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Cyclooxygenase-2 and prostate carcinogenesis
Cancer Letters 191 (2003) 125–135 www.elsevier.com/locate/canlet
Mini-review
Cyclooxygenase-2 and prostate carcinogenesis Tajamul Hussaina, Sanjay Guptab, Hasan Mukhtara,* a
Department of Dermatology, University of Wisconsin, Medical Science Center, 1300 University Avenue, Madison, WI, 53706, USA b Department of Urology, Jim & Eilleen Dicke Research Laboratory, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH, 44106, USA Received 23 August 2002; accepted 29 August 2002
Abstract In recent years a dramatic surge has occurred on studies defining to the role of cyclooxygenase (COX)-2 in causation and prevention of cancer. Prostaglandin (PG) endoperoxidase synthase also commonly referred to as COX is a key enzyme involved in the conversion of arachidonic acid to PGs and other eicosanoids. COX exists as two isoforms, namely COX-1 and COX-2 with distinct tissue distribution and physiological functions. COX-1 is constitutively expressed in many tissues and cell types and is involved in normal cellular physiological functions whereas COX-2 is pro-inflammatory in nature and is inducible by mitogens, cytokines, tumor promoters and growth factors. A large volume of data exists showing that COX-2 is overexpressed in a large number of human cancers and cancer cell lines. The possibility of COX-2 as a candidate player in cancer development and progression evolved from the epidemiological studies which suggest that regular use of aspirin or other non-steroidal antiinflammatory drugs could significantly decrease the risk of developing cancers in experimental animals and in humans. In our recently published study (Prostate, 42 2000 73 – 78), we provided the first evidence that COX-2 is overexpressed in human prostate adenocarcinoma. Many other studies verified our initial observation and reported that compared to normal tissue, COX-2 is overexpressed in human prostate cancer. It should be noted that some recent work has suggested that COX-2 is only upregulated in proliferative inflammatory atrophy of the prostate, but not in prostate carcinoma. In this scenario, COX-2 inhibitors could afford their effects against prostate carcinogenesis by modulating COX-2 activity in other cells in prostate. An exciting corollary to this ongoing work is that selective COX-2 inhibitors may exhibit chemopreventive and even chemotherapeutic effects against prostate carcinogenesis in humans. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cyclooxygenase enzyme; Prostate cancer; Non-steroidal anti-inflammatory drugs; Inflammation; Prostaglandin
1. Introduction The incidence of prostate cancer has strongly increased during the past decades and it has now become the most common malignancy of men in many Western nations [1]. In the USA, among males, prostate cancer is the second leading cause of cancer* Corresponding author. Tel.: þ1-608-263-3927; fax: þ 1-608263-5223. E-mail address: [email protected] (H. Mukhtar).
related deaths next only to lung cancer [2]. According to projections by the American Cancer Society, a total of 189 000 men will be diagnosed with prostate cancer in the USA in the year 2002, and 30 200 prostate cancer-related deaths are predicted this year [3]. Of great concern is that fact that more than 50% of prostate cancer patients present with or develop incurable metastatic disease [4]. Although most patients with advanced prostate cancer initially respond to androgen ablation treatment, relapse to an androgen-indepen-
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3835(02)00524-4
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Fig. 1. Bi-functional role of the COX enzyme in the biosysnthesis of prostaglandins, prostacyclins and thromboxanes and their physiological and pathophysiological effects.
dent state occurs shortly leading to tumor outgrowth [5]. Despite the clinical importance of prostate cancer, the molecular mechanisms underlying the development and progression of this disease are poorly understood. Therefore, much research is needed towards understanding the mechanisms involved in development and progression of prostate cancer and developing new strategies for its prevention and treatment. The present article reviews the role of COX-2 in prostate carcinogenesis. Based on the evidence available, selective COX-2 inhibitors offer promise for prevention and therapy of prostate cancer.
2. Cyclooxygenases and prostaglandins Cyclooxygenase (COX) or prostaglandin (PG) endoperoxide synthase is a rate limiting enzyme in the PG biosynthesis [6]. COX is a bi-functional enzyme containing a COX site that converts arachi-
donic acid to prostaglandin endoperoxide synthases (PGG)2 and a peroxidase site that reduces PGG2 to PGH2 (Fig. 1) [7]. The PGs are a diverse group of autocrine and paracrine hormones that mediate many cellular and physiological processes [6 – 8]. COX exists in two isoforms commonly known as COX-1 and COX-2, the order in which they were identified [8 –10]. Although both isoforms catalyze the same enzymatic reactions and have similar Km and Vmax values for arachidonic acid, significant differences exist between them [11]. COX-1 is constitutively expressed in majority of the cells performing the house keeping functions that require immediate generation of prostanoids related to vascular homeostasis, water reabsorption, gastric acid secretion, platelet aggregation and renal blood flow [10,11]. COX-1 is located on human chromosome 9q32-q33.3 spanning 25 kb in size containing 11 exons and produces 2.8 kb mRNA which synthesizes about 68 kDa protein [10,11]. In contrast, COX-2 is a pro-inflammatory and inducible enzyme
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Fig. 2. Possible involvement of COX-2 and their inhibitors in various stages of prostate cancer.
which can be induced by mitogens, tumor promoters, cytokines and growth factors in different cell types and controlled at both the transcriptional and post-translational levels [10 –12]. COX-2 in involved in differentiative processes, such as inflammation, ovulation, and labor, in situation where only transient PG production is required [9 –11]. COX-2 is an 8 kb gene with 10 exons located on human chromosome 1q25.2 –q25.3 and transcribes a 4.1– 4.5 kb mRNA which encodes a protein of about 68 kDa [9 – 11]. Although, genes for COX-1and COX-2 are located on two separate chromosomes but they are highly related at the DNA, RNA, and protein level. COX-1 and COX2 consist of 576 and 587 amino acids, respectively, and they share approximately 60% primary sequence homology [12,13]. Both these enzymes exist as integral membrane glycoprotein homodimers and are found on the luminal surfaces of the endoplasmic reticulum and nuclear envelop [14,15].
studies have shown that overexpression of COX-2 is sufficient to cause tumorigenesis in animal models and subsequently inhibition of the COX-2 pathway results in reduction in tumor incidence and progression (Fig. 2) [20]. There are some exceptions to this generally accepted observation. One recent study has shown that COX-2 overexpression in the skin of transgenic mice results in suppression of tumor development [21]. In sharp contrast, another recent study has shown that deficiency of COX-2 reduces mouse skin tumorigenesis [22]. Based on these observations it is clear that much additional work with targeted tissue specific over and under expression of COX-2 is required to firmly establish the role of this enzyme in target organ carcinogenesis. However, based on these evidences the potential application of non-steroidal anti-inflammatory drugs (NSAIDs) as well as recently developed COX-2 specific inhibitors in cancer clinical practice has drawn tremendous attention in the past few years as inhibition of COX-2 offers an effective approach in the prevention and treatment of cancer [23,24].
3. COX-2 and cancer In recent years, overexpression of COX-2 has been implicated in the progression of cancer [16,17]. Aberrant or increased expression of COX-2 has been found in most of the cancers of the body sites [9, 16 – 18]. Compelling evidence from genetic and clinical studies indicates that COX-2 upregulation is one of the key steps in carcinogenesis [19]. Recent
4. Diet and prostaglandins in prostate carcinogenesis Epidemiologic studies and nutritional data suggest that androgens and/or environmental exposures, such as diet in particular, dietary fat, play an important role in prostate carcinogenesis [25]. The association
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between diet and prostate cancer has been drawn from the studies where incidence and mortality rates of prostate cancer vary widely between different populations in various regions of the world consuming more dietary fat [26]. Number of studies have reported this link; countries such as the USA, known to have high levels of fat consumption, were also found to have a high mortality rate for prostate cancer, whereas a country such as Japan, with one of the lowest rates of fat consumption in the world, has a low mortality rate for the disease [27]. In addition, migratory studies have further confirmed this observation where it was found that Asian men migrating to the United States acquire a higher clinical incidence of prostate cancer, and subsequent generations of American born Asian men have prostate cancer risks almost equal to those of white Americans [28]. These studies suggest that a change in dietary habits can greatly modify prostate cancer risk. However, the results of most case-control studies demonstrated a significant association of prostate cancer risk with high dietary intake of total fat while some studies did not find a significant association [29,30]. Laboratory studies with experimental animal models further suggest a link between fat content in diet and the risk of prostate cancer [31]. Arachidonic acid and its precursor, linoleic acid, are major ingredients of animal fat and many vegetable oils used in the regions where prostate cancer is more common [32]. Studies have shown that treatment of androgen-unresponsive human prostate carcinoma cells PC-3 with linoleic acid stimulated the growth of these cells [33].The effects of these fatty acids are thought to be caused by their effects on PG synthesis. A study by Hughes-Fulford et al. [34] has demonstrated that linoleic acid, arachidonic acid and the arachidonic acid metabolite prostaglandin (PGE)(2) stimulate prostate tumor growth and alters gene expression in human prostate carcinoma PC-3 cells. Treatment of PC-3 cells with arachidonic acid was shown to result in a dose-dependent increase in the gene expression of c-fos, and COX-2, while the constitutive COX-1 message was not increased. Further effects of dietary fat was examined on the in vivo models, where transplantation of human prostate carcinoma cells DU145 to nude mice fed with high fat diet was shown to result in a significant increase in tumor growth compared to the control group of
animals fed with regular diet [35]. These studies suggest a stimulatory effect of dietary n-6 fatty acid on prostate cancer cell growth which may be critical for the development and progression of prostate cancer. Intake of unsaturated dietary fat may affect PG synthesis, that appear to influence sex hormone levels; this raises the possibility that increased levels of androgens could play an important role in the initiation of prostate cancer [36]. Studies have shown that testosterone is capable of stimulating oxidative stress in prostate carcinoma cells that has been suggested to be a mechanism of initiation of prostate carcinogenesis [37]. However, there is no such study indicating that increase in testosterone levels leads to an increase in COX-2 expression during the development and progression of prostate cancer.
5. COX-2 and prostate cancer Studies on relationship of COX and prostate were initiated in 1993 by O’Neill and Ford-Hutchinson [38] who analyzed COX-1 and COX-2 mRNA expression in various human tissues and reported the highest levels in the prostate where COX-1 and COX-2 transcripts were found to be present in approximately equal levels. In the year 2000, we provided the first evidence that COX-2 is overexpressed in human prostate adenocarcinoma [39]. Employing 12 pairs of unique benign and prostate carcinoma tissue from the same individuals we showed that mean levels of COX-2 mRNA expression was significantly increased in prostate adenocarcinoma. These results were further verified by the COX-2 protein expression, which was significantly higher in cancer tissue compared to their benign counterparts. Many other studies verified our initial observation and reported that compared to normal tissue, COX-2 is overexpressed in human prostate cancer. A study by Yoshimura et al. [40] analyzed tumor specimens obtained from 28 prostate carcinoma patients, eight benign prostatic hyperplasia (BPH) patients, one prostatic intraepithelial neoplasia (PIN) patient, and eight specimens of normal prostate tissue and showed very weak expression of COX-1 and marked expression of immunoreactive COX-2 in prostate tumor cells. The expression of both COX-
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isoforms was found to be very weak in all cases of BPH and in the normal prostate tissues. Further, the extent and intensity of immunoreactive COX-2 polypeptides in tumor cells was statistically much greater than those of cells from BPH. These results were further confirmed by mRNA analysis, where enhanced expression of COX-2, but not COX-1, was observed in prostate cancer tissues. These results lead to the conclusion that human prostate carcinoma cells generated COX-2, and that COX-2 might play an important role in the proliferation of prostate carcinoma cells. Studies by Kirschenbaum et al. [41] analyzed thirty-one specimens of prostate carcinoma and 10 specimens of BPH and showed that COX-1 expression in noncancerous prostatic tissue was predominantly (90% positive staining) seen in the basal epithelial cells of BPH. COX-1 expression was minimal in noncancerous luminal epithelial cells and was found to be upregulated in prostate cancer predominantly in the smooth muscle cells of the prostate. COX-2 was found to be expressed in the basal epithelial cells with 60% BPH, 94% peripheral zone, 75% PIN, respectively. The expression of COX2 in prostate cancer was found to be intense and uniform, with 87% of samples demonstrating immunoreactivity. The results of this study indicated that expression of both COX-1 and COX-2 in human prostate cancer is increased suggesting that COX-1 and COX-2 (and/or their PG products) may play a role in the malignant transformation of the prostate. In another study, Madaan et al. [42] have determined COX-1 and COX-2 expression in 30 BPH and 82 prostate cancer specimens. In this study a significant COX-2 overexpression in tumor cells was found compared to benign glands; however, COX-1 expression in tumor cells was similar to benign glands. A significant positive correlation between COX-2 expression was found with increasing tumor grade suggesting that COX-2 may play an important role in prostate carcinogenesis. These studies are in agreement with the study by Lee et al. [43] where COX-2 was found to be over-expressed in 15 out of 18 (83%) prostate cancer samples and was detected in only 22% (4/18) of paired benign tissues. Further, the intensity of immunostaining correlated with the tumor grading of these specimens. Another recent study by Uotila et al. [44] has compared COX-1 and COX-2 mRNA and protein expression from 12 prostate
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cancer specimens and 13 control prostates. The intensity of COX-2 was found to be significantly stronger in prostate cancer cells than in the nonmalignant glandular epithelium of the control prostates. COX-2 was found to be clearly expressed in the lesions of PIN in control prostates and was also detected in the muscle fibers of the hyperplastic stroma. No significant difference was found in COX-1 expression between control and prostate cancer. The results of this study indicated that the expression of COX-2 is elevated in prostatic adenocarcinoma and in PIN. These experimental data generated from both human prostate tumor tissue specimen and from prostate cancer cell lines demonstrated an upregulation of inducible COX-2 expression and suggested a positive role for COX-2 in prostate carcinogenesis. However, in contrast to these observations a recent study performed immunohistochemical analysis of 144 human prostate cancer cases and found that there was no consistent overexpression of COX-2 in established prostate cancer or high grade PIN, as compared with adjacent normal prostate tissue [45]. Positive staining was only seen in scattered cells in both tumor and normal tissue regions but was much more consistently observed in areas of proliferative inflammatory atrophy, lesions that have been implicated in prostatic carcinogenesis. Studies cited above clearly point to the need of additional studies to examine COX-2 expression in diverse population groups of prostate cancer and in transgenic and knockout models of COX-2 expression in prostate. The expression of COX-2 in human prostate carcinoma cells remains controversial. Either relatively down modulated or undetectable levels of COX-2 expression was observed in prostate cancer cell lines by several investigators. For example in LNCaP, PC-3, DU145 and tumor necrosis factor (TSU) prostate cancer cell lines, COX-2 expression was undetectable under basal conditions but was found to be transiently induced in PC-3 and TSU cells when they were subjected to phorbol ester treatment [45]. Interestingly, in another study basal COX-2 mRNA and protein expression was found to be higher in normal prostate epithelial cells compared to its level of expression in PC-3, LNCaP and DU145 prostate carcinoma cells [46]. Similarly, in one study COX-2 transcripts were found to be absent in LNCaP and PC-3 cells [47]. In another study LNCaP and PC-
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3 prostate carcinoma cells were shown to exhibit trace amounts of COX-1 and COX-2 expression as measured by reverse transcription-polymerase chain reaction and immunoblot analysis [48]. Therefore, there are two significantly contrasting observations exist, one endorsing the marked up regulation of COX-2 and its possible involvement in prostate cancer development and progression, while the other, promoting a COX-2-independent mechanisms for prostate tumor development.
6. Role of COX-2 in prostate carcinogenesis There are multiple mechanisms through which COX-2 may play a role in carcinogenesis and some or all of these may be involved in prostate cancer development and progression [49]. Number of these mechanisms are likely to result from COX-2-induced increase in PG synthesis. Evidence that increased PG synthesis has both growth-promoting and positive feedback effects in prostate cancer was provided in a study by Tjandrawinata et al. [50] where treatment of prostate carcinoma cells PC-3 with exogenous PGE2 was found to result in increased mitogenesis and COX-2 up-regulation. Further, treatment of PC-3 cells with 5 mM flurbiprofen in the presence of exogenous PGE2 inhibited the up-regulation of COX-2 mRNA and cell growth. Another study has demonstrated that PGE2 stimulates PIN cell growth through activation of the interleukin (IL)-6/GP130/signal transducers and activators of transcription (STAT)-3 signaling pathway [51]. PGE2 was found to stimulate soluble IL-6 receptor (sIL-6R) release, gp130 dimerization, STAT-3 protein phosphorylation, and DNA binding activity which led to increased PIN cell growth. This study provides mechanistic evidence that increased expression of COX-2/PGE2 contributes to prostate cancer development and progression via activation of the IL-6 signaling pathway. COX-2 over-expression has been shown to upregulate Bcl2 expression with an associated decrease in apoptosis [52]. Accordingly, in a recent study, the human prostate carcinoma LNCaP cells, which overexpresses COX-2, exhibits apoptosis induction and Bcl2 down-modulation when treated with NS398, a selective inhibitor of COX-2 enzyme function [53]. Inhibition of COX-2 by celecoxib has been shown to
induce apoptosis in both androgen-responsive LNCaP and androgen-unresponsive PC-3 cells by blocking Akt phosphorylation which is independent of Bcl2 [54]. In addition, COX-2 was found to be induced by TNF-a, which underlines the inducibility of COX-2 in response to pro-inflammatory stimulus [46]. These experimental data demonstrate the inflammatory, antiapoptotic and growth stimulating nature of COX-2 in prostate cancer and therefore suggest a positive role for COX-2 in prostate cancer development and progression in humans. Further, Bcl2 expression has been shown to be closely associated with the androgenindependent prostate cancer phenotype and represents a potential pathway through which COX-2 may induce prostate cancer progression to an androgen-independent state [55]. Contrary to well established experimental data that NSAIDs and other COX-2 specific inhibitors exert their chemopreventive action by specific inhibition of COX-2 activity, several recent studies have proposed the existence of COX-2independent mechanism as a mode of action of these agents in their ability to prevent cancer. For example, it has been shown that sulindac derivatives inhibit cell growth and induce apoptosis in prostate cancer cells PC-3 and LNCaP with similar sensitivity despite these cells express COX-2 at varying levels [48]. This similarity in sensitivity to apoptosis by sulindac compounds led to the prediction of involvement of COX-2-independent pathway. Similarly celecoxib, a selective COX-2 inhibitor was reported to induce apoptosis by interfering with functioning of Akt, extracellular-regulated kinases and endoplasmic reticulum Ca2þ-adenosine triphosphatases [54,56]. Disruption of these signaling pathways led to rapid apoptosis which was distinct from the one induced by NSAIDs, suggesting the COX-2-independent mechanism in celecoxib induced apoptosis. In a recent study, using Tet-On antisense COX-2 clones it was demonstrated that celecoxib-induced apoptosis in PC3 and LNCaP prostate carcinoma cells is independent of COX-2 inhibition [57]. Although there is a correlation between the use of NSAIDs and the anticarcinogenic effects, the direct demonstration that these agents inhibit COX-2 activity in their action both in vitro and in vivo remains to be established. Other effects of COX-2 overexpression that may contribute to the malignant phenotype include decreased E-cadherin expression with consequent
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loss of cell-to-cell adhesion, matrix-metalloproteinase overexpression with an associated increase in invasiveness, and modulated production of angiogenic factors by cancer cells [58,59]. A correlation was found between hypoxia-induced COX-2 expression and up regulation of vascular endothelial growth factor (VEGF) in PC-3 and LNCaP prostate carcinoma cells [60]. Moreover, the COX-2-dependent effect on VEGF up-regulation was found to be inhibited by treatment with COX-2 specific inhibitor NS398 and this inhibitory effect was reversed by PGE2 treatment [61]. Since VEGF plays an important role in angiogenesis, its up-regulation by COX-2 expression and inhibition by COX-2 specific inhibitor suggests a positive role of COX-2 in angiogenesis, an important event in prostate metastasis [61]. In another study, PC-3 tumor growth in nude mice was shown to be regressed by administration of COX-2 specific inhibitor and the regression was found to be associated with a significant decrease in VEGF suggesting a role for COX-2 in prostate cancer angiogenesis [62]. Aimed at understanding the role of PG in invasive potential of prostate carcinoma cells, it was been demonstrated that inhibitors of PG synthesis inhibit invasiveness and the secretion of matrix metalloproteinase (MMPs) [63]. This inhibition of prostaglandin biosynthesis has been shown to alter the balance between MMPs and tissue specific inhibitors of matrix metalloproteinases thereby inhibiting tumor invasion in prostate cancer. Since COX-2 reactions involve production of reactive oxygen radicals that can potentially damage biological macromolecules, in another study [64] the possibility that DNA and/or nucleosides can be oxidized during COX reactions was examined. DNA or nucleosides incubated with COX-2 and arachidonic acid resulted in a significant increase in the amount of 8-oxo-2’-deoxyguanosine. This increase was found to be enzyme-dependent and could be prevented by COX-2 inhibitors as well as by antioxidants. These data for the first time indicated that peroxyl radicals or other oxidized species formed during conversion of arachidonic acid to PGG2 might be responsible for increased oxidation of DNA bases. These results suggested that overexpression of COX-2 in inflammatory diseases like prostate cancer places an additional burden on antioxidative defenses of the cell, which might contribute to DNA oxidation and the induction
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of mutations. Further, studies have also shown that overexpression of COX-2 in cancer cells inhibit immune surveillance and increase metastatic potential [65]. These studies need validation in prostate cancer.
7. NSAIDs and prostate cancer NSAIDs have long been known for their analgesic, antipyretic and anti-inflammatory function [66,67]. Since inflammation is closely related to tumor promotion, agents with potent anti-inflammatory activities are anticipated to exert chemopreventive effects. These observations led to a wide variety of investigations to determine whether or not these drugs have an ability to reduce the risk of progression of several human cancers. NSAIDs and their mode of action in cancer chemoprevention has been the focus of many epidemiological and experimental studies, which support the importance of these drugs in cancer chemoprevention [68]. Epidemiological studies have shown a decreased risk of some cancers in people who regularly take aspirin or other NSAIDs [68,69]. Many subsequent studies in several human cancers have established the anti-tumorigenic effect of these drugs. These studies have shown that antitumorigenic action of NSAIDs is mediated by selective inhibition of COX, particularly COX-2. Non-specific NSAIDs such as aspirin, sulindac and indomethacin inhibit not only the enzymatic action of inducible and pro-inflammatory COX-2, but also the constitutively expressed, cytoprotective COX-1 as well. Consequently, non-selective NSAIDs can cause platelet dysfunction, gastrointestinal ulceration, and kidney damage [70 –72]. For this reason, selective inhibition of COX-2 to treat neoplastic proliferation is preferred over non-selective inhibition. Selective COX-2 inhibitors such as melaxicam, celecoxib and rofecoxib are NSAIDs that have been modified chemically to preferentially inhibit COX-2 without affecting COX-1 [73]. The epidemiological evidence for a protective effect of NSAIDs in prostate cancer development and progression is equivocal. A population-based, casecontrolled study from New Zealand, reported a trend towards reduced risk of advanced prostate cancer associated with regular use of NSAIDs while these associations failed to reach statistical significance [74]. Another study by Nelson and Harris [75] has demon-
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strated that regular daily use of ibuprofen or aspirin was associated with a 66% reduction in prostate cancer risk (odds ratio ¼ 0.34, 95% confidence interval ¼ 0.23– 0.58, P , 0:01). The risk of prostate cancer was also significantly reduced in men who were reported to be taking prescription NSAIDs (odds ratio ¼ 0.35, 95% confidence interval ¼ 0.15 – 0.84, P , 0:05). Another recent study [76] employing 1362 white men ranging from 50 –79 year-old from the Olmsted County, Minnesota using prescription and nonprescription NSAIDs has shown that daily use of NSAIDs may be associated with a lower incidence of prostate cancer in men aged 60 years or older. The stronger effect among older men suggests that NSAIDs may prevent the progression rather than early stages of prostate cancer from latent to clinical disease. Overall, these results suggest that NSAIDs may have value in the chemoprevention of prostate cancer. Experimental studies on animal models of prostate cancer have shown that NSAIDs exert chemopreventive as well as therapeutic effects. In a study by Pollard and Luckert [77] where treatment of transplantable rat prostate adenocarcinoma III cells which produce local tumors and osteolytic and osteoplastic lesions and metastasize through defined lymphatic channels to the lungs, with NSAID piroxicam resulted in suppression of tumor growth, bone destruction, and metastasis. In chemically-induced prostate carcinogenesis F344 rat model supplementation with indomethacin in drinking water, exhibited tumor suppressive effects and significant decrease in tissue PGE2 levels [78]. Noble prostate cancer-bearing rats treated with PG modulators viz. indomethacin (a general COX inhibitor), UK 38485 (thromboxane synthase inhibitor), and nafazatron (anti-thrombotic agent which increases prostacyclin), had significantly lower pulmonary metastasis than untreated controls [79,80]. In a rat model, COX-2 inhibitors increased tumor response to radiation treatment without increasing the radiation effects on normal tissues [81]. Therefore, the role of COX-2 inhibitors in prostate cancer prevention and treatment either alone or in combination with chemotherapy or radiotherapy is worthy for further exploration. In a recent study [82] from our group we investigated the effect of human recommended dose of celecoxib, a specific COX-2 inhibitor against prostate carcinogenesis in a transgenic adenocarcinoma of the mouse
prostate (TRAMP) model. In TRAMP, expression of the SV40 early genes (T and t antigen, Tag) are driven by the prostate-specific promoter probasin that leads to cell transformation within the prostate that histologically resembles to human prostate cancer. The basal enzyme activity and protein expression of COX-2 was found to be significantly higher (, 3.2-fold) in the dorso-lateral prostate of TRAMP mice compared to their nontransgenic littermates. This suggested that COX-2 may have a causative role in prostate cancer development in this model. We employed 8 week old TRAMP mice and randomly divided them in groups consuming control diet (AIN 76A) or a custom prepared AIN 76A diet containing 1500 ppm celecoxib ad libitum for 24 weeks. The animals fed control diet developed palpable tumors at 12 – 14 weeks while at these times no palpable tumors were observed in animals fed celecoxibsupplemented diet. Sequential magnetic resonance imaging analysis of celecoxib-fed mice at 16, 24, and 32 weeks of age suggested that each time point prostate volume was lower in celecoxib-fed group compared to control group. These results were consistent with significant decrease in prostate and genito-urinaryweight in celecoxib-fed mice at the termination of the experiment. Feeding celecoxib in the diet was found to result in significant inhibition of distant site metastases to lymph nodes and lungs. These results were further confirmed by the histopathological examination of the tissue. Compared to animals consuming control diet, celecoxib supplemented animals showed reduced proliferation, and down-modulation of COX-2 in the dorso-lateral prostate. Furthermore, celecoxib supplementation resulted in enhanced in vivo apoptosis in the dorso-lateral prostate. Taken together, these studies suggest that NSAIDs and other selective COX-2 inhibitors possess chemopreventive activity against prostate carcinogenesis.
8. Conclusions There is ample evidence that COX-2 and its PG products may play a critical role in prostate cancer development and progression. Significant advances have been made in the past 5 years in understanding the COX-pathway in prostate, however, the relationship between COX-2 and prostate carcinogenesis remains to be more clearly elucidated. Animal models
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of COX-2 overexpression and knockouts specifically in prostate needs to be developed. It will be useful to study the downstream signaling pathways and apoptotic response for the preventive role of COX-2 inhibitors in mouse models of prostate cancer. It will be of great interest to examine if COX-2 is sufficient or need cooperation with other factors to induce prostate carcinogenesis. It will be important to study the specific roles that COX-1 and COX-2 plays during the development of prostate cancer and to examine the key modulator(s) for development of prostate adenocarcinoma: total PG production or the different PG products derived from the COX-1 and COX-2 pathway. Lastly, unraveling the COX-2-dependent versus COX-2-independent effects of the COX-2 inhibitors in prostate cancer may reveal the cellular mechanisms of COX-2 action and its role in prostate cancer development. Answers to these questions will provide a molecular basis for understanding the contributing role of COX-2 in mechanism of prostate carcinogenesis and the effectiveness of the use of COX-2 specific and non-specific inhibitors in different stages of prostate cancer treatment. However, based on available information it appears that COX-2 plays an important role in prostate cancer development and progression and therefore selective COX-2 inhibitors may have promise in prostate cancer management.
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[17]
Acknowledgements Original work from authors laboratory cited in this review is supported by the funds from United States Public Health Service RO3 CA 89739 (to H.M. and S.G.) and Cancer Research Foundation of America (to S.G.).
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Mini-review
Cyclooxygenase-2 and prostate carcinogenesis Tajamul Hussaina, Sanjay Guptab, Hasan Mukhtara,* a
Department of Dermatology, University of Wisconsin, Medical Science Center, 1300 University Avenue, Madison, WI, 53706, USA b Department of Urology, Jim & Eilleen Dicke Research Laboratory, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH, 44106, USA Received 23 August 2002; accepted 29 August 2002
Abstract In recent years a dramatic surge has occurred on studies defining to the role of cyclooxygenase (COX)-2 in causation and prevention of cancer. Prostaglandin (PG) endoperoxidase synthase also commonly referred to as COX is a key enzyme involved in the conversion of arachidonic acid to PGs and other eicosanoids. COX exists as two isoforms, namely COX-1 and COX-2 with distinct tissue distribution and physiological functions. COX-1 is constitutively expressed in many tissues and cell types and is involved in normal cellular physiological functions whereas COX-2 is pro-inflammatory in nature and is inducible by mitogens, cytokines, tumor promoters and growth factors. A large volume of data exists showing that COX-2 is overexpressed in a large number of human cancers and cancer cell lines. The possibility of COX-2 as a candidate player in cancer development and progression evolved from the epidemiological studies which suggest that regular use of aspirin or other non-steroidal antiinflammatory drugs could significantly decrease the risk of developing cancers in experimental animals and in humans. In our recently published study (Prostate, 42 2000 73 – 78), we provided the first evidence that COX-2 is overexpressed in human prostate adenocarcinoma. Many other studies verified our initial observation and reported that compared to normal tissue, COX-2 is overexpressed in human prostate cancer. It should be noted that some recent work has suggested that COX-2 is only upregulated in proliferative inflammatory atrophy of the prostate, but not in prostate carcinoma. In this scenario, COX-2 inhibitors could afford their effects against prostate carcinogenesis by modulating COX-2 activity in other cells in prostate. An exciting corollary to this ongoing work is that selective COX-2 inhibitors may exhibit chemopreventive and even chemotherapeutic effects against prostate carcinogenesis in humans. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cyclooxygenase enzyme; Prostate cancer; Non-steroidal anti-inflammatory drugs; Inflammation; Prostaglandin
1. Introduction The incidence of prostate cancer has strongly increased during the past decades and it has now become the most common malignancy of men in many Western nations [1]. In the USA, among males, prostate cancer is the second leading cause of cancer* Corresponding author. Tel.: þ1-608-263-3927; fax: þ 1-608263-5223. E-mail address: [email protected] (H. Mukhtar).
related deaths next only to lung cancer [2]. According to projections by the American Cancer Society, a total of 189 000 men will be diagnosed with prostate cancer in the USA in the year 2002, and 30 200 prostate cancer-related deaths are predicted this year [3]. Of great concern is that fact that more than 50% of prostate cancer patients present with or develop incurable metastatic disease [4]. Although most patients with advanced prostate cancer initially respond to androgen ablation treatment, relapse to an androgen-indepen-
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3835(02)00524-4
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Fig. 1. Bi-functional role of the COX enzyme in the biosysnthesis of prostaglandins, prostacyclins and thromboxanes and their physiological and pathophysiological effects.
dent state occurs shortly leading to tumor outgrowth [5]. Despite the clinical importance of prostate cancer, the molecular mechanisms underlying the development and progression of this disease are poorly understood. Therefore, much research is needed towards understanding the mechanisms involved in development and progression of prostate cancer and developing new strategies for its prevention and treatment. The present article reviews the role of COX-2 in prostate carcinogenesis. Based on the evidence available, selective COX-2 inhibitors offer promise for prevention and therapy of prostate cancer.
2. Cyclooxygenases and prostaglandins Cyclooxygenase (COX) or prostaglandin (PG) endoperoxide synthase is a rate limiting enzyme in the PG biosynthesis [6]. COX is a bi-functional enzyme containing a COX site that converts arachi-
donic acid to prostaglandin endoperoxide synthases (PGG)2 and a peroxidase site that reduces PGG2 to PGH2 (Fig. 1) [7]. The PGs are a diverse group of autocrine and paracrine hormones that mediate many cellular and physiological processes [6 – 8]. COX exists in two isoforms commonly known as COX-1 and COX-2, the order in which they were identified [8 –10]. Although both isoforms catalyze the same enzymatic reactions and have similar Km and Vmax values for arachidonic acid, significant differences exist between them [11]. COX-1 is constitutively expressed in majority of the cells performing the house keeping functions that require immediate generation of prostanoids related to vascular homeostasis, water reabsorption, gastric acid secretion, platelet aggregation and renal blood flow [10,11]. COX-1 is located on human chromosome 9q32-q33.3 spanning 25 kb in size containing 11 exons and produces 2.8 kb mRNA which synthesizes about 68 kDa protein [10,11]. In contrast, COX-2 is a pro-inflammatory and inducible enzyme
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Fig. 2. Possible involvement of COX-2 and their inhibitors in various stages of prostate cancer.
which can be induced by mitogens, tumor promoters, cytokines and growth factors in different cell types and controlled at both the transcriptional and post-translational levels [10 –12]. COX-2 in involved in differentiative processes, such as inflammation, ovulation, and labor, in situation where only transient PG production is required [9 –11]. COX-2 is an 8 kb gene with 10 exons located on human chromosome 1q25.2 –q25.3 and transcribes a 4.1– 4.5 kb mRNA which encodes a protein of about 68 kDa [9 – 11]. Although, genes for COX-1and COX-2 are located on two separate chromosomes but they are highly related at the DNA, RNA, and protein level. COX-1 and COX2 consist of 576 and 587 amino acids, respectively, and they share approximately 60% primary sequence homology [12,13]. Both these enzymes exist as integral membrane glycoprotein homodimers and are found on the luminal surfaces of the endoplasmic reticulum and nuclear envelop [14,15].
studies have shown that overexpression of COX-2 is sufficient to cause tumorigenesis in animal models and subsequently inhibition of the COX-2 pathway results in reduction in tumor incidence and progression (Fig. 2) [20]. There are some exceptions to this generally accepted observation. One recent study has shown that COX-2 overexpression in the skin of transgenic mice results in suppression of tumor development [21]. In sharp contrast, another recent study has shown that deficiency of COX-2 reduces mouse skin tumorigenesis [22]. Based on these observations it is clear that much additional work with targeted tissue specific over and under expression of COX-2 is required to firmly establish the role of this enzyme in target organ carcinogenesis. However, based on these evidences the potential application of non-steroidal anti-inflammatory drugs (NSAIDs) as well as recently developed COX-2 specific inhibitors in cancer clinical practice has drawn tremendous attention in the past few years as inhibition of COX-2 offers an effective approach in the prevention and treatment of cancer [23,24].
3. COX-2 and cancer In recent years, overexpression of COX-2 has been implicated in the progression of cancer [16,17]. Aberrant or increased expression of COX-2 has been found in most of the cancers of the body sites [9, 16 – 18]. Compelling evidence from genetic and clinical studies indicates that COX-2 upregulation is one of the key steps in carcinogenesis [19]. Recent
4. Diet and prostaglandins in prostate carcinogenesis Epidemiologic studies and nutritional data suggest that androgens and/or environmental exposures, such as diet in particular, dietary fat, play an important role in prostate carcinogenesis [25]. The association
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between diet and prostate cancer has been drawn from the studies where incidence and mortality rates of prostate cancer vary widely between different populations in various regions of the world consuming more dietary fat [26]. Number of studies have reported this link; countries such as the USA, known to have high levels of fat consumption, were also found to have a high mortality rate for prostate cancer, whereas a country such as Japan, with one of the lowest rates of fat consumption in the world, has a low mortality rate for the disease [27]. In addition, migratory studies have further confirmed this observation where it was found that Asian men migrating to the United States acquire a higher clinical incidence of prostate cancer, and subsequent generations of American born Asian men have prostate cancer risks almost equal to those of white Americans [28]. These studies suggest that a change in dietary habits can greatly modify prostate cancer risk. However, the results of most case-control studies demonstrated a significant association of prostate cancer risk with high dietary intake of total fat while some studies did not find a significant association [29,30]. Laboratory studies with experimental animal models further suggest a link between fat content in diet and the risk of prostate cancer [31]. Arachidonic acid and its precursor, linoleic acid, are major ingredients of animal fat and many vegetable oils used in the regions where prostate cancer is more common [32]. Studies have shown that treatment of androgen-unresponsive human prostate carcinoma cells PC-3 with linoleic acid stimulated the growth of these cells [33].The effects of these fatty acids are thought to be caused by their effects on PG synthesis. A study by Hughes-Fulford et al. [34] has demonstrated that linoleic acid, arachidonic acid and the arachidonic acid metabolite prostaglandin (PGE)(2) stimulate prostate tumor growth and alters gene expression in human prostate carcinoma PC-3 cells. Treatment of PC-3 cells with arachidonic acid was shown to result in a dose-dependent increase in the gene expression of c-fos, and COX-2, while the constitutive COX-1 message was not increased. Further effects of dietary fat was examined on the in vivo models, where transplantation of human prostate carcinoma cells DU145 to nude mice fed with high fat diet was shown to result in a significant increase in tumor growth compared to the control group of
animals fed with regular diet [35]. These studies suggest a stimulatory effect of dietary n-6 fatty acid on prostate cancer cell growth which may be critical for the development and progression of prostate cancer. Intake of unsaturated dietary fat may affect PG synthesis, that appear to influence sex hormone levels; this raises the possibility that increased levels of androgens could play an important role in the initiation of prostate cancer [36]. Studies have shown that testosterone is capable of stimulating oxidative stress in prostate carcinoma cells that has been suggested to be a mechanism of initiation of prostate carcinogenesis [37]. However, there is no such study indicating that increase in testosterone levels leads to an increase in COX-2 expression during the development and progression of prostate cancer.
5. COX-2 and prostate cancer Studies on relationship of COX and prostate were initiated in 1993 by O’Neill and Ford-Hutchinson [38] who analyzed COX-1 and COX-2 mRNA expression in various human tissues and reported the highest levels in the prostate where COX-1 and COX-2 transcripts were found to be present in approximately equal levels. In the year 2000, we provided the first evidence that COX-2 is overexpressed in human prostate adenocarcinoma [39]. Employing 12 pairs of unique benign and prostate carcinoma tissue from the same individuals we showed that mean levels of COX-2 mRNA expression was significantly increased in prostate adenocarcinoma. These results were further verified by the COX-2 protein expression, which was significantly higher in cancer tissue compared to their benign counterparts. Many other studies verified our initial observation and reported that compared to normal tissue, COX-2 is overexpressed in human prostate cancer. A study by Yoshimura et al. [40] analyzed tumor specimens obtained from 28 prostate carcinoma patients, eight benign prostatic hyperplasia (BPH) patients, one prostatic intraepithelial neoplasia (PIN) patient, and eight specimens of normal prostate tissue and showed very weak expression of COX-1 and marked expression of immunoreactive COX-2 in prostate tumor cells. The expression of both COX-
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isoforms was found to be very weak in all cases of BPH and in the normal prostate tissues. Further, the extent and intensity of immunoreactive COX-2 polypeptides in tumor cells was statistically much greater than those of cells from BPH. These results were further confirmed by mRNA analysis, where enhanced expression of COX-2, but not COX-1, was observed in prostate cancer tissues. These results lead to the conclusion that human prostate carcinoma cells generated COX-2, and that COX-2 might play an important role in the proliferation of prostate carcinoma cells. Studies by Kirschenbaum et al. [41] analyzed thirty-one specimens of prostate carcinoma and 10 specimens of BPH and showed that COX-1 expression in noncancerous prostatic tissue was predominantly (90% positive staining) seen in the basal epithelial cells of BPH. COX-1 expression was minimal in noncancerous luminal epithelial cells and was found to be upregulated in prostate cancer predominantly in the smooth muscle cells of the prostate. COX-2 was found to be expressed in the basal epithelial cells with 60% BPH, 94% peripheral zone, 75% PIN, respectively. The expression of COX2 in prostate cancer was found to be intense and uniform, with 87% of samples demonstrating immunoreactivity. The results of this study indicated that expression of both COX-1 and COX-2 in human prostate cancer is increased suggesting that COX-1 and COX-2 (and/or their PG products) may play a role in the malignant transformation of the prostate. In another study, Madaan et al. [42] have determined COX-1 and COX-2 expression in 30 BPH and 82 prostate cancer specimens. In this study a significant COX-2 overexpression in tumor cells was found compared to benign glands; however, COX-1 expression in tumor cells was similar to benign glands. A significant positive correlation between COX-2 expression was found with increasing tumor grade suggesting that COX-2 may play an important role in prostate carcinogenesis. These studies are in agreement with the study by Lee et al. [43] where COX-2 was found to be over-expressed in 15 out of 18 (83%) prostate cancer samples and was detected in only 22% (4/18) of paired benign tissues. Further, the intensity of immunostaining correlated with the tumor grading of these specimens. Another recent study by Uotila et al. [44] has compared COX-1 and COX-2 mRNA and protein expression from 12 prostate
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cancer specimens and 13 control prostates. The intensity of COX-2 was found to be significantly stronger in prostate cancer cells than in the nonmalignant glandular epithelium of the control prostates. COX-2 was found to be clearly expressed in the lesions of PIN in control prostates and was also detected in the muscle fibers of the hyperplastic stroma. No significant difference was found in COX-1 expression between control and prostate cancer. The results of this study indicated that the expression of COX-2 is elevated in prostatic adenocarcinoma and in PIN. These experimental data generated from both human prostate tumor tissue specimen and from prostate cancer cell lines demonstrated an upregulation of inducible COX-2 expression and suggested a positive role for COX-2 in prostate carcinogenesis. However, in contrast to these observations a recent study performed immunohistochemical analysis of 144 human prostate cancer cases and found that there was no consistent overexpression of COX-2 in established prostate cancer or high grade PIN, as compared with adjacent normal prostate tissue [45]. Positive staining was only seen in scattered cells in both tumor and normal tissue regions but was much more consistently observed in areas of proliferative inflammatory atrophy, lesions that have been implicated in prostatic carcinogenesis. Studies cited above clearly point to the need of additional studies to examine COX-2 expression in diverse population groups of prostate cancer and in transgenic and knockout models of COX-2 expression in prostate. The expression of COX-2 in human prostate carcinoma cells remains controversial. Either relatively down modulated or undetectable levels of COX-2 expression was observed in prostate cancer cell lines by several investigators. For example in LNCaP, PC-3, DU145 and tumor necrosis factor (TSU) prostate cancer cell lines, COX-2 expression was undetectable under basal conditions but was found to be transiently induced in PC-3 and TSU cells when they were subjected to phorbol ester treatment [45]. Interestingly, in another study basal COX-2 mRNA and protein expression was found to be higher in normal prostate epithelial cells compared to its level of expression in PC-3, LNCaP and DU145 prostate carcinoma cells [46]. Similarly, in one study COX-2 transcripts were found to be absent in LNCaP and PC-3 cells [47]. In another study LNCaP and PC-
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3 prostate carcinoma cells were shown to exhibit trace amounts of COX-1 and COX-2 expression as measured by reverse transcription-polymerase chain reaction and immunoblot analysis [48]. Therefore, there are two significantly contrasting observations exist, one endorsing the marked up regulation of COX-2 and its possible involvement in prostate cancer development and progression, while the other, promoting a COX-2-independent mechanisms for prostate tumor development.
6. Role of COX-2 in prostate carcinogenesis There are multiple mechanisms through which COX-2 may play a role in carcinogenesis and some or all of these may be involved in prostate cancer development and progression [49]. Number of these mechanisms are likely to result from COX-2-induced increase in PG synthesis. Evidence that increased PG synthesis has both growth-promoting and positive feedback effects in prostate cancer was provided in a study by Tjandrawinata et al. [50] where treatment of prostate carcinoma cells PC-3 with exogenous PGE2 was found to result in increased mitogenesis and COX-2 up-regulation. Further, treatment of PC-3 cells with 5 mM flurbiprofen in the presence of exogenous PGE2 inhibited the up-regulation of COX-2 mRNA and cell growth. Another study has demonstrated that PGE2 stimulates PIN cell growth through activation of the interleukin (IL)-6/GP130/signal transducers and activators of transcription (STAT)-3 signaling pathway [51]. PGE2 was found to stimulate soluble IL-6 receptor (sIL-6R) release, gp130 dimerization, STAT-3 protein phosphorylation, and DNA binding activity which led to increased PIN cell growth. This study provides mechanistic evidence that increased expression of COX-2/PGE2 contributes to prostate cancer development and progression via activation of the IL-6 signaling pathway. COX-2 over-expression has been shown to upregulate Bcl2 expression with an associated decrease in apoptosis [52]. Accordingly, in a recent study, the human prostate carcinoma LNCaP cells, which overexpresses COX-2, exhibits apoptosis induction and Bcl2 down-modulation when treated with NS398, a selective inhibitor of COX-2 enzyme function [53]. Inhibition of COX-2 by celecoxib has been shown to
induce apoptosis in both androgen-responsive LNCaP and androgen-unresponsive PC-3 cells by blocking Akt phosphorylation which is independent of Bcl2 [54]. In addition, COX-2 was found to be induced by TNF-a, which underlines the inducibility of COX-2 in response to pro-inflammatory stimulus [46]. These experimental data demonstrate the inflammatory, antiapoptotic and growth stimulating nature of COX-2 in prostate cancer and therefore suggest a positive role for COX-2 in prostate cancer development and progression in humans. Further, Bcl2 expression has been shown to be closely associated with the androgenindependent prostate cancer phenotype and represents a potential pathway through which COX-2 may induce prostate cancer progression to an androgen-independent state [55]. Contrary to well established experimental data that NSAIDs and other COX-2 specific inhibitors exert their chemopreventive action by specific inhibition of COX-2 activity, several recent studies have proposed the existence of COX-2independent mechanism as a mode of action of these agents in their ability to prevent cancer. For example, it has been shown that sulindac derivatives inhibit cell growth and induce apoptosis in prostate cancer cells PC-3 and LNCaP with similar sensitivity despite these cells express COX-2 at varying levels [48]. This similarity in sensitivity to apoptosis by sulindac compounds led to the prediction of involvement of COX-2-independent pathway. Similarly celecoxib, a selective COX-2 inhibitor was reported to induce apoptosis by interfering with functioning of Akt, extracellular-regulated kinases and endoplasmic reticulum Ca2þ-adenosine triphosphatases [54,56]. Disruption of these signaling pathways led to rapid apoptosis which was distinct from the one induced by NSAIDs, suggesting the COX-2-independent mechanism in celecoxib induced apoptosis. In a recent study, using Tet-On antisense COX-2 clones it was demonstrated that celecoxib-induced apoptosis in PC3 and LNCaP prostate carcinoma cells is independent of COX-2 inhibition [57]. Although there is a correlation between the use of NSAIDs and the anticarcinogenic effects, the direct demonstration that these agents inhibit COX-2 activity in their action both in vitro and in vivo remains to be established. Other effects of COX-2 overexpression that may contribute to the malignant phenotype include decreased E-cadherin expression with consequent
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loss of cell-to-cell adhesion, matrix-metalloproteinase overexpression with an associated increase in invasiveness, and modulated production of angiogenic factors by cancer cells [58,59]. A correlation was found between hypoxia-induced COX-2 expression and up regulation of vascular endothelial growth factor (VEGF) in PC-3 and LNCaP prostate carcinoma cells [60]. Moreover, the COX-2-dependent effect on VEGF up-regulation was found to be inhibited by treatment with COX-2 specific inhibitor NS398 and this inhibitory effect was reversed by PGE2 treatment [61]. Since VEGF plays an important role in angiogenesis, its up-regulation by COX-2 expression and inhibition by COX-2 specific inhibitor suggests a positive role of COX-2 in angiogenesis, an important event in prostate metastasis [61]. In another study, PC-3 tumor growth in nude mice was shown to be regressed by administration of COX-2 specific inhibitor and the regression was found to be associated with a significant decrease in VEGF suggesting a role for COX-2 in prostate cancer angiogenesis [62]. Aimed at understanding the role of PG in invasive potential of prostate carcinoma cells, it was been demonstrated that inhibitors of PG synthesis inhibit invasiveness and the secretion of matrix metalloproteinase (MMPs) [63]. This inhibition of prostaglandin biosynthesis has been shown to alter the balance between MMPs and tissue specific inhibitors of matrix metalloproteinases thereby inhibiting tumor invasion in prostate cancer. Since COX-2 reactions involve production of reactive oxygen radicals that can potentially damage biological macromolecules, in another study [64] the possibility that DNA and/or nucleosides can be oxidized during COX reactions was examined. DNA or nucleosides incubated with COX-2 and arachidonic acid resulted in a significant increase in the amount of 8-oxo-2’-deoxyguanosine. This increase was found to be enzyme-dependent and could be prevented by COX-2 inhibitors as well as by antioxidants. These data for the first time indicated that peroxyl radicals or other oxidized species formed during conversion of arachidonic acid to PGG2 might be responsible for increased oxidation of DNA bases. These results suggested that overexpression of COX-2 in inflammatory diseases like prostate cancer places an additional burden on antioxidative defenses of the cell, which might contribute to DNA oxidation and the induction
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of mutations. Further, studies have also shown that overexpression of COX-2 in cancer cells inhibit immune surveillance and increase metastatic potential [65]. These studies need validation in prostate cancer.
7. NSAIDs and prostate cancer NSAIDs have long been known for their analgesic, antipyretic and anti-inflammatory function [66,67]. Since inflammation is closely related to tumor promotion, agents with potent anti-inflammatory activities are anticipated to exert chemopreventive effects. These observations led to a wide variety of investigations to determine whether or not these drugs have an ability to reduce the risk of progression of several human cancers. NSAIDs and their mode of action in cancer chemoprevention has been the focus of many epidemiological and experimental studies, which support the importance of these drugs in cancer chemoprevention [68]. Epidemiological studies have shown a decreased risk of some cancers in people who regularly take aspirin or other NSAIDs [68,69]. Many subsequent studies in several human cancers have established the anti-tumorigenic effect of these drugs. These studies have shown that antitumorigenic action of NSAIDs is mediated by selective inhibition of COX, particularly COX-2. Non-specific NSAIDs such as aspirin, sulindac and indomethacin inhibit not only the enzymatic action of inducible and pro-inflammatory COX-2, but also the constitutively expressed, cytoprotective COX-1 as well. Consequently, non-selective NSAIDs can cause platelet dysfunction, gastrointestinal ulceration, and kidney damage [70 –72]. For this reason, selective inhibition of COX-2 to treat neoplastic proliferation is preferred over non-selective inhibition. Selective COX-2 inhibitors such as melaxicam, celecoxib and rofecoxib are NSAIDs that have been modified chemically to preferentially inhibit COX-2 without affecting COX-1 [73]. The epidemiological evidence for a protective effect of NSAIDs in prostate cancer development and progression is equivocal. A population-based, casecontrolled study from New Zealand, reported a trend towards reduced risk of advanced prostate cancer associated with regular use of NSAIDs while these associations failed to reach statistical significance [74]. Another study by Nelson and Harris [75] has demon-
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strated that regular daily use of ibuprofen or aspirin was associated with a 66% reduction in prostate cancer risk (odds ratio ¼ 0.34, 95% confidence interval ¼ 0.23– 0.58, P , 0:01). The risk of prostate cancer was also significantly reduced in men who were reported to be taking prescription NSAIDs (odds ratio ¼ 0.35, 95% confidence interval ¼ 0.15 – 0.84, P , 0:05). Another recent study [76] employing 1362 white men ranging from 50 –79 year-old from the Olmsted County, Minnesota using prescription and nonprescription NSAIDs has shown that daily use of NSAIDs may be associated with a lower incidence of prostate cancer in men aged 60 years or older. The stronger effect among older men suggests that NSAIDs may prevent the progression rather than early stages of prostate cancer from latent to clinical disease. Overall, these results suggest that NSAIDs may have value in the chemoprevention of prostate cancer. Experimental studies on animal models of prostate cancer have shown that NSAIDs exert chemopreventive as well as therapeutic effects. In a study by Pollard and Luckert [77] where treatment of transplantable rat prostate adenocarcinoma III cells which produce local tumors and osteolytic and osteoplastic lesions and metastasize through defined lymphatic channels to the lungs, with NSAID piroxicam resulted in suppression of tumor growth, bone destruction, and metastasis. In chemically-induced prostate carcinogenesis F344 rat model supplementation with indomethacin in drinking water, exhibited tumor suppressive effects and significant decrease in tissue PGE2 levels [78]. Noble prostate cancer-bearing rats treated with PG modulators viz. indomethacin (a general COX inhibitor), UK 38485 (thromboxane synthase inhibitor), and nafazatron (anti-thrombotic agent which increases prostacyclin), had significantly lower pulmonary metastasis than untreated controls [79,80]. In a rat model, COX-2 inhibitors increased tumor response to radiation treatment without increasing the radiation effects on normal tissues [81]. Therefore, the role of COX-2 inhibitors in prostate cancer prevention and treatment either alone or in combination with chemotherapy or radiotherapy is worthy for further exploration. In a recent study [82] from our group we investigated the effect of human recommended dose of celecoxib, a specific COX-2 inhibitor against prostate carcinogenesis in a transgenic adenocarcinoma of the mouse
prostate (TRAMP) model. In TRAMP, expression of the SV40 early genes (T and t antigen, Tag) are driven by the prostate-specific promoter probasin that leads to cell transformation within the prostate that histologically resembles to human prostate cancer. The basal enzyme activity and protein expression of COX-2 was found to be significantly higher (, 3.2-fold) in the dorso-lateral prostate of TRAMP mice compared to their nontransgenic littermates. This suggested that COX-2 may have a causative role in prostate cancer development in this model. We employed 8 week old TRAMP mice and randomly divided them in groups consuming control diet (AIN 76A) or a custom prepared AIN 76A diet containing 1500 ppm celecoxib ad libitum for 24 weeks. The animals fed control diet developed palpable tumors at 12 – 14 weeks while at these times no palpable tumors were observed in animals fed celecoxibsupplemented diet. Sequential magnetic resonance imaging analysis of celecoxib-fed mice at 16, 24, and 32 weeks of age suggested that each time point prostate volume was lower in celecoxib-fed group compared to control group. These results were consistent with significant decrease in prostate and genito-urinaryweight in celecoxib-fed mice at the termination of the experiment. Feeding celecoxib in the diet was found to result in significant inhibition of distant site metastases to lymph nodes and lungs. These results were further confirmed by the histopathological examination of the tissue. Compared to animals consuming control diet, celecoxib supplemented animals showed reduced proliferation, and down-modulation of COX-2 in the dorso-lateral prostate. Furthermore, celecoxib supplementation resulted in enhanced in vivo apoptosis in the dorso-lateral prostate. Taken together, these studies suggest that NSAIDs and other selective COX-2 inhibitors possess chemopreventive activity against prostate carcinogenesis.
8. Conclusions There is ample evidence that COX-2 and its PG products may play a critical role in prostate cancer development and progression. Significant advances have been made in the past 5 years in understanding the COX-pathway in prostate, however, the relationship between COX-2 and prostate carcinogenesis remains to be more clearly elucidated. Animal models
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of COX-2 overexpression and knockouts specifically in prostate needs to be developed. It will be useful to study the downstream signaling pathways and apoptotic response for the preventive role of COX-2 inhibitors in mouse models of prostate cancer. It will be of great interest to examine if COX-2 is sufficient or need cooperation with other factors to induce prostate carcinogenesis. It will be important to study the specific roles that COX-1 and COX-2 plays during the development of prostate cancer and to examine the key modulator(s) for development of prostate adenocarcinoma: total PG production or the different PG products derived from the COX-1 and COX-2 pathway. Lastly, unraveling the COX-2-dependent versus COX-2-independent effects of the COX-2 inhibitors in prostate cancer may reveal the cellular mechanisms of COX-2 action and its role in prostate cancer development. Answers to these questions will provide a molecular basis for understanding the contributing role of COX-2 in mechanism of prostate carcinogenesis and the effectiveness of the use of COX-2 specific and non-specific inhibitors in different stages of prostate cancer treatment. However, based on available information it appears that COX-2 plays an important role in prostate cancer development and progression and therefore selective COX-2 inhibitors may have promise in prostate cancer management.
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Acknowledgements Original work from authors laboratory cited in this review is supported by the funds from United States Public Health Service RO3 CA 89739 (to H.M. and S.G.) and Cancer Research Foundation of America (to S.G.).
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