Eicosanoid Biosynthetic Pathway
Arachidonic acid, a polyunsaturated fatty acid, is almost exclusively found as an ester at the 2-position of membrane phospholipids (Dempke et al. 2001). Arachidonic acid is produced through hydrolysis of phospholipids, which is catalyzed by phospholipase A2. The next two steps in this pathway, COX reaction and peroxidation reaction, both of which are catalyzed by COX, produce unstable bicyclic peroxide intermediate, PGG2, and thereafter PGH2, respectively. PGH2 is the common precursor from which all other prostanoids such as PGE2, PGD2, PGF2a, PGI2, HHT, TXA2, etc., are synthesized in reactions catalyzed by specific synthetases.
There are at least two isoenzymes in COX, COX1 and COX2, of which genes are located on different chromosomes. COX1 and COX2 genes are located on chromosome 9 (22 kb containing 11 exons) and 1 (8.3 kb with 10 exons), respectively.
Proteins with Functional Moieties
PGs are arachidonate metabolites which have diverse biologic activities, including vasoconstriction, vasodilation, stimulation or inhibition of platelet aggregation, and immumomodulation, primarily immunosuppression (Dempke et al. 2001). The wide implication of PGs in critical physiologic roles in tissue homeostasis and function made it possible to focus on modulation of COXs for controlling various clinical situations. For example, the inhibition of COX in platelets reduces the production of TXA2, which contributes to rising of bleeding time and delay of platelet aggregation (Nandakishore et al. 2014). There are important differences in the physiologic roles of the two COX isoforms, COX1 and COX2.
COX1 is constitutively expressed in most human cells and known to have “housekeeping” functions under basal conditions (Yu et al., 2016). In the stomach, it produces PGI2 and PGE2 which reduce gastric acid secretion and stimulate mucus production with vasodilation forming a protective layer. In the kidney, PGs such as PGI2, PGE2, and PGD2 exert vasodilation and maintain renal blood flow enhancing organ perfusion. In the central nervous system, most abundantly in the forebrain, PGs may be involved in complex, integrative functions (Bertolini et al. 2002). COX1 is also important for establishing and maintaining healthy pregnancy given the expression in the uterine epithelium and fetal and amniotic cells, and even sustaining the integrity of platelet function.
On the other hand, COX2 was known to be only induced specifically in the site of inflammation by cytokines, growth factors, mitogen, and hormones, and primarily involved in producing PGs in response to environmental and internal stimuli (Lee et al. 2013). However, accumulating evidence shows that COX2 also plays physiologic roles in homeostasis (Bertolini et al. 2002). COX2, like COX1, constitutively expressed in kidneys, heart, brain, and the central nervous system, where COX2 is involved in the degeneration of neuronal connections in cases of stress, fever response, protection of intestinal cells during inflammatory conditions, relaxation of vascular smooth muscles, and platelet constriction.
COX3, an alternative splice isoform of COX1, functions in conditions of pain and fever. However, the exact role of COX3 is yet to be defined.
Multiple Roles in Inflammation and Carcinogenesis
Chronic inflammation and cancer have a lot in common. Dvorak’s postulation, “tumors are wounds that never heal,” explains these characteristics well. Both inflammation and cancer tissues involve angiogenesis and tissue-infiltrating leukocytes, which may trigger cellular stress by secreting reactive oxygen species (ROS). Inflammatory immune cells surrounding a developing tumor create a local and systemic environment of the chronic inflammatory state by releasing numerous proinflammatory mediators, including PGs (Surh and Kundu 2007). Chronic inflammation characterized by elevated levels of proinflammatory mediators often excessively produce ROS and reactive nitrogen species which are potentially damaging to cellular macromolecules, such as DNA, proteins, and lipids (Surh and Kundu, 2007). Epidemiologic studies consistently show that more than 20% of cancers are associated with the inflammatory response which is a prominent feature in all types of solid tumors (Qiao and Li 2014). About 5% of all human colorectal cancer is associated with ulcerative colitis. Inflammatory cytokines and chemokines might activate signaling molecules involved in inflammation and carcinogenesis such as COX2 and nuclear factor-kappa B (NF-κB), which induce preneoplastic mutation, stumulation of angiogenesis, and resistance to apoptosis (Kim et al. 2013). Cytokines from inflammatory cells can also promote the outgrowth of neoplastic cells (Yu et al. 2016). Aberrant COX2 expression in cancers was recognized as one of the hallmarks of chronic inflammation-associated malignancies (Kim et al. 2008).
Mechanism of Action
There are abundant evidence suggesting the potential contribution of COX2 to the development and/or progression of various types of cancers. Inappropriate over-expression of COX2 has been implicated in the pathogenesis of various types of cancers such as colorectal, breast, gastrointestinal, pancreatic, and head/neck cancers (Surh and Kundu 2007). Prognostic association of COX2 expression was also found in colorectal, gastrointestinal, thyroid, breast, oral cavity, ovary, and lung cancers (Yu et al., 2016; Lee et al. 2013). For colorectal cancer, multiple lines of literature demonstrated a 40–50% decrease in relative risk for colorectal cancer in regular users of NSAIDs including aspirin (Dubois et al. 1998). Interestingly, a selective COX2 inhibitor could reduce cell growth only in colorectal cancer cell lines with high level of COX2 expression, however, not in cell lines without COX2 expression (Blobaum and Marnett 2007). Increasing adhesion to extracellular matrix making intestinal epithelial cells resistant to apoptosis and promoting angiogenesis were also included suggested mechanisms of tumorigenic potential of COX2 overexpression. For uterine cervical cancer, COX2 upregulation through NF-κB and activator protein-1 (AP-1) was shown to be a mechanism underlying human papillomavirus 16 E5 oncoprotein-mediated carcinogenesis (Kim et al. 2009). Jo et al. reported DNA hypermethylation of COX2 gene might be a potential prognostic marker in early stage cervical cancer of uterus (Jo et al. 2007). Transgenic mice overexpressing COX2 in mammary glands, skin, or stomach develop malignancies of these organs, whereas knocking out the COX2 gene suppresses the development of intestinal tumors and skin papillomas (Surh and Kundu 2007).
Molecular Regulation of COX2 Gene
Although COX2 expression is aberrantly increased in colorectal adenoma and cancer, higher in carcinoma than adenoma, the mechanisms underlying increased COX2 expression in colorectal carcinomas is still unclear. Colorectal adenomas and cancers frequently express high levels of a variety of tumor promoters and receptor-mediated signals triggered by interleukins, transforming growth factor (TGF)-β, and ligands of epidermal growth factor receptor (EGFR), which could stimulate constitutive COX2 expression (Dempke et al. 2001). There is another evidence of regulation of COX2 gene by hypoxic cellular conditions. Hypoxia induces COX2 expression via the NF-κB in vascular endothelial cells. NF-κB-response elements exist in the promoter of the COX2 gene, underlying that the transcription factor NF-κB may control COX2 in terms of hypoxia-induced angiogenesis (Dempke et al. 2001). NF-κB binds to specific DNA sequences and activates target genes that regulate apoptosis and stimulate metastasis (Yu et al. 2016). For example, NF-κB has an important transcription function in MMP-2/9 and VEGF gene expression. Other than NF-κB, there are lots of other transcriptional factors that regulate COX2 expression, including CCAAT/enhancer binding protein (C/EBP), polyoma enhancer activator 3 (PEA3), nuclear factor for activating T cells (NFAT), cyclic AMP-response element binding proten (CREB), activator protein (AP)-1 and 2, and SP-1 .
Inhibition of COX
NSAID is one of the classes of COX inhibitors that directly targets PG synthesis; therefore, it provides antipyretic and analgesic effects as well as anti-inflammatory effect at its higher dose (Nandakishore et al. 2014). This anti-inflammatory effect of NSAIDs were shown to be through inhibition of PGG and PGH synthesis from arachidonic acid mediated by the catalysis of COXs, both COX1 and COX2 (Vane and Botting 2003). NSAIDs also show their antipyretic effect by inhibiting PGE2 synthesis, which is responsible for increase in body temperature by triggering the hypothalamus during inflammation (Nandakishore et al., 2014). While the anti-inflammatory and analgesic effects of traditional NSAIDs are caused by COX2 inhibition, the ulcerogenic side effects of COX inhibitors are due to COX1 inhibition (Blobaum and Marnett 2007). For reducing gastrointestinal toxicity profiles of traditional NSAIDs, selective inhibitors of COX2 were developed in the late 1990s (Dempke et al. 2001). Although the additional chemopreventive effect of selective COX2 inhibitors proven in many studies, particularly in studies of colorectal cancer, many of them (rofecoxib, valdecoxib, or lumiracoxib) were withdrawn from the worldwide market due to increased cardiovascular adverse events, such as death, myocardial infarction, and stroke (Blobaum and Marnett 2007; Nandakishore et al. 2014). Given the finding that COX2 inhibitors reduce the urinary excretion of the major metabolite of PGI2 and PGE2, COX-dependent products of vascular endothelial cells regulating vascular tone and atherosclerosis, decreased level of PGI2 by COX2 inhibitors appear to explain how to COX2 inhibition leads to cardiovascular toxicity (Blobaum and Marnett 2007). A randomized controlled trial showed that administration of celecoxib for median 14.2 months could increase risk of hypertension in participants with pre-existing cardiovascular risk factors compared with placebo (Thompson et al. 2016). On the other hand, a recently published systematic review also concluded adverse events between the COX2 inhibitors and placebo were uncertain (Miao et al. 2014).
Clinical Implication in Chemoprevention
COX2 is thought as an interface between inflammation and cancer (Surh and Kundu 2007). A recent randomized controlled trial showed that the administration of celecoxib just for limited duration (median 14.2 months) could prevent adenoma recurrence in patients with prior high-risk adenomas (relative risk = 0.23, 95% confidence interval = 0.07 to 0.80, p = 0.02) (Thompson et al. 2016). Although the detailed mechanism of the role of COX2 in the development of cancer is still not fully understood, a recent review summarized that the role of COX2 plays a major role in the progression of colorectal cancer (Liu et al. 2016).
Proinflammatory stimuli trigger the activation of an intracellular signal transduction network comprising proline-directed serine/threonine kinases and their downstream transcription factors, resulting in an inappropriate induction of COX2 (Surh and Kundu 2007). Considering chronic inflammation-associated abnormal cellular signaling as a critical factor in carcinogenesis, certain types of malignancy, at least, chronic inflammation-associated malignancies can be prevented by intervention with anti-inflammatory therapy (Surh and Kundu 2007). Therefore, anti-inflammatory natural products, so-called, phytochemicals have been highlighted as a cancer preventive method based on their underlying molecular mechanisms with a focus on representative transcription factors and upstream kinases responsible for COX2 induction (Surh and Kundu 2007).
There are many pytochemicals proven to possess anticancer property, such as resveratrol (in red wine), curcumin (in rhizomes of turmeric), capsaicin (in hot chilli pepper), genistein (in soy products), and epigallocatechin gallate (EGCG). For example, EGCG, an ingredient from green tea, was shown to suppress COX2 expression in human prostate and colorectal cancer cells. The inhibitory effects of EGCG on the production of PGE2 suggested that not only the expression but also the activity of COX2 was inhibited by EGCG. EGCG also shows downregulatory effect on upstream kinases and transcription factors such as AP-1 and NF-κB, which might leads to the inhibition of upstream mitogen-activated protein (MAP) kinases (Surh and Kundu, 2007). The other anti-inflammatory phytochemicals were also found to have an inhibitory impact on COX2 expression in variety of cancer cells through their inhibition of inflammatory signaling mediated by AP-1, NF-κB, and their upstream kinases (Fig. 3) (Surh and Kundu 2007). Therefore, the modulation of the aforementioned components of inflammatory signaling pathway by these phytochemicals appeared to be the molecular details of cancer prevention by dietary constituents via COX2 inhibition without significant adverse events.
Current Status Selective COX2 Inihibitors
COX2 selective inhibitors have lost the cardiovascular protective effects of nonselective NSAIDs, effects which are mediated through COX1 inhibition. In addition, COX2 has a role in sustaining vascular PGI2 production (Bertolini et al. 2002). At last, COX2 selective inhibitors were withdrawn from many parts of world due to their potential side effects which is caused by the imbalance between PGI2 synthesis and TXA2. Selective inhibition of COX2 results in relative elevation of TXA2, which increases platelet aggregation and vasoconstriction, and in turn increases cardiovascular risks. After withdrawal of rofecoxib in 2004, other selective COX2 inhibitors including celecoxib have been more focused in clinical trials of cancer prevention. Because the cardiovascular side effects of the selective COX2 inhibitors are not universal for all classes of COX2 inhibitors and not significant in certain groups of users, it is important to identify patients with low cardiovascular risk and balance the risk and benefit case by case.
COX1 is expressed in most tissues but variably. It is described as a “housekeeping“ enzyme, regulating normal cellular processes such as gastric cytoprotection, vascular homeostasis, platelet aggregation, and kidney function, and is stimulated by hormones or growth factors. COX2 is usually undetectable in most tissues; its expression is increasing during states of inflammation, or experimentally in response to mitogenic stimuli. COX2 is constitutively expressed in the brain, kidney, bone, and probably in the female reproductive system. Inducible isoform of COX2, which appears to be devoid of gastrointestinal toxicity, spares mucosal PG synthesis. COX2 definitely plays an important role in multiple diseases including cancer through its role in the pathogenesis of inflammation. Therefore, selective COX2 inhibitors remain attractive options for several major unmet medical needs including rheumatoid arthritis, cancer, and neurodegeneration despite potential cardiovascular side effects.
- Miao XP, Li JS, Ouyang Q, Hu RW, Zhang Y, Li HY. Tolerability of selective cyclooxygenase 2 inhibitors used for the treatment of rheumatological manifestations of inflammatory bowel disease. Cochrane Database Syst Rev. 2014;CD007744.Google Scholar
- Thompson PA, Ashbeck EL, Roe DJ, Fales L, Buckmeier J, Wang F, et al. Celecoxib for the prevention of colorectal adenomas: results of a suspended randomized controlled trial. J Natl Cancer Inst. 2016;108.Google Scholar