Cyclooxygenase (COX) which has two isoforms, COX-1 and COX-2 is the enzyme that catalyzes the rate-limiting step in prostaglandin synthesis, converting arachidonic acid into prostaglandin H₂, which is then further metabolized to prostaglandin E2 (PGE₂), PGF_2α, PGD₂ and other eicosanoids 56. COX-1 is constitutively expressed in many tissues and plays a role in tissue homeostasis. COX-2, which can be expressed in a variety of cells and tissues, is an inducible isoform the expression of which is stimulated by growth factors, cytokines, and tumor promoters. Despite the structural similarity between the two isoforms, COX-1 and COX-2 differ substantially in the regulation of their expression and their roles in tissue biology and disease 56. In the past decade, tremendous progress has been made in understanding the functional roles of COX in cancer development and growth. COX-2 is up-regulated in many cancer types, including the colon, breast, lung, pancreas, and esophagus as well as squamous cell carcinoma of the head and neck 7891011. COX-2 specific inhibitors inhibit cell growth in a number of tumors including skin, colonic, gallbladder, esophageal and pancreatic cancer cells 7891011. Studies from both COX-2 transgenic and COX-2 knockout mice confirm that COX-2 plays a key role in colonic cancer development 12. However, a recent study in COX-1 deficient mice, showed that lack of COX-1 also significantly reduced intestinal tumorigenesis in min mice, a phenotype similar to that of COX-2^-/- mice 12. Furthermore, there may be other forms of COX enzymes, making things even more complicated. Simmons and colleagues, recently identified an enzyme that they have called cyclooxygenase-3, or COX-3. COX-3 is an isoform of COX-1, but is expressed in cells in an inducible manner 13. COX-3 is selectively inhibited by different NSAIDs and has a high sensitivity to acetaminophen. However, whether COX-3 is involved in tumorigenesis is unknown 13.

Lipoxygenases possess regiospecificity during interaction with substrates and on this basis have been designated as arachidonate 5-, 8, 12-, 15-lipoxygenase (5-LOX, 8-LOX, 12-LOX, and 15-LOX) 51314151617. The four distinct enzymes insert oxygen at carbon 5, 8, 12 or 15 of arachidonate acid. The primary products are 5S-, 12S-, or 15S-hydroperoxyeicosatetraenoic acid (5-, 8-, 12-, or 15-HPETE), which can be further reduced by glutathione peroxidase to the hydroxy forms (5-, 8-, 12-, 15-HETE) respectively 51314151617. 5-LOX represents a dioxygenase that possesses two distinct enzymatic activities leading to the formation of LTA₄. First it catalyzes the incorporation of molecular oxygen into arachidonic acid (oxygenase activity), producing HPETE and subsequently forms the unstable epoxide LTA₄ (LTA₄ synthase activity) 56. This is followed by the insertion of molecular oxygen at position C-5, converting LTA₄ to either 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetranoic acid (5-HETE) or leukotrienes. Five Lipoxygenase Activating Protein (FLAP), which is a 18 kDa membrane-bound protein, plays an important role in mediating the arachidonic catalytic activity of 5-LOX 56. FLAP activity can be blocked by the FLAP inhibitor, MK886 56. Hydrolytic attack of LTA₄ by leukotriene A₄ hydrolase yields LTB₄, a potent neutrophil chemoattractant and stimulator of leukocyte adhesion to endothelial cells. LTC₄ synthase catalyzes the conjugation of LTA₄ with glutathione to form LTC₄ at the nuclear envelope. The peptide moiety of LTC₄ is subject to extracellular metabolism, forming LTD₄ and LTE₄ 56. These three leukotrienes comprising the cysteinyl leukotrienes, before their structures were identified were known as the "slow-reacting substance of anaphylaxis" named for their slow and sustained smooth muscle contracting abilities 56. Accumulating evidence suggests that the 5-LOX pathway has profound influence on the development and progression of human cancers 18. 5-LOX inhibitors have chemopreventive effects in animal lung carcinogenesis 19. Furthermore, the 5-LOX pathway interacts with multiple intracellular signaling pathways that control cancer cell proliferation 9. Blockade of 5-LOX inhibits prostate cancer cell proliferation while the 5-LOX metabolite, 5-HETE stimulates prostate cancer cell growth 20. Furthermore, the FLAP inhibitor, MK886 exerts a similar growth inhibitory effect on cancer cell proliferation to 5-LOX inhibitors, further supporting a role for 5-LOX as a mediator of cancer cell proliferation 21.

Three forms of 12-lipoxygenase have been identified, including the leukocyte-type and platelet-type (both 12(S) LOXs) as well as an epidermal form (also called 12(R) LOX), with differences in tissue localization, substrate specificities, immunogenicities and sequence homology 1415. The "platelet-type" 12-LOX converts arachidonic acid to 12-(S)-HETE while the "leukocyte type" 12-LOX metabolizes arachidonic acid or linoleic acid to either 12(S)-HETE or 15(S)-HETE 1415. Both the leukocyte-type and platelet-type12-LOX have been found in different cancer tissues, including melanoma, prostate and epidermal cancers 18. Evidence indicates that 12-LOX is involved in both cancer cell proliferation and survival 15. Inhibition of 12-LOX with either 12-LOX inhibitors or a 12-LOX antisense oligonucleotide inhibits proliferation and induces apoptosis in carcinosarcoma cells, while adding back the 12-LOX metabolite, 12(S)-HETE prevents 12-LOX inhibitor-induced apoptosis 22. Expression of 12-LOX is also correlated with tumor cell metastasis. 12(S)-HETE directly stimulates prostate cancer cell migration. Clinically, the degree of 12-LOX expression in human prostate cancer correlates with the tumor grade and stage and 12-LOX expression level is higher in metastatic prostate cancers than in nonmetastatic ones 23.

Another arachidonic acid-metabolizing enzyme is 8-lipoxygenase, which was cloned recently. This enzyme is expressed in the skin after irritation or treatment with tumor promoters 25. To understand the function of the product of 8-lipoxygenase, 8-hydroxyeicosatetraenoic acid transgenic mice have been generated 26. These animals show enhanced differentiation of epidermal keratinocytes along with enhanced proliferation of the basal cell population. While the function of 8-HPETE and 8-HETE are unknown, their importance in tumor promoter-elicited events is suggested by the finding that application of the lipoxygenase inhibitor, eicosatetraenoic acid maximally inhibits tumor promotion when applied at the time of maximum induction of 8-lipoxygenase 2627. Compared with other LOX enzymes, 8-LOX has received little attention for its role in carcinogenesis and cancer growth. Expression and function of 8-LOX in pancreatic cancer have not been investigated to date even though 8-LOX cDNA and antibody are available 27.

15-lipoxygenase, which is distributed widely in tissues, converts arachidonic acid to 15-HPETE which is then reduced by glutathione peroxidase to 15-HETE 16172428. There are two 15-LOX isoenzymes, 15-LOX-1 and 15-LOX-2 28. The preferred substrate for 15-LOX-1 is linoleic acid and for 15-LOX-2 is arachidonic acid. 15-LOX-1 metabolizes linoleic acid to 13-S-HODE, which differs from 15-LOX-2 which, as expected, converts arachidonic acid to 15-HETE 28. The role of 15-LOX in cancer development is unclear and somewhat controversial 28. A series of studies from one group suggest that the 15-LOX-1 product, 13-S-HODE enhances colonic tumorigenesis. 13-S-HODE enhances cell proliferation and potentiates the mitogenic response to EGF in different cell types 2930. These proposed effects, however, are not consistent with other reports that 13-S-HODE did not enhance EGF-dependent DNA synthesis 28. Indeed, Lippman et al reported that 13-S-HODE induces apoptosis and cell cycle arrest in colorectal cancer cells 31. Evidence also indicates that 15-S-HETE may have anti-tumorigenic effects by antagonizing other LOX products, such as LTB₄ and 12-S-HETE 28. 15-HETE inhibits LTB₄ production and reduces LTB₄ binding to its receptors and thereby blocks cellular responses to LTB₄. 15-HETE also blocks platelet-type 12-LOX activity and reduces 5-LOX activity in rat basophilic leukemia cells 2832. Different studies have suggested that 15-S-HETE may suppress apoptosis, or have no effect on apoptosis in cancer cells 28. A recent study has shown that 15-S-HETE inhibits proliferation in PC3 prostate carcinoma cells, possibly through activation of PPARγ 33. Therefore, further studies are required to clarify the role of 15-LOX in tumorigenesis and cancer growth.

Tumorigenesis is a complex process and understanding its mechanisms is the key to designing effective targeted therapies. Several epidemiologic studies have shown an inverse correlation between the incidence of colon cancer and the use of nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit prostaglandin synthesis 8. Because COX-2 is frequently over-expressed in premalignant lesions and neoplasms, specific COX-2 inhibitors have been investigated as chemo-preventive and potential chemotherapeutic agents 678. New preclinical and early clinical data suggest, that inhibitors of COX-2 may protect against colon, breast, lung, esophageal, and oral tumors 8. Even though epidemiological studies on the role of NSAIDs in pancreatic cancer incidence are not as detailed as in colonic cancer, evidence does suggest that NSAIDs might decrease the incidence of pancreatic cancer 34. Recently, Anderson and colleagues 34 from University of Minnesota investigated the association between pancreatic cancer and aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) in 28283 participants. Multivariate analysis suggested that women who used aspirin had a 43% lower risk of pancreatic cancer compared with those who did not use aspirin. Furthermore, the association appeared to be related to the dose; women who took aspirin 2–5 times a week had a 53% lower risk, and those who took aspirin six or more times a week had a 60% lower risk than women who never took aspirin. In contrast to COX inhibitors, epidemiological evidence regarding the effect of LOX inhibitors on pancreatic cancer incidence is lacking, since specific LOX inhibitors have only recently become available in the clinic.

COX-2 is only expressed in pancreatic islets and has no expression in normal exocrine pancreatic tissues 35. A considerable amount of evidence from several clinical studies in the United States and Japan supports that COX-2 is up-regulated in pancreatic adenocarcinoma. Studies that demonstrate this have employed RT-PCR, quantitative RT-PCR, in situ hybridization, western blotting and immunohistochemistry 36373839404142. One report showed that levels of COX-2 mRNA were more than 60-fold increased in pancreatic cancer compared to adjacent non-tumor tissue 36. The COX-2 protein was detected in 90% of pancreatic adenocarcinomas 36. In general, studies suggest that COX-2 is weakly expressed in benign pancreatic adenoma, but dramatically up-regulated in pancreatic adenocarcinoma, even though extent of COX-2 expression differs from one study to another 36373839404142. Expression of COX-2 has been demonstrated in several pancreatic cancer cell lines, although once again there is a lack of consistency between different studies 3843. Nzeako and Gores, analyzed possible reasons for the inconsistent reporting of COX-2 expression by different groups 44. Firstly, the lack of correlation in immunohistochemical staining intensity for COX-2 may result from working with previously fixed and embedded tissue, the fixation methods used and the duration of fixation. The process of fixing and embedding can destroy or denature cellular proteins, thereby altering antigenic recognition by immunohistochemistry 44. Under such circumstances, attempts at correlating staining intensity with other variables are very likely to yield spurious results. In this situation, more reliable results may be obtained using frozen tissues with a standardized fixation procedure. Secondly, the use of polyclonal antibodies for immunohistochemistry may diminish reliability, since other tissue proteins may be inadvertently stained 44. Despite these inconsistencies, all of these studies indicate that COX-2 is over-expressed in pancreatic cancer. A recent study showed, that perineural invasion was associated with COX-2 expression in pancreatic cancer and increased COX-2 expression was more common in the glandular component than the solid component of the tumors 41. Regarding expression of COX-2 in early neoplastic lesions of the pancreas, researchers from John Hopkins immunohistochemically examined expression of COX-2 in adenocarcinomas, pancreatic intraepithelial neoplasia [PanIN] and normal pancreatic ducts with an automated platform 42. The overall percentage of positive cells was 47.3% in adenocarcinomas, 36.3% in PanINs and 19.2% in normal ducts. COX-2 was expressed in more than 20% of cells in 23 adenocarcinomas (77%), 42 PanINs (65%), and 12 normal ducts (40%). Significant differences in COX-2 expression were demonstrable in both adenocarcinomas vs normal ducts and PanINs vs normal ducts 42. The up-regulation of COX-2 in a subset of noninvasive precursor lesions makes it a potential target for chemoprevention using selective COX-2 inhibitors.

Expression of LOX is also up-regulated in both adenocarcinomas and early neoplastic lesions of the pancreas. Reverse transcriptase-polymerase chain reaction revealed expression of 5-LOX mRNA in all of the commonly used pancreatic cancer cell lines, including PANC-1, AsPC-1, and MiaPaCa2 cells, but not in normal pancreatic ductal cells 45. The expression of the 5-LOX protein in pancreatic cancer cell lines was confirmed by western blotting 46. 5-LOX up-regulation in human pancreatic cancer tissues was verified by immunohistochemistry, which revealed intense positive staining in cancer cells 46. Staining of the 5-LOX protein was particularly evident in the ductal components of the more differentiated tumors but, in contrast, ductal cells in normal pancreatic tissues from organ donors did not stain. Immunohistochemistry also revealed strong staining of cancer tissues with an antibody to the receptor of the downstream 5-LOX metabolite, leukotriene B4 46. It also appears that levels of 5-LOX in hepatic metastases of pancreatic cancer express more 5-LOX than the primary tumors 46. These findings provide further evidence of up-regulation of this pathway in pancreatic cancer and suggest that LOX inhibitors are likely to be valuable for the treatment or prevention of this dreadful disease.

There is also RT-PCR evidence of 12-LOX expression in pancreatic cancer cell lines 45. Expression of 12-LOX in pancreatic islets has been reported and it is not yet clear, whether 12-LOX is expressed or up-regulated in pancreatic cancer 47. Whether 15-LOX and 8-LOX are expressed in pancreatic cancer or in normal pancreatic tissues has not been investigated.

The effects of COX inhibition on pancreatic cancer growth has been examined both in vitro and in vivo, using both non-specific NSAIDs and specific COX-2 inhibitors. The effect of these inhibitors depends, at least in part, on the COX-2 pathway in cultured pancreatic cancer cell lines 48. One study showed that COX inhibitors significantly suppressed proliferation of cells that have high levels of COX-2 expression, but exerted minimal effects on proliferation in cells with a significantly lower level of COX-2 48. We have reported that the non-specific COX inhibitor, indomethacin is equally potent in inhibiting pancreatic cancer cell proliferation compared with the specific COX-2 inhibitor, NS398 43.

Another important action of the COX-2 enzyme is inhibition of apoptosis, which in many cases constitutes another mechanism to promote tumor cell growth. Several studies have revealed that COX-2-specific and non-specific NSAIDs induce apoptosis in a variety of cancer cells, including colon, gastric, glioma, and lung cancer cells 678. Recently, we and others have demonstrated that COX inhibitors significantly induce apoptosis in pancreatic cancer cells 43. Non-specific NSAIDs and COX-2 specific inhibitors were also equipotent for inducing apoptosis, suggesting that the COX-2 pathway plays a key role in preventing apoptosis and that NSAIDs reverse this effect by inhibiting the pathway 43. Pancreatic cancer growth is inhibited by COX-2 in animal models as well as in vitro. Our data show that the COX-2 inhibitor, celecoxib significantly decreases both volume and weight of pancreatic tumors xenografted subcutaneously into nude mice.

Compared with the investigations on the role of COX in the development and growth of cancers, information regarding the role of lipoxygenases in the regulation of pancreatic cancer growth is limited. Studies conducted by Anderson et al showed inhibition of pancreatic cancer cell proliferation following treatment with the 5-lipoxygenase activating protein (FLAP) inhibitor, MK886 495051. Coincident with growth inhibition, changes in expression of several genes, including tumor suppressor genes and growth factor receptors were induced by MK886 50. Anderson et al reported that MK886 induces an "atypical" apoptosis in PANC-1 pancreatic cancer cells which is enhanced by gamma linolenic acid 51. We recently explored the effect of LOX inhibition on pancreatic cancer cell proliferation and survival using pharmacological LOX inhibitors and a FLAP inhibitor, as well as antisense oligonucleotides targeting 5-LOX, 12-LOX or 15-LOX expression 4552. Blockade of either 5-LOX or 12-LOX markedly inhibited cell proliferation and induced apoptosis in all of the pancreatic cancer cell lines studied. Apoptosis induced by blockade of either the 5-LOX or 12-LOX enzyme was documented by propidium iodide DNA staining, demonstration of DNA fragmentation by electrophoresis as well as by the TUNEL assay 4552. Antisense oligonucleotides directed against both 5-LOX and 12-LOX had similar inhibitory effects on pancreatic cancer cell proliferation to the small drug LOX inhibitors. In contrast, 15-LOX does not appear to play any important role in pancreatic cancer cell growth, since transfection of 15-LOX antisense oligonucleotide did not affect DNA synthesis of pancreatic cancer cells in our studies 45. These studies support the potential use of LOX inhibitors in pancreatic cancer treatment or prevention. Animal experiments conducted in our laboratory indicate that blockade of LOX pathways also inhibits growth of pancreatic cancers xenografted into athymic nude mice 5354. While the study conducted by Anderson et al showed that blockade of 5-LOX induced only an "atypical" apoptosis, our data clearly show that apoptosis is induced by LOX enzyme inhibition 52. The reasons for these different results are not clear. To confirm that apoptosis occurs in pancreatic cancer cells following LOX inhibitor treatment, we conducted further studies that showed that LOX inhibitors reduce expression of Bcl-2 and Mcl-1 and induce activation of caspase-3 and -9 as well as cleavage of the caspase-3 target, PARP 5354.

Pancreatic carcinoma can be induced in hamster models by administering N-nitrosobis(2-oxopropyl)amine (BOP). This animal model mimics human pancreatic cancer biologically and genetically, including mutated k-Ras, desmoplasia and insulin resistance 555657. Takahashi et al treated hamsters with BOP and then subsequently treated them with the non-selective COX inhibitors, indomethacin or aspirin 58. Animals in the indomethacin group developed significantly fewer tumors than the control group. Aspirin also had a tendency to decrease the incidence of pancreatic cancers 58. Using the same pancreatic cancer model, Wenger et al reported that the specific COX-2 inhibitor, celebrex also prevented pancreatic cancer 59.

Several excellent studies support the use of COX inhibitors for preventing cancer of different organs including colon, lung, breast and pancreas, while exploration of the potential for LOX inhibitors as chemopreventive agents is just beginning. It was recently reported that 5-LOX pathway inhibitors, accolate, MK-886, zileuton, and combinations of zileuton with either accolate or MK-886 reduced lung tumor multiplicity by 37.8, 29.5, and 28.1%, respectively following injection of lung cancer carcinogen vinyl carbamate into mice 60. These inhibitors also decreased the size of the tumors and the yield of carcinomas 60. An extract from the sea cucumber, Cucumaria frondosa which is a potent LOX inhibitor, attenuates pancreatic cancer development. In our recent study, hamsters were fed with an extract of sea cucumber for one week before pancreatic cancer cells were transplanted orthotopically into the pancreas. The extract decreased pancreatic cancer incidence as well as tumor size.

Evidence suggests that the prostaglandin products of the COX pathway, especially PGE2 enhance cell proliferation in either normal and cancer cells through specific receptors 61626364656667. Prostaglandins exert their effects locally in both an autocrine and paracrine manner. PGE₂ receptors are a family of G-protein-coupled receptors, namely, EP₁, EP₂, EP₃, and EP₄ 68. Functional studies on the EP receptors have relied on use of EP knockout mice. The deficiency of the prostaglandin receptor EP₂ gene reduced the size and number of intestinal tumors in APC⁷¹⁶ mice, similar to deficiency of the COX-2 gene 69. Besides prostaglandin E2 and its receptor, thromboxane A2 is also involved in cancer development 707172737475. Thromboxane A2 enhances cancer cell proliferation, while its antagonist blocks cancer cell growth. We have shown that both 5-LOX and 12-LOX are also involved in pancreatic cancer cell proliferation. Both 5-HETE and 12-HETE enhance DNA synthesis in pancreatic cancer cells by activating multiple intracellular signal pathways 767778. Our most recent study suggests that LTB4 receptors are up-regulated in human pancreatic cancer tissues and coincidentally, pancreatic cancer cells produce LTB4 46. This autocrine loop might be important for pancreatic cancer cell growth, as LTB4 itself stimulates pancreatic cancer cell growth while the LTB4 receptor antagonist, LY293111 blocks growth of pancreatic cancer both in vitro and in vivo 79. The signaling pathways that respond to COX and LOX-derived metabolites in cancer cells include ERK1/2, PI3 kinase/AKT cascade, tyrosine kinases, STAT, c-Myc and others which are summarized in Table 1.

Inhibition of either COX or LOX induces apoptosis of pancreatic cancer cells while forced expression of COX-2 prevents apoptosis. Multiple cellular proteins are involved in this process and mitochondria are the key organelles for COX or LOX inhibition-induced apoptosis 83848586. Treatment of cancer cells with COX inhibitors induces cytochrome C release from mitochondria, which in turn activates caspase-9 and then caspase-3 83848586. Cytochrome C release seems to be directly related to COX inhibition since addition of prostaglandin E2 can prevent NS398-induced cytochrome C release, caspase activation and PARP cleavage 83848586. Other studies have shown that over-expression of COX-2 increases Bcl-2 expression, which might be responsible for preventing cytochrome C release from mitochondria 87. A recent study showed that death receptors are also involved in COX-regulated cancer cell survival 888990. Forced COX-2 expression significantly attenuated TRAIL-induced apoptosis and was associated with transcriptional repression of death receptor-5 and up-regulation of Bcl-2 89. Over-expression of COX-2 also reduced caspase-8, caspase-3, and caspase-9 activation relative to corresponding parental cells 89. In contrast, COX inhibitors were able to restore death receptor-5 expression. Several lines of evidence suggests that NFκB and PI3 kinase are involved in NSAID-induced cancer cell apoptosis 89. NSAIDs inhibit NFκB and PI3 kinase activity, while restoration of NFκB or PI3 kinase activity blocks NSAID-induced apoptosis 919293. Inhibition of COX-2 causes an increase in tissue concentrations of ceramide. Since ceramide is an effective inducer of apoptosis, metabolism of arachidonic acid by COX-2 might prevent apoptosis by reducing intracellular ceramide concentrations. Therefore, an imbalance between PGE2 and ceramide may contribute to the apoptosis-preventing effects of COX-2 in cancer cells 9495. The mechanisms involved in COX-mediated cancer cell survival are complex and it is likely that even more pathways involved in these effects will be identified in the future.

To date, three signal cascades have been shown to be linked to LOX-regulated pancreatic cancer cell survival. The first pathway is the Bcl protein family-release of cytochrome C from mitochondria-caspase cascade 5354969798. Treatment of pancreatic cancer cells with either 5-LOX or 12-LOX inhibitors, dramatically upsets the balance between the anti-apoptotic proteins (such as Bcl-2, Mcl-1) and the pro-apoptotic protein (Bax). This increased pro/anti-apoptotic protein ratio triggers cytochrome C release from mitochondria, which in turn activates caspase cascade and results in apoptosis. The second cascade is the extracellular regulated kinase cascade (MEK/ERK). It is known that activation of the MEK/ERK signal transduction pathway prevents apoptosis. Our studies show that the LOX metabolites, 5-HETE, 12-HETE and LTB4 stimulate MEK/ERK phosphorylation 76777880. PI3 kinase/AKT protects cells from apoptosis by phosphorylation of Bad. Bad is a pro-apoptotic member of the Bcl-2 family that can displace Bax from binding to Bcl-2 and Bcl-xL, resulting in cell death. Phosphorylation of Bad results in its binding to 14-3-3 proteins and prevents it binding to Bcl-2 and Bcl-xL. We have shown that both 5-HETE and LTB4 activate the PI3 kinase/AKT cascade, which constitutes the third pathway which links LOX activation to the prevention of apoptosis in pancreatic cancer cells 787980.

It is well documented that COX inhibitors reduce cancer cell migration, cell adhesion and tumor invasiveness 6799. In contrast, constitutive COX-2 expression leads to enhanced cell invasiveness 100101. The biochemical changes associated with over-expression of COX-2 include increased expression and activation of metalloproteinase-2 (MMP-2) and reduced expression of E-cadherin 102103104. We speculate that there are likely to be several pathways that mediate COX-2-promoted cell invasion, which may be cell-type dependent. The multiple effects of COX-2 on cell motility and cell adhesion suggest a key role for COX-2 in tumor invasiveness and angiogenesis.

Both non-specific and specific COX-2 inhibitors significantly inhibit angiogenesis, a process that involves inactivation of the MAPK pathway. COX-2 induces the production of vascular endothelial growth factor (VEGF), a proangiogenic growth factor. This induction is blocked by a COX-2 specific inhibitor, NS-398 105106107. Recently, the molecular mechanism for COX-2-mediated VEGF expression has been elucidated. A large amount of PGE₂ is generated when COX-2 expression is induced or over-expressed in pancreatic cancer. Activation of the PGE₂ surface membrane receptors increases intracellular cAMP, in turn stimulating VEGF production and thereby promoting neovascularization and tumor angiogenesis 108. Separate studies suggest that other products of the COX pathway, thromboxane A₂ and PGI₂ also promote angiogenesis 7275109110. The thromboxane A2 analogue, U46619, stimulates endothelial cell migration 72. On the other hand, basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF), which are angiogenic, increase thromboxane A2 synthesis 111112113. In contrast, inhibition of thromboxane A2 synthesis reduces HUVEC migration stimulated by VEGF or bFGF 75114. Furthermore, the thromboxane A2 receptor antagonist inhibits VEGF- or bFGF-stimulated endothelial cell migration and bFGF-induced angiogenesis in vivo 75114.

LOX activity is required for serum- and bFGF-stimulated endothelial cell proliferation and for modified low-density lipoprotein-induced monocyte binding to endothelial cells 115. 12(S)-HETE can directly stimulate endothelial cell mitogenesis, migration and surface expression of integrin 115116117118. In addition to its role in endothelial cell migration, 12-LOX is also involved in endothelial cell differentiation, seen as tube formation 115. Pretreatment of endothelial cells with the 12-LOX inhibitor, BHPP inhibits the formation of these vessel-like structures in Matrigel. Exogenous 12(S)-HETE can induce a reversible retraction of endothelial cell monolayers cultured on collagen by regulating PKC 115116117118. The involvement of 12-LOX in endothelial cell angiogenic responses seen in vitro has been confirmed in vivo, by the observation that the 12-LOX inhibitor, BHPP significantly reduces bFGF-stimulated angiogenesis 115. The role of 12-LOX in endothelial cell proliferation, together with the finding that 12-LOX is involved in endothelial cell migration and differentiation, implicate the mobilization of arachidonic acid and the generation of 12(S)-HETE in endothelial cells as an early event in the intracellular cascade of angiogenic responses. We have reported that levels of 5-LOX in metastatic pancreatic cancer are higher than in non-metastatic tumor. However, there is little information on the role of 5-LOX on the regulation of angiogenesis and cancer metastasis.

Individuals at high risk for pancreatic cancer include smokers, and persons with all forms of chronic alcoholic, metabolic, tropical or hereditary pancreatitis. The duration of exposure to inflammation seems to be the major factor involved in the transition from benign to malignant condition 119120121122. Cytokine-mediated up-regulation of COX-2 contributes to increased synthesis of PGs in inflamed tissues 119120121122. The occurrence and growth of various tumors are associated with immune suppression and prostaglandin E2 itself is a potent immunsuppressor 119120121. Not only do cancer cells themselves produce prostaglandins, but factors released from tumor cells activate monocytes and macrophages to synthesize PGE₂. In turn, PGE₂ inhibits the production of immune regulatory lymphokines, T-cell and B-cell proliferation, and the cytotoxic activity of natural killer cells, thus favoring tumor growth 119120121. PGE₂ also inhibits the production of tumor necrosis factor-α and induces the production of IL-10, an immunosuppressive cytokine 119120121122123. Recently, more molecular mechanisms for PGE₂-mediated immunosuppression have been revealed. For example, PGE2 suppressed IL-15-mediated human natural killer cell function 123.

Nitric oxide (NO) is a short-lived molecule required for many physiological functions which is produced from L-arginine by NO synthases (NOS) 124. It is a free radical, producing many reactive intermediates that account for its bioactivity 124. Sustained induction of the inducible form of NOS (iNOS) in chronic inflammation may be mutagenic, either through NO-mediated DNA damage or by hindering DNA repair. Thus sustained NO production is potentially carcinogenic 124. Progression of the majority of human and experimental tumors appears to be stimulated by NO resulting from activation of iNOS or constitutive NOS 124. NO can stimulate tumor growth and metastasis by promoting migration and invasion, which may also be triggered by activation of cyclooxygenase 125126. Furthermore, iNOS is induced by COX-2 and there is a significant correlation between positive COX-2 and iNOS expression in cancer tissues 125126.

Both COX and LOX enzymes are peroxidases. It has been proposed that the peroxidase activity of these enzymes may catalyze the conversion of procarcinogens into carcinogens 127128129130131132133134. In the liver, such oxidative reactions are catalyzed principally by cytochrome P-450s. However, pancreatic tissues and other organs frequently have low concentrations of P-450s and, therefore, significant amounts of xenobiotics may be co-oxidized to mutagens by the peroxidase activities of COX and LOX 126. In addition to catalyzing the synthesis of mutagens, both COX-2 and LOX can be induced by carcinogens, such as NNK and BOP 131132133134. Our unpublished data show that both 5-LOX and 12-LOX are induced in pancreatic ductal cells following treatment with BOP or NNK. Other studies showed that carcinogens induce COX-2 expression and COX-2 in turn catalyzes the oxidation of procarcinogens to produce a highly reactive and strongly mutagenic compounds 132127133. Taken together, these findings suggest a role for COX-2 or LOX inhibitors in preventing carcinogen-induced DNA damage, thereby preventing cancer development.