-1, while COX-2 is barely detectable [94]. These findings support the notion that estrogen enhances COX-1 and reduces COX-2 expression [94]. Furthermore, in cultured epithelial endometrial cells, estradiol decreases and progesterone increases COX-2 mRNA and PGE2 levels [95]. Rat endometrium expresses COX-2 during the

Cyclooxygenase-2 in neoplasia IT

three later stages of a five-stage estrus cycle. The timing coincides with the peak in the serum estradiol level, indicating either that estrogen induces COX-2 [96] or that cyclical progestins have started to take effect. Estrogen-independent breast carcinoma cells (MDA- MB-231) exhibit high levels of COX-2 and low levels of COX-1 [94]. In these cells, COX-2 expression is apparently independent of estrogen. The extent of PGE2 production in this highly invasive cell line corresponds well with COX-2 expression, supporting a role for PGE2 in the invasive growth.

The chromosome region lq25 further harbors the gene for the intracellular cholesterol esterifying enzyme [97], acyl coenzyme Archolesterol acyltransferase (ACAT, EC 2.3.1.26) (Fig. 4). By esterifying intracellular free cholesterol, it reduces the availability of free cholesterol. The steroid acute regulatory protein (StAR) regulates uptake of cholesterol by mitochon­dria. TG F-p inhibits StAR and thereby regulates the mitochondrial synthesis of sex steroids. O f these, estrogen and progesterone are most important in the regulation of EGF and EGFR respectively (Fig. 4).

COX-2 expression is part of the processes of ovulation and uterine implantation [98]. The essential role of COX-2 expression in female reproductive functions is evidenced by disruption of the COX-2 gene which renders mice infertile [99]. A study of the peri-implantation mouse uterus suggests that while ovarian steroids regulate the COX-1 gene, the implanting blastocyst regulates expression of COX-2 [45]. Fiuman chorionic gonadotropin stabilizes COX- 2 transcripts during differentiation o f hum an endom etrial strom al cells in to decidua [100]. Remarkably, COX-2 is involved in male reproductive function as well. Androgen induces COX-2 in the vas deferens. The prostaglandins produced may play a role in erection [101]. For that reason, impotence might be a consequence of therapeutic, selective COX-2 inhibition.

COX-2 is also expressed in carcinogen-induced neoplasia. As an example, treatm ent w ith the carcinogen azoxymethane (AOM) induces COX-2 - expressing colonic tumors in rats. In a murine study all of 6 such tumors showed markedly increased COX- 2 mRNA and protein levels in the tumors compared with paired normal mucosa (Fig. 4) [102], In contrast, the intensity of COX-1 mRNA transcripts in normal

mucosa and tumors were similar in all of the specimens. NSAIDs significantly reduce the risk of colon cancer in carcinogen-treated rodents [102], Nimesulide suppresses azoxymethane-induced colon carcinogenesis [103]. Aspirin [104] and the selective COX-2 inhibitors celecoxib (SC-58635) [105] and NS-398 [106] reduce the incidence of colon tumors and can suppress overall azoxymethane-induced colon tumor burden in rats.

PGE2 can tem porarily induce its own synthesizing enzyme, suggesting the presence of a limited, latching positive feedback loop. Evidence in support of this view was found in the human prostate carcinoma cell line PC-3 in which exogenous dimethyl- prostaglandin E2 (dmPGE2) increased COX-2 mRNA steady-state levels (Fig. 4). In add ition , cell proliferation, total DNA content, and endogenous PGE2 concentration were elevated indicating that PGE2 has a specific role in the maintenance of cancer growth [107]. Likewise, the effect was observed in other human cancer cell lines, such as breast cancer cells (MDA-MB-134), androgen-dependent prostate carcinoma cells (LNCaP), and colon carcinoma cells (DiFi) [107]. Addition of 5 |iM of the COX-1 selective NSAID flurbiprofen [24] to cultured PC-3 cells exposed to exogenous PGE2 inhibits the upregulation of COX-2 mRNA and cell growth [108]. The use of flurbiprofen in the above experiment hampers interpretation of the role of PGE2 in cell proliferation. The carboxyl group of flurbiprofen decouples oxida­tive phosphorylation (OXPHOS) that lowers ATP generation and reduces cell proliferation (Fig. 5) [4]. Furthermore, the decoupling of OXPHOS generates H 2O2 and induces BAX, which enhances apoptosis. This effect might actually point to an alternate way in which flurbiprofen and similar NSAIDs inhibit cancer growth.

Pathophysiology

The most convincing evidence of a pivotal role of COX-2 in neoplasia comes from gene transfection and gene knockout experiments. For example, an en­hanced tumorigenic potential is a consequence of COX-2 experimental overexpression. Such phenoty­pic alterations occur in permanently transfected rat intestinal epithelial (RIE) cells. A COX-2 expression

12 Annals o f Clinical & Laboratory Science

Fig. 5. Schematic illustration of evidence-based and hypothetical effect o f COX-2 overexpression in neoplasia. Overexpression of cyclooxygenase (COX)-2 contributes to cancer growth by inhibiting programmed cell death (apoptosis) and enhancing metastatic potential and angiogenesis. COX-2 increases prostaglandin (PG) E2 (PGE2) synthesis and bcl- 2 synthesis. Bcl-2 closes mitochondrial pores (black ellipses) and reduces apoptosis (black filled arrows). COX-2-induced tumor invasion and metastatic spread are associated with enhanced PGE2, interleukin-6 (IL-6), and haptoglobin synthesis. Numbers in small boxes in the figure are indicated in legend by [ ]. Nonselective NSAIDs inhibit both COX-2 [1] and COX-1 [3] enzymatic activity. Selective COX-1 inhibitors, e.g. VSA [4], are used to differentiate COX-1 and COX-2 effects. Nonselective NSAIDs and selective COX-2 inhibitors, eg NS-398 [2], reduce the formation of PGE2 and bcl-2, thereby opening mitochondrial pores and increasing apoptosis (shaded arrows). Nonselective NSAIDs harboring a COOH- group restore apoptosis through additional mechanisms (illustrated with dotted background). They uncouple oxidative phosphorylation (OXPHOS), which increases H 2O 2 and BAX levels that open mitochondrial pores. Mitochondrial cytochrome c escapes and initiates apoptosis. Inhibition of the lipoxygenase (LO) [5] also induces BAX that opens mitochondrial pores by complexing with bcl-2. As more tumor cells succumb to apoptosis, tumor growth is inhibited or reversed.

vector oriented in the sense direction elevates the levels of COX-2 protein and the levels of the anti-apoptotic protein bcl-2 (Fig. 5) [109]. The apoptosis is reduced and the cells resist butyrate-induced apoptosis. TGF- RII levels are reduced (Fig. 4). E-cadherin, a protein essential in cell adhesion, becomes undetectable. Loss o f cadherin function in tumors results in rapid progression of adenoma to invasive, metastatic carcinomas [110]. The NSAID sulindac sulfide inhibits the transfected RIE cell growth by markedly increasing apoptosis. O f particular importance, even a metabolite of sulindac sulfide, sulindac sulfone, which essentially lacks prostaglandin synthesis inhibitory

activity, strongly induces apoptosis of the cells [111].It appears possible that, when cyclooxygenases are

overexpressed in cancer cells, some overlap will occur between COX-1- and COX-2-induced pathophysio­logies. By comparison, the two isoforms of cyclo­oxygenase have separate functions in normal cells. As examples, in human and bovine endothelial cells and murine 3T3 adipocytes, COX-1 functions predom­inantly in the endoplasmic reticulum (ER), whereas COX-2 functions in the ER and the nuclear envelope. Similar observations were made in bovine endothelial cells and murine 3T3 adipocytes [112]. However, immortalized endothelial cells transfected with

Cyclooxygenase-2 in neoplasia 13

COX-1 express high levels of functional COX-1 protein in both the endoplasmic reticulum and the nucleus [113]. Most important, the cells proliferate aggressively and form tumors in athymic nude mice.

Surprisingly, indomethacin does not inhibit the growth of the COX-1-induced tumors. These findings hint at a nonenzymatic, nonprostanoid, nuclear function of the COX-1 protein [113]. Similarly, in human colon carcinoma HCA-7-cells the TGF-OC induces COX-2 and translocates COX-2 protein to the nucleus [114]. EGFR levels are increased and promote mitogenesis. From these experiments, it appears quite possible that the COX-2 protein can also induce genes involved in cell proliferation. Furthermore, other similarities are evident. For example, transfection of the human colon carcinoma cell line COLO 320DM by either a COX-1 or a COX- 2 eukaryotic expression vector just about doubles the growth rate compared with mock transfected cells [115]. Either COX expression vector markedly increases EGF receptor (EGFR) expression.

COX-2 expression induces resistance to apoptosis in many different cell types (Fig. 5) [116,117]. That NSAIDs restore apoptosis is illustrated by the in vitro apoptosis induced by the NSAID indomethacin on colon carcinoma Colo320, THRC and S/SK [ 118] cells (Fig. 2 and Fig. 5). The COX-2 selective inhibitor (2- cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) [88] increases apoptosis of Colo320, THRC [119], and HT29 cancer cells [119]. It increases apoptosis of THRC and Colo320 cells about 7- and9-fold compared to nontreated cells respectively [118]. Interestingly, S/SK cells contain no detectable COX-2 expression. It is quite remarkable that NS-398, a selective COX-2 inhibitor, induces apoptosis in cells lacking COX-2 expression (Fig. 3) [119]. Evidently, NS-398, which does not inhibit COX-1 enzymatic activity, m ust also induce apoptosis through mechanisms independent of prostaglandin synthesis. In contrast, meloxicam, a COX-2 selective inhibitor[23], has no effect on the growth of HCT-116 cells, which do not express COX-2 [120].

Aspirin, indomethacin, and ibuprofen inhibit cyclooxygenases and decreases non-small cell lung cancer cell proliferation [121] in vitro. The NSAID meloxicam significantly inhibits colony growth of the 2 colon cancer cell lines, HCA-7 and Moser-S, that

both express COX-2 [120]. SC-58125 inhibits serum stimulated cell proliferation of both a continuously proliferating rat small intestinal cell line IEC-18 and a mouse colon cancer cell line W B-2054 [122], Valerylsalicyclic acid (VSA), a selective COX-1 inhibitor (Fig. 5) does not inhibit the proliferation as measured using H-thymidine incorporation and cell counting. Interestingly, SC-58125, VSA, and indo­methacin all inhibited PGE2 formation. However, PGE2 production did not correlate with the inhibition of proliferation by the selective COX-2 inhibitor therapy, distinguishing the two processes as unrelated in these experiments [122]. Aspirin and indomethacin inhibit proliferation of human gastric cancer cells, in part through apoptosis [123].

SC-58125 and NS-398 inhibit COX-2 catalytic activity, reduce PGE2 synthesis, downregulate bcl-2, and induce apoptosis in cultured prostate carcinoma LNCaP cells (Fig. 5) [124]. A clue to the mechanisms of apoptotic induction by NS-398 is provided by its effect on human fetal prostate fibroblasts. In these cells, NS-398 does not alter cell viability, nuclear function, or morphology. Such data suggest that induction of apoptosis in the carcinoma cells does indeed occur as a result of inhibition of COX-2 enzymatic activity (Fig. 5). However, since COX-2 has been detected in the nucleus of malignant cells, it cannot be definitely excluded that COX-2 protein interacts directly at the nuclear gene level. At low, physiologically relevant concentrations, NSAIDs typically inhibit COX-2, but in high in vitro con­centrations, some, such as diclophenac, can induce both apoptosis and COX-2 [125]. In comparison, high concentrations of staurosporine or acetaminophen can induce apoptosis without inducing COX-2.

Experim ents dem onstrate tha t COX-2 overexpression in neoplasia enhances cancer-induced angiogenesis [126] and invasiveness [127] (Fig. 5). The increased PGE2 synthesis is associated with tumor métastasés and IL-6 expression with invasion (Fig. 5) [128,129]. For instance, colon carcinoma cells that overexpress COX-2 stimulate endothelial cell migration and tube formation in vitro. NS-398, aspirin, and antibodies against proangiogenic factors inhibit the angiogenesis. Notably, while COX-2 apparently regulates angiogenesis in cancer cells, COX-1 regulates angiogenesis in endothelial cells [126]. Caco-2 cells

14 Annals o f Clinical & Laboratory Science

permanently transfected with a COX-2 transfection vector acquire increased invasiveness compared with parental cells or cells transfected with the identical vector lacking the COX-2 insert [127]. Inhibition of COX with sulindac sulfide reverses the increase in prostaglandin synthesis and the enhanced invasiveness.

In vitro studies provide clues to how COX-2 expression modulates the cell cycle. For example, rat intestinal epithelial cells permanently transfected with the COX-2 gene exhibit a phenotype similar to that which follows supplementation of cultured cells with the biphasic growth factor T G F-p [78]. It strongly induces COX-2 and lowers cyclin D1 levels. Therefore, the G1 cell cycle progression is delayed. Transfection of the cells with a COX-2 antisense expression vector or a cyclin D 1 gene expression vector restores cyclin D 1 levels and reverses the G 1 delay [78]. Transforming growth factor beta (T G F -P ) characteristically stimulates growth at low concentrations and inhibits growth at higher concentrations [130] via activation of TGF—p Type I and Type II receptors respectively in vitro. Nonetheless, during human colon carcinoma progression TGF—p i switches from an inhibitor of tumor cell growth to a stimulator of growth and invasion [131]. As examples, in the highly metastatic mouse colon carcinoma CT26 cell line, TGF—p signaling is essential for cell invasiveness and metastasis[80]. U9 colon cancer cells xenografted to nude mice form tumors that metastasize to the liver and skin [132]. Significantly, transfection of U9 cells with T G F -p l antisense vector lowers T G F -P l protein levels and reduces metastatic spread.

Animal model studies corroborate in vitro findings of the anti-neoplastic effects of selective COX- 2 inhibitors. As an illustration, COX-2—expressing HCA-7 colon carcinoma cells xenografted to nude mice formed tumors [120]. Four weeks of meloxicam treatment reduced tumor size by 51% compared with animals with xenografted HCA-7 cells not receiving therapy with meloxicam. Post-treatment analysis of HCA-7 tumor lysates revealed only a slight decrease in COX-2 protein. COX-1 protein was not detected.

NS-398 reduces experimental lung tum or multiplicity by 34% [133]. The tumors were induced in the A/J mouse by the administration of the tobacco- specific carcinogen 4-(m ethylnitrosam ino)-l-(3-

pyridyl)-l-butanone (NNK). Sulindac, but not naproxen, also curtails N N K —induced lung tumorigenesis [134], In contrast, sulindac does not affect benzo[a]pyrene-induced lung tumorigenesis [134]. These findings again point to different effects of an NSAID in different types of tumors and different effects of different NSAIDs on the same tumor. Furthermore, interpretation of cell culture results is limited by findings that in vivo COX selectivity of NSAIDs might differ from in vitro selectivity [135].

Knocking out the COX-2 gene in the murine model of human familial adenomatous polyposis (APC) dramatically reduces the number of polyps [136,137]. Further evidence for a central role of COX- 2 in this disease, although less direct, is provided by NSAID treatment in the APC Min mouse model. NSAIDs have a protective effect even if given 14 weeks after administration of the carcinogen [22]. The effect of 200 ppm of piroxicam in the APC Min mouse is rapid, with over 90% reduction in multiplicity after 1 week of treatment [138]. However, there is a strain- related effect on chemosuppression. The results suggest the existence of genetic elements that might affect NSAID chemosensitivity [138]. The effectiveness of NSAIDs in preventing APC-related tumor formation depends on which type of APC mutation is present [139].

Chromosome region lq21-32 is significant for evolution of certain tumors [140]. For instance, all of 35 patients with hematological malignancies, either acute leukemia, polycytemia vera, or myelofibrosis, carried trisomy for band lq25 [141] that could cause overexpression of COX-2. However, there is presently no direct proof of alterations at lq25 causing elevated COX-2 expression in cancer. For instance, a study of human HT29 adenocarcinoma cells failed to detect any mutation of the COX-2 promoter [142], A search for a mutation of the COX-2 gene in an attenuated (A) form of APC (AAPC) was also futile [143]. AAPC patients exhibit fewer colorectal polyps and a later age of onset of colorectal cancer than regular APC patients do. Striking variations in colorectal polyp numbers occur among patients carrying identical AAPC mutations, suggesting that alleles of another gene modify the expression of the APC disease phenotype. It was suspected that loss of function of COX-2 results in a decreased tumor burden in AAPC patients who

Cyclooxygenase-2 in neoplasia 15

develop very few colorectal polyps. However, no mutation of the COX-2 gene was detected.

COX-2 is expressed in certain norm al physiological conditions. For instance, it is highly expressed in trophoblastic cells during implantation [144]. As noted above, PGE2 induces interleukin-6 (IL-6), which induces haptoglobin. Haptoglobin is associated not only with implantation [145] but also with angiogenesis [146,147], which is important in tumor growth, but also plays a central role in normal wound healing.

Prostanoid production depends on an arachidonic acid—liberating stimulus [148]. Interestingly, like the COX-2 gene, the gene for cPLA2, the enzyme that supplies the arachidonic acid is located on chro­mosome region lq 2 5 , suggesting coordinated expression of COX-2 and cPLA2 (Fig. 5) [149]. The gene for the secretory type II phospholipase (sPLA2) is located on chromosome Ip35-p36 [150], a region that is the target of frequent deletions in colonic cancer. Surprisingly, apoptosis in the mouse WB-2054-M4 colon carcinom a cell-line was enhanced and proliferation inhibited only by specific inhibition of sPLA2 but not by inhibition of cPLA2 [151].

When the COX-2 pathway is inhibited, substrate accumulates and increases the rate of conversion through the lipoxygenase pathway (Fig. 5). Studies of the colorectal carcinoma cell-line Caco-2 that expresses low levels of COX-1 but high levels of COX-2 illustrate the importance of this pathway in apoptosis [152]. In these cells, exogenous sodium butyrate (NaBT) induces a 72-kDa 15-lipoxygenase (15-LOX), which then becomes the major enzyme of the arachidonic acid cascade. Furthermore, lipoxygenase (LOX) inhibitors or LOX- antisense oligos dramatically reduce bcl-2 protein and the bcl-2/BAX ratio, and induce apoptosis in rat Walker 256 (W256) carcinosarcoma cells [153]. For instance, the LOX-inhibitor nordihydroguaiare- tic acid (NDGA) causes rapid apoptosis of W256 cells (Fig. 5) [154], Interestingly, exogenous arachidonic acid, or the polyunsaturated fatty acids alpha-linolenic and linoleic acid, can suppress NDGA induction of apoptosis. The inhibitor 3-[l-(p-chlorobenzyl)-5- (isopropy l)-3 -tert-bu ty lth io indo l-2 -y l]-2 , 2- dimethylpropanoic acid (MK886) blocks and reverses m em brane translocation and activation o f 5- lipoxygenase [155]. MK886 [156], as well as the

specific 5-lipoxygenase inhibitor SC41661A [157], induce apoptosis in hormone-responsive LNCaP and -nonresponsive PC3 prostate carcinoma cells.

Inhibition of alternate pathways of arachidonic acid metabolism to disrupt carcinogenesis may be important. For instance, human colorectal epithelial cancer cells highly express 15-lipoxygenase [158]. Remarkably, lipoxygenase inhibitors reduced in vitro growth of twenty different human cancer cell-lines even more than selective COX-2 inhibitors did [159]. Verification of a similar in vivo effect on tumor growth by selective lipoxygenase inhibitors is still pending. Combined inhibition of COX-2 and the lipoxygenase pathway should be promising. Some new NSAID derivatives lacking COX-2 inhibitory activity might also be chemopreventive [11], suggesting alternate pathways of inhibition of tumor growth. Finally, COX- 2 selective inhibitors that inhibit cells lacking detect­able COX-2 may inhib it an alternate form of cyclooxygenase [125].

Discussion and Conclusions

To date, there is no evidence that the COX-2 gene is mutated in neoplasia. Nonetheless, three strong argum ents support a causative role o f COX overexpression in the development of cancer. First, COX-2 gene-transfection experiments demonstrate that COX-2 overexpression alone can induce the tumorigenic phenotype. Some cancers carry trisomy of lq25, the chromosome region where the COX-2 gene is located; however, COX-2 levels were not investigated in those tumors. Second, and quite surprisingly, it appears that genetically engineered COX-1 overexpression can have the same effect as COX-2 overexpression. Third, COX-2 gene knockout inhibits experimental carcinogenesis.

The m ost im portan t effect o f COX-2 overexpression in neoplastic cells is the inhibition of apoptosis. Both nonselective and selective inhibition of COX-2 restores apoptosis. However, selective inhibition avoids gastrointestinal side effects. The mechanism of growth regulation by selective COX-2 inhibitors of COX-2-negative cells is unknown. One possibility is that they induce apoptosis independently of COX-2-regulated pathways. Furthermore, there is recent evidence of the existence of a variant and

differently regulated COX-2 protein, inducible by apoptosis-inducing concentrations of diclophenac [125]. Acetaminophen, but not aspirin, inhibits the variant enzyme. It is also less sensitive to inhibition 9by potent COX-1 and COX-2 inhibitors such as flurbiprofen or tolfenamic acid. Its response to NS- 398 is unknown. The existence of such a third COX 10'enzyme (COX-3) might account for some of the observed variation of the inhibitory effects of NSAIDs on different cancer cell types. 11-

Celecoxib (SC-58635) is the first selective COX- 2 inh ib ito r approved by the Food and D rug Administration for the treatment of rheumatoid 13arthritis [160]. It will soon be known if any significant side effects occur. However, faced with limited choices in the treatment of lethal cancers, the possible benefits of treatment with selective COX-2 inhibitors probably will outweigh their side effects. O f particular interest 15.is the finding that a combination of a selective COX- 2 inhibitor and an antioxidant, which suppresses COX- 2 expression, causes regression of cancer in animal ^models. Finally, therapeutic inhibition of iNOS and PLA2 and the lipoxygenase pathway most likely will enhance the anti-neoplastic effects of selective COX- 17•2 inhibition and contribute to cancer regression.

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