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The Role of Cyclooxygenase-2 in Cell Proliferation and Cell Death in Human Malignancies

The Role of Cyclooxygenase-2 in Cell Proliferation and Cell Death in Human Malignancies Hindawi Publishing Corporation International Journal of Cell Biology Volume 2010, Article ID 215158, 21 pages doi:10.1155/2010/215158 Review Article TheRoleofCyclooxygenase-2inCellProliferationand Cell Death in Human Malignancies 1 1 1 2 1 Cyril Sobolewski, Claudia Cerella, Mario Dicato, Lina Ghibelli, and Marc Diederich LaboratoiredeBiologieMol´eculaire et Cellulaire du Cancer, Hopital ˆ Kirchberg, 9 rue Edward Steichen, 2540 Luxembourg, Luxembourg Dipartimento di Biologia, Universita ` di Roma di Roma Tor Vergata, Via Ricerca Scientifica snc, 00133 Rome, Italy Correspondence should be addressed to Marc Diederich, marc.diederich@lbmcc.lu Received 16 July 2009; Accepted 18 December 2009 Academic Editor: Simone Fulda Copyright © 2010 Cyril Sobolewski et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. It is well admitted that the link between chronic inflammation and cancer involves cytokines and mediators of inflammatory pathways, which act during the different steps of tumorigenesis. The cyclooxygenases (COXs) are a family of enzymes, which catalyze the rate-limiting step of prostaglandin biosynthesis. This family contains three members: ubiquitously expressed COX- 1, which is involved in homeostasis; the inducible COX-2 isoform, which is upregulated during both inflammation and cancer; and COX-3, expressed in brain and spinal cord, whose functions remain to be elucidated. COX-2 was described to modulate cell proliferation and apoptosis mainly in solid tumors, that is, colorectal, breast, and prostate cancers, and, more recently, in hematological malignancies. These findings prompt us to analyze here the effects of a combination of COX-2 inhibitors together with different clinically used therapeutic strategies in order to further improve the efficiency of future anticancer treatments. COX- 2 modulation is a promising field investigated by many research groups. 1. Introduction: Inflammation and ago [3], only recently, this link has been further investigated, thus evidencing that the incidence of several cancers is Cancer are Linked tightly associated to inflammation such as colon, breast, and Inflammation is the major reaction of natural immunity with prostate cancers [4–6]. This hypothesis is supported by the the goal to defend the organism against pathogens. It can be findings that the tumor microenvironment is characterized induced upon bacterial infections by compounds including by the infiltration with different types of immune cells (i.e., lipopolysaccharides, as well as by viruses, which are detected dendritic cells, lymphocytes, and macrophages) responsible by Toll-like receptors (TLRs), expressed by immune cells for the release of cytokines [1]. The role of these cytokines like macrophages. Besides, inflammation can be triggered in tumor incidence has been established in many studies. by physical injuries (i.e., UV) or chemical compounds (i.e., For example, the overexpression of TNFα in transgenic reactive oxygen species) [1]. The activation of specific recep- mice bearing a lung tumor is associated with an increase tors triggers intracellular signals (i.e., NFκB, p38, or MAPKs- of the size of the tumor [7]. Moreover, a chronic intake mediated), which regulate pro-inflammatory cytokine expre- of nonsteroidal antiinflammatory drugs (NSAIDs) leads to ssion, such as interleukin 1 beta (IL1β), tumor necrosis factor a significant reduction in the incidence of such tumors. alpha (TNFα), interleukin 6 (Il6), together with chemokines Colorectal cancer (CRC), which remains an important cause and cell adhesion proteins [1], in turn, leading to the of death in the industrialized world, is one of the most recruitment and the activation of immune cells. characterized types of tumor that benefits from treatment by Several diseases are associated to chronic inflamma- NSAIDs [8]. Interestingly, chronic use of aspirin is reported tion, such as osteoarthritis, Crohn’s disease, and cancer to reduce the relative risk of CRC by about 50% [9]. Familial [2]. Although the first evidence of a connection between adenomatous polyposis, an inherited form of colon cancer, inflammation and cancer dates back to more than a century is characterized by the development of preneoplastic polyps. 2 International Journal of Cell Biology At the molecular level, this disease is caused with a mutation conversion of arachidonic acid released from the plasma of a tumor suppressor gene called Adenomatous polyposis membrane by phospholipase A2 to prostaglandin G2 by coli (APC). It has been shown that the use of NSAIDs, like the cyclooxygenase activity. The second reaction is mediated sulindac, as a chemopreventive treatment, is able to decrease by the peroxidase activity and leads to the conversion the incidence of polyp formation [10]. Similar results were of prostaglandin G2 to prostaglandin H2. Then, different obtained with celecoxib [11], which is now approved by the synthases convert prostaglandin H2 to prostaglandin D2, Food and Drug Administration’s Oncologic Drugs Advisory F2α, E2, I2, and thromboxane A2 (Figure 1). Committee as an adjuvant in FAP therapy. Prostanoids (prostaglandins and thromboxanes) are A body of evidence indicates a role for inflammation immediately released from the cells, where it is believed in the development/modulation of different steps of cancer that they act locally in an autocrine and paracrine manner progression. Inflammation may play a role in tumor initia- through different receptors activating different intracellular tion by triggering the production of reactive oxygen species pathways still to be completely elucidated (Figure 1)[22]. (ROS), responsible for DNA damage, thus increasing the Prostaglandins, specifically, are important for physiological rate of mutations [12]. It may also be implicated in tumor functions like vasodilatation (PGD2, PGE2, PGI2), gastric promotion, where inflammation triggers the secretion of cytoprotection (PGI2), maintenance of renal homeostasis, growth factors, such as the epithelial (EGF) and fibroblast and platelet aggregation. Besides, prostaglandins play a growth factors (FGF). These, in turn, favor the proliferation major role in mediating fever (PGE2), pain sensitivity, and of initiated tumor cells by determining an imbalance between inflammation [21]. cell proliferation and cell death stimuli [6], due to the So far, three isoforms of COXs have been identified. activation of different cell survival pathways [7]. Cyclooxygenase-1 (COX-1) is a glycoprotein of 71kDa, which Besides, the different cytokines produced during inflam- is constitutively expressed in different tissues. COX-1 is mation (i.e., TNFα,IL1β, IL6, and IL8) can also activate encoded by a gene on chromosome 9 and plays a role in tissue several survival pathways, thus leading to an escape of tumor homeostasis by modulating several cellular processes ranging cells from cell death. Well known is the case of TNFα, from cell proliferation to angiogenesis or platelet aggregation produced by tumor and immune cells, which leads to the due to thromboxane production [21]. survival of cancer cells by the upregulation of antiapoptotic Cyclooxygenase-2 (COX-2) is the inducible isoform, proteins, that is, Bcl-2 [13–15], via the activation of the which is regulated by growth factors and different cytokines nuclear factor kappa B (NFκB) [16]. The modulation of such as IL1β,IL6,orTNFα [23], therefore overexpressed pro-survival pathways or anti-apoptotic proteins makes the during inflammation. The COX-2 gene is located on chro- expression/activation of such proinflammatory mediators mosome 1 and its promoter displays an NFκBresponse also a determining factor in chemoresistance. A constitu- element as well as other cytokine-dependent (i.e., IL6) tive activation of such proinflammatory factors has been response elements [21]. The protein shows a 60% homology frequently found in many cancers, such as hepatocellular with COX-1 [24]; in addition, COX-2 presents a C-terminal carcinoma [17], prostate cancer [18], as well as chronic and extension and a different binding site for NSAIDs, which acute myeloid leukemia [19], where it is frequently associated makes COX-2 a preferential target compared to COX-1, thus with a bad prognosis. In these instances, the modulation being specifically inhibited at lower doses [25]. of Bcl-2 anti-apoptotic family members has been frequently Finally, COX-3 has been identified as a splice variant of shown [13–15, 20]. COX-1, and it is present mainly in brain and spinal cord Amongst the different mediators of inflammation, the [26, 27]. Currently, the role of COX-3 is not known. Some cyclooxygenases (COXs) clearly appear to be implicated in pieces of evidence suggest a possible role in pain sensitivity, cancer. This review focuses on COX-2, the inducible form, based on studies focused on the mechanism of action of normally induced and implicated in inflammation, and acetaminophen (paracetamol), recently evoked as a selective intends to analyze what is currently known about the link inhibitor of COX-3 [28]. However, this hypothesis is debated between COX-2 and cancer, in terms of effects on cell pro- because other findings argue that acetaminophen targets at liferation and cell death. In this view, we will focus our the same time COX-2 [29]. attention on studies analyzing the effects of COX-2 inhibitors on cancer cells, when used alone as well as in combination 3. COX-2 As a Tumor Promoter and a Good with therapeutic approaches, including radiotherapy, chem- Candidate for Cancer Therapy otherapeutic agents, and photodynamic therapy. Finally, we will consider the relevance of COX-2-independent effects. Overexpression of COX-2 has been detected in a number of tumors,suchascolorectalbreastaswellaspancreatic and lung cancers [2, 30–32], where it correlates with a 2. The Cyclooxygenase Enzyme Family poor prognosis. Moreover, overexpression of COX-2 has Cyclooxygenases (or prostaglandin H synthases), commonly been reported in hematological cancer models such as referred to as COXs, are a family of myeloperoxidases located RAJI (Burkitt’s lymphoma) and U937 (acute promonocytic at the luminal side of the endoplasmic reticulum and nuclear leukemia) [33, 34]aswellasinpatient’s blastcells [32, 34]. membrane [21], which catalyze the rate-limiting step of Data suggested that COX-2 may play a role in different steps prostaglandin biosynthesis from arachidonic acid [21]. These of cancer progression, by increasing proliferation of mutated enzymes act by two coupled reactions. The first one is the cells [30], thus favoring tumor promotion as well as by International Journal of Cell Biology 3 Arachidonic acid COX-2 NSAIDs, COX-2 selective PGG2 inhibitors COX-2 PGH2 Isomerases PGF2α PGD2 PGJ2 PGE2 PGI2 EP 1, 2, 3, 4 FP receptor DP receptor PPARγ IP receptor PPARδ Figure 1: Metabolism of arachidonic acid by COX-2 and receptors implicated in response to prostaglandins (according to Chandrasekharan et al. [21]). Prostaglandins act through different receptors to mediate their effects. PGE2 is able to bind four receptors (EP1, 2, 3, and 4). These receptors do not possess the same ligand affinity and their expression is tissuedependent. The different receptors are associated with different intracellular pathways. Most of these receptors are localized in the plasma membrane but nuclear receptors PPARγ can also bind PGJ2. Abbreviation: COX-2, cyclooxygenase-2; PG, prostaglandin; FP, prostaglandin F receptor; DP, prostaglandin D receptor; EP, prostaglandin E receptor; IP, prostaglandin I receptor; PPAR, peroxisome proliferator-activated receptor; NSAIDs, nonsteroidal anti-inflammatory drugs. affecting programmed cell death and affecting the efficacy are mostly described for adherent tumors while this link of anticancer therapies [35–39] to be, finally, implicated in is poorly understood for hematopoietic malignancies such metastasis formation, for example, by affecting apoptosis as leukemia or lymphoma. Indeed, several papers have induced by loss of cell anchorage (anoikis) [40]. reported that PGE2 is the most important prostaglandin COX-2 induction or overexpression is associated with an produced during colorectal carcinogenesis [44]. Moreover, it increased production of PGE2, one of the major products of is known that the level of PGE2 increases in a size-dependent COX-2 which is known to modulate cell proliferation, cell manner in Familial Adenomatous Polyposis (FAP) patients death, and tumor invasion in many types of cancer including [45], suggesting a correlation between tumor growth and colon, breast, and lung. Prostaglandin E2 acts through prostaglandin biosynthesis. Tumorigenesis is characterized different membrane receptors called EP receptors (EP1, EP2, by a disequilibrium between cell proliferation and cell death. EP3, and EP4) [41]. Thesereceptors areall locatedon PGE2 is able to inhibit apoptosis in human colon cancer the cell surface and characterized by seven-transmembrane cells. It has been demonstrated that PGE2 can upregulate domains, and rhodopsin-type G protein-coupled receptors, the level of the anti-apoptotic protein Bcl-2 in HCA-7 cells but trigger different signaling pathways. Thus, it is known (adenocarcinoma), which produce significant amounts of that EP1 signaling acts through phospholipase C/inositol PGE2. This paper described a modulation of the MAPK triphosphate signaling, leading to intracellular mobilization pathway that precedes the upregulation of Bcl-2 [46]. PGE2 of calcium. EP2 and EP4 receptors are coupled with G can mediate its effect through EGF receptor, leading to proteins which activate adenylate cyclase, leading to an MAPK activation. The ability of PGE2 to modulate tumor increase of intracellular cAMP [41]. cAMP is then able to progression in colorectal cell has been shown in other activate kinases such as protein kinase A (PKA) or PI3K for models of colon cancer such as HT-29 cells that express example, and also GSK3 leading to an activation of β-catenin, EP receptors. In this cell type, PGE2 is associated with an a pathway regulating cell proliferation [42, 43]. In contrary increase of cAMP through EP4 receptor. The effect can be to EP2 and EP4, EP3 is coupled with Gi protein, leading reversed by L-161982, an antagonist of EP4 [47]. Moreover, to an inhibition of adenylate cyclase, and thus a decrease PGE2 transactivates EGFR by triggering the release of of cAMP inside the cells [41]. The differential expression amphiregulin, a well-known EGFR ligand [48]. SC-236, an of these different receptors according to the cell type may inhibitor of COX-2, is able to inhibit cell proliferation of explain the diverse and antagonist effects of PGE2 described HT-29 cells and this effect is greater in combination with an in literature. amphiregulin neutralizing antibody [47]. In this cell line, the Until now, there are multiple evidences about the role expression of amphiregulin is correlated to the expression of of PGE2 in tumorigenesis in some cancers. These evidences COX-2. 4 International Journal of Cell Biology The transactivation of EGFR by PGE2 can lead also to AKT Alltogether these data together suggest that PGE2 and, activation, which is a well-known survival pathway [49]. thus, COX-2 play an important role in tumor progression This effect was well described in a study by Tessner et al. by enhancing cell proliferation, cell survival, and tumor [50] demonstrating that 16,16-dimethyl PGE2 (dmPGE2) invasion. The diversity of PGE2 receptors and their different inhibits radiation-induced apoptosis in the mouse intestinal signaling pathways suggest that the protumorigenic effect epithelium. Using HCT-116 cell line as a model to reflect of PGE2 depends on the cell type and the type of receptor the effect on mouse small intestine, it has been shown expressed. Until now, many signaling pathways associated that the anti-apoptotic effect of dmPGE2, which is known with tumor progression are linked to PGE2 and this could to bind EP2, was tightly related to AKT phosphorylation explain why the use of COX-2 inhibitors is a good strategy through activation of EGFR and leads to an inhibition of in cancer therapy. However, the signaling pathways of EP Bax translocation in mitochondria, an important step for receptors are not completely characterized and their precise apoptosis [51]. roles in the different cancers remain to be elucidated before a PGE2 modulates also tumor growth of lung cancer. This clinical application. effect has been described by Yamaki et al. [52] showing COXs may be targets of several compounds that may that PGE2 activates Src kinase in A549 cells, leading to inhibit their functions. Combination of such preferential or an induction of cell growth. These cells express EP3 that selective COX-2 inhibitors with anti-cancer agents already activates Src (sarcoma) kinase. This study has demonstrated used in clinics were tested with the goal to improve the that the activation of Src leads to an activating phosphory- efficiency of anti-cancer protocols. lation of STAT3, a transcription factor known to regulate COX-2 is the preferential target of several NSAIDs cyclin D1 transcription, an important positive regulator (Figure 2)[64, 65]. Historically, NSAIDs used for clinical and of cell proliferation. Apoptosis can be inhibited because anti-inflammatory purposes were represented by the nonse- STAT3 regulates the transcription of Bcl-XL, a well-known lective COX-2 inhibitors, to which belong aspirin, sulindac anti-apoptotic protein [53]. Moreover, Src phosphorylates acid and, more recently, agents such as nimesulide, ibuprofen p27, a protein known to inhibit cell cycle progression and naproxen. As their definition well reflects, this first especially at the G1/S transition [54]. However, it has been generation of NSAIDs may affectbothmainCOXsisoforms, recently shown that this protein plays a dual role as the even if preferentially COX-2 (see above). Their mechanisms unphosphorylated form of p27 inhibits the cell cycle, and of action are not all completely elucidated, complicated by thus cell proliferation. If phosphorylation occurs on T157 the fact that different agents seem to act in different ways. and T198 by PI3K (phosphoinositide 3-kinase), it triggers For example, different NSAIDs bind the active site of COX- cell cycle transition by stabilizing the cyclin D1/cdk4 complex 2. Commonly, binding occurs by a reversible competitive [55]. Thus phosphorylation of S10 appears to be important inhibition (i.e., ibuprofen, naproxen, and indomethacin). for other phosphorylation steps and it has been hypothesized In contrast, aspirin is able to acetylate the active site of that Src kinase can play this role [55]. Moreover, it is COX at a serine residue, leading to an irreversible inhibition known that phosphorylation of p27 is responsible also for (see Figure 2, summarizing the classification of COX-2 its degradation by the proteasome [56]. All together these inhibitors mentioned in this review). Considerable side data suggest that PGE2 increases cell proliferation via p27 effects generated by the interference with homeostatic func- phosphorylation through EP4 receptors. tions modulated by COX-1 include increased incidence of Nonsmall lung cancer is characterized by a Ras mutation gastrointestinal hemorrhage and ulceration upon chronic or correlated with a poor prognosis [57]. Activation of Ras leads long-time intake [66]. A novel generation of COX-2-selective to an upregulation of COX-2 resulting in increased PGE2 inhibitors NSAIDs termed “Coxibs” was then developed. production [58]. PGE2 increases cell proliferation of A549 These compounds promised to be much less gastrotoxic. cells (adenocarcinoma) and this effect is associated with an They act as competitive inhibitors of the active site of activation of Ras pathway via EP4 receptor. In this case, PGE2 COX-2 and present indeed a higher specificity. However, mediates its effect by the release of amphiregulin, the most concerns related to a long-time/chronic intake of these abundant ligand in A549 cells [59]. EGFR activation leads to drugs raised quite soon, following some clinical reports, activation of MAPK pathway that regulates cell proliferation suggest a correlation between an increased risk of myocardial by transactivating several oncogenes such as c-myc [60]. infarction and their consumption [67]. This has lead to PGE2 is also important for tumor invasion. A study by the voluntary withdrawal of some of these agents, that is, Ma et al. [61] described that PGE2 can increase the number rofecoxib and valdecoxib [68], and drastic regulatory advices of metastasis. This effect has been demonstrated in a model regarding the use of the other ones, thus opening a discussion in which murine mammary tumor cells 66.1 were injected on the real benefitsversussideeffects of their use in clinics. in syngenic immune competent BALB/CByJ mice. All these Consequently, studies focused on the use of traditional cell lines express EP1, 2, 3, and 4. The use of EP4 antagonists versus COX-2-selective NSAIDs, frequently associated to the (AH23848 and AH6809) decreased surface tumor colonies elaboration of economical models, have been performed in and reduced tumor invasion. Another study has revealed that these latest years, with the aim to evaluate the real risks PGE2 increases the level of VEGF in granuloma [62]. VEGF together with the costeffectiveness and, possibly, identify is an important factor of angiogenesis, and thus of tumor classes of users/patients where regular NSAIDs intake may be progression by enhancing the vascularization of the tumors beneficial. Although, further analyses need to be performed, [63]. a number of reports suggest that Coxibs may really increase International Journal of Cell Biology 5 O O HO H C CH CH Aspirin COOH Ibuprofen COOH Sulindac sulphone Cl Sulindac sulphide OH NH Cl OH Nabumetone Naproxen Cl Diclofenac OH N N H CO CH 3 S Indomethacine Piroxicam O O CH COOH NH 2 O O S O NO H C CH CF H C S 3 NH H N S Valdecoxib NS-398 CF Br H C 3 Celecoxib Rofecoxib F C 3 SC-236 O O S DUP697 O S CH Cay10404 NH 2 O OH NO O 2 CH NH CH O NH Meloxicam Nimesulide O Figure 2: COX-2 inhibitor classification. COX-2 is the target of many compounds. COX-2 inhibitors described in this review are classified according to their ability to inhibit COX-2: nonselective (green), selective (pale blue), and preferential (grey). O 6 International Journal of Cell Biology cardiovascular risks only in patients presenting a positivity transition, which requires activation of the cyclin B-cdk2 to other cardiovascular factor risks, as high blood pressure complex. This complex accumulates during the previous step and altered lipid metabolism [69–73]. These results suggest of the cell cycle but is inactivated by a phosphorylation that their use should be limited to patients with a low risk at tyrosine 15 and threonine 14 by Wee 1 and Myt 1. of cardiovascular complications after analysis of multiple These phosphate groups are removed by the phosphatase biomarkers [Chaiamnuay et al., 2006, clinical reviews]. CDC25A when cells enter mitosis. In the case of DNA Therefore, the future perspective in the pharmacological damages, p53 is activated and increases the level of p21 use of preferential versus selective COX-2 inhibitors is the that is directly inhibiting cdk2. Moreover, 14-3-3 protein, identification of a panel of interesting biomarkers, helping a transcriptional target of p53, leads to a sequestration of in defining individual biological risk factors and limiting the cdk2 in the cytoplasm [83]. Other mechanisms involved in use of a specific class of COX-2 inhibitors to the appropriate the regulation of the G2 checkpoint or the mitotic spindle responders [74, 75]. This approach will have a considerable checkpoint are reviewed by Stewart et al. [54]. implication in therapy as well as in chemoprevention of Cancer cells are characterized by deregulation of the cell inherited forms of colon cancer. cycle via alteration of cell cycle controllers (cyclins) and cell It is interesting to mention that recent alternative cycle regulators (p53) [54], resulting in a perturbation of cell approaches have been considered. Strillacci et al. [76]and cycle checkpoints. Chan et al. suggested RNA interference using adenoviral Currently, there is evidence that prostaglandins produced vehicles. Moreover, other selective COX-2 inhibitors have by COX-2 intervene in tumor cell proliferation as NSAIDs been developed and experimentally used: SC-558 [35], and selective COX-2 inhibitors inhibit proliferation of DUP-697 [77], SC-58125 [78], and NS-398 [8]. Some of different cancer cell types expressing COX-2 [30]. NS-398, them induce an irreversible inhibition. This is the case for a COX-2 specific inhibitor, was described to reduce cell NS-398, which acts by inducing a conformational change proliferation of MC-26 cell line, a highly invasive mouse of COX-2 [25](Figure 2). Another strategy discussed in CRC cell model expressing constitutively COX-2 [8]. This literature could be the use of EP receptor antagonists. Indeed, effect was associated with a reduction of cyclin D level, a it has been demonstrated that EP antagonists can decrease key protein involved in G1-S transition [54], and PCNA, cell proliferation and cell invasion [47, 61, 79]. This could be thus increasing the processivity of DNA polymerase [82]. a more specific strategy that could limit the other side effects NS-398 and COX-2 specific inhibitor nabumetone reduced of classic COX-2 inhibitors. cell proliferation of U937 (acute promonocytic leukemia) and ML1 (human myeloblastic leukemia), thus leading to an accumulation in G0/G1 phase [33]. Interestingly, meloxicam 4. COX-2 As a Regulator of Cell Proliferation was also able to downregulate PCNA and cyclin A in HepG2 Cell cycle is regulated by different serine-threonine kinase cell line (hepatocellular carcinoma cells), leading to an proteins called cyclin-dependent kinase (Cdk). These pro- inhibition of the cell proliferation and an accumulation of teins regulate the different steps of cell cycle progression the cells in G0/G1 phase of cell cycle [84]. Alternatively, by phosphorylating many substrates (i.e., nuclear lamins) the link between COX-2 and CRC has been demonstrated by the fact that prostaglandin E2 (PGE2) derivating from [54]. These proteins are regulated by phosphorylation and dephosphorylation. Thus, Cdks can be activated by phos- COX-2-mediated arachidonic acid metabolism increased the phatases such as CDC25C (cell division cycle 25 homolog proliferation of colorectal cancer cells [85]. The inhibitory effect of NSAIDs on cell proliferation C) for CDK1 or kinase like CAK (Cdk activating kinase). The activity of cdks is also regulated by cyclins, which form of CRC has been also observed in ovarian cancer. Indeed, heterodimers with cdks leading to an activation of Cdks by treatment of OVCAR-3 tumors xenotransplanted in nu/nu conformational change [54, 80]. mice (nude mice) with aspirin and piroxicam (NSAIDs) and Cell cycle is under the control of other factors, implicated the selective COX-2 inhibitor meloxicam led to a reduction in the regulation of cell cycle transition. These regulatory of tumor growth [86]. mechanisms form checkpoints where the cell cycle can be It has been estimated that 40% of breast cancers show stopped after cellular damage in order to allow repair and an overexpression of COX-2, which is associated with a bad prognosis [5]. Indomethacin (NSAIDs), celecoxib, rofecoxib to maintain cellular integrity or, alternatively, to eliminate mutated and potentially dangerous cells. The INK4 family and nimesulide have been shown to able to inhibit cell prolif- (p16, p15, p18, and p19) and the Cip/Kip family (p21, p27, eration of these cells [5]. Moreover, prostaglandins were able and p57) [54, 80, 81] are key regulators of G1/S transition. to increase cell proliferation of hormonal-dependent breast For example, after DNA damage, p53, a tumor suppres- cancer by increasing transcription of CYP19 aromatase sor gene, activates transcription of p21, which inhibits implicated in estrogen biosynthesis [87]. cyclin E phosphorylation leading to hypophosphorylation Several studies revealed that inhibition of COX-2 by of retinoblastoma protein (pRb) [81]. INK4 family inhibits celecoxib in Burkitt’s lymphoma cell lines RAJI, BjAB, (Epstein-Barr virusnegative), and BL41 led to a reduction Cdk4 and Cdk6, whereas Cip/Kip family inhibits all Cdks. Retinoblastoma protein needs to be phosphorylated in order of cell proliferation [34]. NS-398 and celecoxib were able to to release transcription factor E2F activating genes involved reduce proliferation of pancreatic cancer cell line, Panc-1 in a dose-dependent manner [88]. Treatment with celecoxib of in the S phase-like PCNA (proliferating cell nuclear antigen) [82]. p53 is also important for the regulation of the G2/M these cells implanted into nude mice led to a reduction of International Journal of Cell Biology 7 Cip1 p21 kip1 p27 CDC25 NS-398 (MC26) Growth factors Celecoxib (K562) Cyclin B E2F 1-3 DUP-697 (K562) Genotoxic CDK1 stress Cyclin B Cyclin D pRB CDK1 CDK 4/6 Wee1 G2 E2F 1-3 Celecoxib(K562) DUP-697(K562) G1 p15, p16, p18, p19 p53 NS-398 (MC-26) meloxicam (HepG2) Cyclin E CDK2 PCNA S Cip1 Meloxicam p21 PP (HepG2) Cyclin A p21,p27 CDK2 E2F1/2, pRB PCNA cyclin A/E Pol α ··· P P p21 E2F AAAAA p27 Figure 3: Effects of COX-2 inhibitors on cell proliferation. Cell cycle is divided into different steps: G1, S, G2, and M (mitosis). This process is regulated by cyclin proteins, which activate cyclin-dependent kinase (cdk) and phosphatase (i.e., CDC25) or kinase like cyclin-dependent kinase inhibitors such as p16, p15, p18, p19, p21, and p27 [54]. Selective COX-2 inhibitors are able to modulate some cell cycle checkpoints. In this picture, some examples of this link have been shown for different cell types: MC26, colorectal cancer; HepG2, hepatocellular carcinoma; K562, chronic myeloid leukemia. Cdk; cyclin-dependent kinase; pRb, retinoblastoma protein; PCNA, proliferating cell nuclear antigen. the volume of the tumor [88]. Other studies have shown that pathway, also called the mitochondrial or stress-induced celecoxib is able to reduce cell proliferation of the chronic apoptotic pathway, is activated in response to damaging myeloid leukemia (CML) cell line K562, which expresses stresses, such as DNA damage. Typical hallmarks of this path- COX-2 at the mRNA and protein level [89]. This effect way are mitochondrial outer membrane permeabilization was accompanied by an accumulation of cells in G0/G1. (MOMP), accompanied by a collapse of the mitochondrial Moreover, the inhibition of cell proliferation was correlated membrane potential [51]. These events lead to the release to a downregulation of cyclin D1, cyclin E, and pRb and the of cytochrome c into the cytosol, which is an indispensable upregulation of p16 and p27 [89]. Similar results were found component of the apoptosome, the death complex formed on this cell type with the other selective COX-2 inhibitor also by APAF-1, and procaspase-9. Once recruited, this DUP-697 [77]. Different effects are recapitulated in Figure 3. protease is cleaved to its activated form (caspase-9) to further activate the executor caspase-3 and, finally, to finalize the apoptotic program. 5. Implication of COX-2 in Cell Death Alternatively, the extrinsic, or physiological, apoptotic pathway (Figure 4) can be triggered upon binding of specific 5.1. Apoptosis. Apoptosis (type I cell death) is important for the development and maintenance of tissue homeostasis ligands to death receptors characterized by the presence of of multicellular organisms [90, 91]. This active form of a death effector domain [94]. Ligands include cytokines, such as TNFα, tumor necrosis factor-related apoptosis- cell death is characterized by the occurrence of typical cell alterations including plasma membrane blebbing, cell inducing ligand-induced apoptosis (TRAIL), or FAS. After shrinkage, chromatin condensation and nuclear fragmen- binding, death inducing silencing complex (DISC) is formed. tation, and, finally, formation of apoptotic bodies, which The DISC is composed by the adaptors proteins TRADD can be phagocyted by macrophages [92]. Deregulation of (TNF receptor-associated death domain) and FADD (Fas- associated death domain) and is able to recruit and activate apoptosis is linked to several pathophysiological disorders, including autoimmune disorders, Alzheimer’s disease, and pro-caspase-8. Finally, caspase-8 activates caspase-3 in order cancer [93]. to trigger the final steps of apoptosis (Figure 4). Two major cascades of intracellular events are commonly Cross-talks between the two pathways take place. The involved in mediating apoptosis (Figure 4). The intrinsic extrinsic apoptotic pathway can activate the intrinsic 8 International Journal of Cell Biology Increase of TRAIL receptor Sulindac TNFα, indomethacine by NS-398 and CAY10404 in TRAIL, SC-236 (HT29) SK-Hep 1 and HLE cells DR5 clustering in FasL celecoxib (MG-63) cholesterol-rich domain by DUP-697 in HT29 cells 14-3-3 PI3K/PKB GSH depletion Bad Bax Bad DD Bak Bax Bcl-2 Cyt Bax Bid Cyt Cyt Cyt Diablo Bak Cyt Pro Bak Bak Bcl-2 Pro-casp-8 Casp9 Bcl-x DUP-697 Bax Cyt Cyt (K562) Diclofenac Bak (HT29; HCT-15) Cyt Cyt Bcl-x L Casp-8 Cyt Cyt Cyt Bcl-2 APAF Cyt NS-398 IAPs CAY10404 Apoptosome (SK-Hep; HLE) NS-398 Diablo LAP Casp-9 Casp-3 CAY10404 (SK-Hep1; HLE) Celecoxib DNA damage (neuroblastoma) Activation Gelsolin Tap 73 transcription Bax, Bid, Lamin A NFkB casp-9 β-catenin Figure 4: Effects of COX-2 inhibitors on apoptosis. Apoptosis can be mainly mediated by two pathways: the mitochondrial, intrinsic, or stress-induced apoptosis, which is activated in response to damaging stresses and the extrinsic pathway, triggered by the binding of ligands to specific death receptors [51]. COX-2 inhibitors are able to modulate stress-induced apoptosis as well as extrinsic apoptosis in several cell types. In this picture, some examples of these interaction discussed in the text are presented for different cell types: LNCaP, prostate cancer; K562, chronic myeloid leukemia; HT29, colorectal cancer; SK-Hep 1 and HLE, human hepatocarcinoma cells; HepG2, hepatocarcinoma; Be17402, hepatocarcinoma; SMMC-7402, hepatocarcinoma; MG-63, osteosarcoma. Abbreviation: AIF: apoptosis-inducing factor; Bcl-2, B cell lymphoma 2; Bid, Bcl-2 interacting domain; Casp, caspase; Cyt, cytochrome C; DD, death domain; DED, death effector domain; DISC, death-inducing silencing complex; PI3K/PKB, phosphatidyl inositol-3 kinase/protein kinase B; FADD, Fas-associated death domain; GSH, glutathione; PTP, transition permeability pore; TNF, tumor necrosis factor; TRAIL, TNF-related-inducing-apoptosis-ligand. pathway via truncation of the BH3-only protein Bid (t- same role [99]. In contrast to Bax, Bak is already present Bid) by caspase-8. t-Bid interacts with mitochondria, by at the surface of mitochondria, normally sequestered in its favoring the activation of the proapoptotic Bcl-2 family active monomeric form by the Bcl-2 anti-apoptotic members members Bak and Bax, thus leading to MOMP and caspase- Bcl-xL and Mcl-1 (see Burlacu for a general overview of 9activation[51, 95](Figure 4). The intrinsic apoptotic the Bcl-2 family members modulation involved in Bax/Bak pathway may, in turn, activate caspase-8, downstream to activation [51]). caspase-3 [96](Figure 4). Cross-talks represent an important Apoptosis is regulated in order to maintain tissue home- strategy of amplification loops carried out by dying cells to ostasis. This regulation implicates protein-protein inter- ensure/potentiate cell death. actions, with some of them counteracting apoptosis. In this view, the interaction between Bcl-2 family pro- and antiapoptotic members represents a crucial and delicate step. 5.1.1. Involvement of COX-2 in Intrinsic Apoptosis. When cells are damaged by a variety of chemicals or physical stress Bcl-2 is the best described member of this family preventing (i.e., reactive oxygen species, UV, and ionizing radiation), Bax activation [51]. Bax can form also a complex with the anti-apoptotic protein Bcl-xL [53] and Mcl-1 [14]. Similarly, they undergo apoptosis by triggering the intrinsic apoptotic pathway (Figure 4). This pathway may be associated with a Bak activity is monitored by the anti-apoptotic members Bcl- xL and Mcl-1 [51]. The interaction between Bax/Bak and the redox disequilibrium, mediated by depletion of glutathione (GSH) [94, 97, 98], required for the activation and transloca- Bcl-2 family anti-apoptotic members is carefully regulated by tion to mitochondria of the Bcl-2 pro-apoptotic member Bax the BH-3-only proteins. Another carefully regulated down- stream checkpoint of the apoptotic pathway is the activation [98], which, in turn, forms complexes (oligomers) mediating MOMP and cytochrome c release. As Bax, Bak may play the of caspases. Inhibitor of apoptosis (IAP) family, by directly PTP DED FADD DED FADD DISC International Journal of Cell Biology 9 interacting with caspases (i.e., XIAP, survivin [100]) controls is anti-apoptotic and lacks the transactivation domain. and prevents their activity once cleaved. IAPs monitoring DeltaNp73 is overexpressed in neuroblastoma, leading to function can be, in turn, counteracted by the pro-apoptotic chemotherapy resistance [109]. It has been shown that SMAC/DIABLO, a mitochondrial heterodimer, which is celecoxib was able to upregulate Tap73 and downregulate released from mitochondria when MOMP is affected [101]. DeltaNp73. These data suggest the use of COX-2 inhibitors as This interaction favors the induction of apoptosis. p73 modulators in order to improve efficiency of chemother- Imbalance between cell proliferation and apoptosis apy [110]. observed in cancer can be tightly related to an altered The apoptotic effect of COX-2 inhibitors has been also function of pro-apoptotic proteins as well as to an up- observed for other tumor cell types, such as in the chronic regulation of anti-apoptotic proteins (i.e., Bcl-2 or IAPs) myeloid leukemia model K562 where DUP-697 induced or a downregulation of tumor suppressor genes (i.e., p53). apoptosis by cell cycle arrest and caspase-8 activation [77]. In addition, the activation of prosurvival pathways (i.e., COX-2 inhibitors can also activate prosurvival pathways. PI3K/Akt) may be implicated upstream. Inflammation can The PI3K/Akt pathway is a survival pathway, frequently contribute to this imbalance via cytokines secreted in the activated in cancer cells [49]. PI3K produces PIP3 (phos- tumor microenvironment able to activate survival path- phatidylinositol 3,4,5 triphosphate) that activates PDK1 ways. For example, TNFα can induce NFκB, leading to (pyruvate deshydrogenase kinase). This protein phosphory- an inhibition of apoptosis [38]. COX-2 seems also to play lates and activates PKB (protein kinase B), which, in turn, is a role in this process because it is known that COX-2 responsible for the phosphorylation of several targets playing inhibition is correlated to an increase of apoptosis in several a modulator function in apoptosis. An anti-cancer effect of cancer models. NS-398 downregulated Bcl-2 expression in an celecoxib due to the inhibition of Akt signaling [111]was androgen-sensitive human prostate cancer cell line LNCaP observed in a gastric cancer model. Celecoxib triggered also that exhibited a high constitutive level of COX-2 [102]. apoptosis in osteosarcoma cells (MG-63) through down- Similar results have been observed in human colorectal regulation of Bcl-2, survivin and PI3K (phosphoinositide 3- cancer cells (HCA-7 cell line which expresses COX-2) where kinase) pathway [112]. Similarly, Hsu et al. [113] found that PGE2 was able to inhibit apoptosis induced by SC58125, inhibition of Akt phosphorylation by celecoxib in prostate a selective COX-2 inhibitor, and increase Bcl-2 expression cancer models (LNCaP and PC3 cell lines which express [46]. Different mechanisms are supposed to explain how constitutively COX-2) led to apoptosis, but in this case COX-2 inhibitors may trigger apoptosis. In a number of without affecting Bcl-2 level. studies, COX-2 inhibition was linked to a concomitant The PI3K pathway is negatively regulated by PTEN increase of intracellular arachidonic acid. In HT-29 human (phosphatase and TENsin homolog), which converts PIP3 colon adenocarcinoma cell this accumulation led to the in PIP2, preventing PKB activation and Bad phosphory- induction of apoptosis [103]. The arachidonic acid-induced lation/sequestration. Thus PTEN is considered as a tumor apoptosis was inhibited by Bcl-2 transfection, indicating suppressor gene. It has been shown that NS-398 was able to a role of arachidonic acid in affecting Bcl-2 intracellular increase the level of PTEN in human gastric carcinoma cell levels [103]. Accumulation of arachidonic acid can affect line MKN45 [114]. apoptosis by mediating an increase of pro-apoptotic intra- One of the PKB targets is Bad, a BH3-only member [51]. cellular ceramides caused by activation of sphingomyelinase The nonphosphorylated form of Bad plays a pro-apoptotic [104, 105]. Sulindac sulphide, a metabolite of sulindac, also role, by binding Bcl-xL or Bcl-2 and, thus, preventing their activates sphingomyelinase and enhances the ceramide level interactions with Bak and Bax. The activation of PI3K/Akt in the two human colorectal carcinoma cell lines HCT116 pathway may lead to the phosphorylation of Bad, which and SW480 [106]. is consequently sequestrated in the cytoplasm by 14-3- COX-2 reduces pro-apoptotic nitric oxide (NO) levels in 3 protein and, in this way, inhibited in its pro-apoptotic cancer cells downstream of prostaglandin production [30] function [51]. It has been reported that sulindac sulphone, (see Brune ¨ et al. [107] for an overview on nitric oxide role indomethacine, and SC-236 were able to induce apoptosis in apoptosis). Chang et al. reported that PGE2 prevented via Bad activation, by inhibiting 14-3-3 expression in a apoptosis induced by NGF (nerve growth factor) withdrawal dose- and time-dependent manner in HT-29 cells [115]. by increasing the level of dynein light chain, an inhibitor This effect was tightly related to PPARδ. It is known, of neuronal NO synthase in pheochromocytoma of the indeed, that 14-3-3 protein contains PPRE recognized and rat adrenal medulla PC12 cells, thus leading to decreased bound by PPARδ [115]. COX-2 can mediate the synthesis of intracellular NO levels [108]. prostaglandin I2, which can bind and activate PPARδ [41]. More recently, connections between COX-2 inhibitors Thus, it has been suggested that the inhibition of COX-2, and p53 family members have been described. For example, leading to a decrease of PGI2, impaired PPARδ activation, celecoxib was shown to able to modulate different isoforms of which, in turn, was responsible for a downregulation of p73, a p53 family member in neuroblastoma cell lines [109]. 14-3-3 protein, thus allowing Bad to play its pro-apoptotic p73 encodes many isoforms with different roles. Tap73 is functions [115]. pro-apoptotic and contains a transactivation domain. This NFκB is a most important transcription factor involved isoform is considered as a tumor suppressor gene because in survival by enhancing transcription of anti-apoptotic it seemstobeinvolvedincellcycle regulationaswellas proteins such as Bcl-2 [14, 15]. Sulindac inhibits NFκBin in apoptosis induction [109, 110]. In contrast, DeltaNp73 two colon cancer cell lines (human colon adenocarcinoma 10 International Journal of Cell Biology HCT-15 and HT29 cell lines) [116]. Similar results were Altogether, these results encourage the perspective that obtained with diclofenac, which was able to inhibit nuclear COX-2 inhibitors could be used in future as a therapeutic accumulation of NFκB[117]. In the same study, PGE2 was strategy to sensitize tumor cells to apoptosis induced by demonstrated to increase the transcriptional activity of NFκB physiological stimuli. p65/p50 dimer in CACO-2 cells (human epithelial colorectal adenocarcinoma cells), transfected with a luciferase con- 5.2. Involvement of COX-2 in Other Types of Cell Death struct containing NFκB response elements [117]. It is known that activation of prostaglandin receptors 5.2.1. Anoikis. Anoikis is a form of apoptosis mediated by the induces an increased cAMP level which in turn can activate loss of cell anchorage. This pathway plays a fundamental role protein kinase A (PKA) [58]. Studies have suggested that during development and maintenance of tissue homeostasis PKA, like PKB, phosphorylates Bad [118], leading to its by killing damaged cells or detached cells in order to main- sequestration and inhibition of apoptosis. Some of the pro- tain tissue architecture. For example, the inner endodermal or anti-apoptotic mediators affected by COX-2 inhibitors are cells undergo anoikis after the loss of anchorage to the matrix schematized in Figure 4. during development [124]. It is known also that intestinal epithelial cells loose anchorage when located at the luminal surface, leading to anoikis [42]. As a form of apoptosis, 5.1.2. Implication of COX-2 in Extrinsic Apoptotic Cell Signal- anoikis is dependent on caspase activation and cytochrome ing Mechanisms. Studies reported that COX-2 inhibitors are c release by mitochondria and is regulated by Bcl-2 family also associated with a sensitization of tumor cells to extrinsic members [42]. apoptosis. Thus, DUP-697 sensitized HT29 colon cancer It has been shown that anoikis is prevented in cancer cell line to TRAIL-induced apoptosis. This effect was due cells, thus favoring tumor progression with the formation to an accumulation of arachidonic acid inside the cells, of metastasis [42]. Accordingly, modulation of anoikis is which activates sphingomyelinase, triggering a clustering of considered a promising target for anti-cancer strategies. deathreceptor(DR)5receptorsinceramideand cholesterol- Cell anchorage is due to cell-cell and cell-matrix interac- rich domains [119]. Alternatively, the expression of COX- tions. Cell-cell interactions are mainly mediated by integrins 2 has been frequently associated with a modulation of the which are transmembrane receptors located at the cell expression of death receptors, thus leading to an upstream surface and composed of alpha and beta chains [125]. Many control of the extrinsic apoptotic pathway. Tang et al. [120] intracellular signals can act downstream to integrins, which, showed that COX-2 overexpression in human colon cancers correctly switched on, can ensure cell survival. Some of them cells led to an inhibition of DR5 expression and a resistance are mediated by kinases such as Focal-adhesion-kinase (Fak) to TRAIL-induced apoptosis. Accordingly, COX-2 specific or integrin-linked kinase (ILK) [42]. Fak is phosphorylated inhibitors, NS-398 and CAY10404, are sensitizing human upon integrin adhesion, leading to activation of other sig- hepatocarcinoma cells (SK-Hep1 and HLE) to TRAIL- naling pathways like PI3K, MAPK. ILK is a serine/threonine induced apoptosis. This effectwas duetoanupregulation kinase that directly phosphorylates PKB. of TRAIL receptors (TRAIL R2/DR5 and TRAIL-R1/DR4), Together with cell-cell and cell-matrix interactions, para- together with an ability of the compounds to induce a down- crine factors could be important for the regulation of anoikis. regulation of the anti-apoptotic proteins survivin (IAP) and It has been shown that E-cadherin (epithelial cadherin) Bcl-xL [121]. In hepatocellular carcinoma models (HepG2, can activate COX-2 [23]. It is possible that prostaglandins Bel7402, and SMMC-7402), Li et al. [84] showed that COX- produced by COX-2, which act in an autocrine and a 2 inhibition with meloxicam led to an upregulation of Fas- paracrine manner, favor cell survival. A study from Joseph et mediated apoptosis. In vivo studies performed on transgenic al. [126] showed that PGE2 inhibited anoikis in IEC-18 cells mice constitutively expressing human COX-2 confirmed an (rat intestine ileum cells). This effect was suggested to be due increased resistance to Fas-induced apoptosis in liver, as to cAMP signaling because prostaglandin E2 receptors are coupled to adenylate cyclase, which converts AMP to cAMP shown by the preservation of liver architecture in COX- 2-expressing mice compared to wild type [122]. Similarly, [126]. another study performed on human extrahepatic bile duct Other studies demonstrated that COX-2 inhibits anoikis carcinoma cell line showed that COX-2 induction led to the via activation of PI3K/Akt pathway, as the case of a human inhibition of Fas-induced apoptosis, whereas the inhibition bladder cancer cell line expressing COX-2 [40]. A link of COX-2 with NS-398 in cytokine-treated cells exacerbated between COX-2 and anoikis has been described, further- apoptosis induced by CH-11, an agonist of Fas receptor more, in uterine endometrial carcinoma [127]. COX-2 is [123]. over-expressed in this type of cancer and this is associated AKT pro-survival pathway may play a role also in with tumor aggressiveness. In addition, a recent report based the modulation of extrinsic apoptosis. The human gastric on HEC-1B and RL95-2 (two human endometrial cancer carcinoma cell line MKN45, which expresses COX-2, was cell lines) showed that the treatment of these cells with sensitized to Fas-induced apoptosis by NS-398. The COX- hepatocyte growth factor (HGF) led to an up-regulation of 2 inhibitor, indeed, was able to increase the level of PTEN, COX-2. Hepatocyte growth factor interacts with its tyrosine leading to a decrease of Akt phosphorylation and activation kinase receptor c-Met. This interaction is responsible for of Bad [114]. Some effects of COX-2 inhibitors on extrinsic tumor progression. Overexpression of HGF/c-Met has been apoptosis are summarized in Figure 4. described in different tumors such as breast cancer [128] International Journal of Cell Biology 11 as well as head and neck cancer [129], also in endometrial Polyposis (for celecoxib) [134, 135] and also in breast cancer carcinoma [130]. It has been demonstrated that HGF [136]. This property of COX-2 inhibitors could be useful for inhibited anoikis and treatment of HEC-1B and RL95-2 patients with a high risk to develop cancer such as people cells with the COX-2 selective inhibitor meloxicam prevented with Li-Fraumeni syndrome, for example [137]. The fact HGF-mediated anoikis resistance [127]. Similar results were that many reports in literature suggest that COX-2 inhibitors obtained in head and neck squamous cell carcinoma [131]. are responsible for an inhibition of cell proliferation and Altogether these data suggest that COX-2 may be impli- apoptosis induction in a number of different cancer cell cated in the inhibition of anoikis and that COX-2 inhibitors models prompts to consider a possible use of COX-2 may play a role in inhibiting tumor progression (metastasis), inhibitors in future therapeutical protocols, administered by sensitizing tumor cell to anoikis. alone as well as in combination with anti-cancer clinical protocols in order to improve tumor cell death. 5.2.2. Autophagy. Autophagy is a process triggering cells 6.1. COX-2 Inhibitors in Combination with Radiotherapy. to degrade intracellular constituents, ranging from proteins Radiation therapy is a common treatment used for the up to entire organelles. It represents an important process treatment of solid tumors, such as breast, prostate, colorectal, ensuring the turnover of long-lived cellular components, and lung cancers. It is known that the anti-cancer properties which can be activated also by stress conditions like nutrient of ionizing radiation are due to pleiotropic mechanisms. starvation in order to avoid cell death. The process starts Radiation leads to the formation of DNA doubled-strand with the formation of doubled membrane-bound vacuoles breaks in proliferating cells, which triggers the activation of corresponding to autophagosomes that entrap parts of the DNA damage pathways (i.e., p53), followed by the induction cytoplasm or organelles (i.e., mitochondria). Then, these of apoptosis [36]. The importance of Bcl-2 family members structures are fused with lysosomes (autolysosomes), thus during apoptosis [51] suggests that prosurvival proteins (i.e., leading to the degradation of the intracellular parts previ- Bcl-2, Bcl-xL) play an important role in radioprotection of ously enclosed. Together with apoptosis, when exacerbated, tumor cells. The NFκB pathway seems to be implicated, autophagy contributes to the modulation of homeostasis, by being required in regulating expression of the anti-apoptotic eliminating damaged and potentially dangerous cells (type II Bcl-2 family members like Bcl-xL [36]. Moreover, it is well cell death) [132]. However, the relationship between apopto- established that NFκB regulates the level of COX-2, suggest- sis and autophagy is currently still poorly understood [132] ing that COX-2 may play a role in radiotherapy resistance because in some cases autophagy permits an adaptation of [21]. Similarly, nimesulide could increase radiation efficiency the cells to stress (i.e., nutrient starvation), thus counteract- in nonsmall cell lung cancer in vivo (nude mice) and in ing apoptosis, whereas, in other cases, autophagy is a process vitro (A549 cell line) as shown by Grimes et al. [138]. This triggering downstream apoptosis [132]. Indeed, similar effect was due to a down-regulation of MnSOD (superoxide stimuli can induce both apoptosis or autophagy [132]. dismutase containing manganese (Mn) and localized in This process is implicated in pathologies such as Alz- mitochondria), a primary antioxidant protein and survivin, heimer’s disease and cancer, suggesting a promising field in an anti-apoptotic protein (IAPS family member). These two therapy. By considering that COX-2 is supposed to play a proteins are regulated by NFκB. It is well known that during role in apoptosis and a link between apoptosis and autophagy radiation therapy NFκB can be upregulated due to reactive exists, it is conceivable that COX-2 plays a role also in this oxygen species release and inflammation (i.e., PGE2). This process. Currently, not many studies aimed at investigating report suggests that nimesulide may act on NFκB to inhibit a possible link between COX-2 and autophagy have been MnSOD and survivin. published. Nevertheless, one study revealed that sulindac Melanoma is known to be very resistant to conven- sulphide (NSAIDs) induced apoptosis of the colon cancer tional radiotherapy and chemotherapy. Irradiation of two HT29 cell line. This effect was increased by treatment of melanoma cell lines WM35 and LU1205 in the presence of the cells with 3 methyl-adenine, a well-known inhibitor NS-398, a selective COX-2 inhibitor, strongly exacerbated the of autophagy [133]. Moreover, the extent of apoptosis in G2/M arrest as well as the induction in apoptosis. Accord- Q204L cells (a clone of HT-29 cells in which 3 methyl- ingly, the down-regulation of COX-2 by RNA interference in adenine-sensitive autophagic sequestration is impaired) was these cell lines was followed by an upregulation of p53 and less than in HT29. These data suggest that autophagy can G2/M arrest [36], thus confirming that the effect of NS-398 delay sulindac sulphide-induced apoptosis [133]. is due to its role on COX-2 inhibition. Other studies have shown that the radiosensitivity of PC3 6. COX-2 Inhibitors in Cancer Therapy (human prostate carcinoma cells) and Hela (human cervical Despite the latest progress in cancer research and the differ- carcinoma cells) was enhanced after silencing of COX-2 by ent strategies to kill cancer cells, several tumors are resistant siRNA. NS-398 was able to increase radiosensitivity of PC3 to conventional therapeutics treatment (i.e., radiotherapy, cells expressing COX-2, but not in PC3 silenced for COX-2. chemotherapy, and photodynamic therapy). In contrast, NS-398 enhanced radiosensitivity of Hela cells, COX-2 inhibitors play an important role in cancer irrespective to the level of COX-2 [37]. prevention. Indeed, the chronic intake of NSAIDs is able However, combination of COX-2 inhibitors with radi- to consistently reduce the appearance and incidence of ation therapy can also lead to a reduction of efficiency of many types of cancer as described in Familial Adenomatous the radiotherapy. In one report, it has been shown that the 12 International Journal of Cell Biology selective COX-2 inhibitor nimesulide decreased radiation a decrease of cell proliferation in a dose-dependent manner efficiency of two head-and-neck cancer cells lines (SCC9 and [34]. SCC25) which are COX-2 positive [139]. This suggests that NS-398 was able to increase the cytotoxicity of gemc- the sensitization of tumor cells to radiation might be strongly itabine, an analog of the antimetabolite nucleoside deoxycy- dependent on tumor cell type. tidine, used for treatment of nonsmall cell lung carcinoma, in A549ACA cell line (lung adenocarcinoma cell line) by 6.2. COX-2 Inhibitors in Combination with Chemotherapy. enhancing apoptosis [144]. The combination of NS-398 and Many types of cancer are treated with chemotherapeutic gemcitabine is also associated with an inhibition of cell proliferation with an accumulation of the cells in G0/G1 agents leading to inhibition of cell proliferation or induction of apoptosis [140]. phase of cell cycle and an increase of p21 [144]. One of the major causes of chemotherapy failure is the All of these data suggest that COX-2 is implicated in survival and/or development of multidrug resistant cancer anti-apoptotic and MDR pathways and that selective COX-2 cells. This resistance is mediated by many mechanisms inhibitors could be used to improve chemotherapy efficiency. including over-expression of proteins involved in inhibition of apoptosis (i.e, Bcl-2), leading to insensitivity of tumor cells to apoptotic stimuli; an up-regulation of DNA repair; 6.3. COX-2 Inhibitors in Combination with Photodynamic alteration of the target; up-regulation of detoxification Therapy. An alternative therapeutic approach to treat can- enzymes (i.e., Glutathione S-transferases); and extrusion of cers is photodynamic therapy. This procedure is particularly chemotherapeutic drugs by overexpression of ATP-binding used for such solid tumors including skin, bladder, and cassette family proteins, such as MRP (multidrug resistant- head and neck cancers in addition to other diseases like associated protein) BCRP (breast cancer resistance protein or age-related macular degeneration and psoriasis [145]. The mitoxantrone resistance protein) because these proteins reg- treatment consists in the administration of a photosensitizer, ulate absorption, distribution, and excretion of various phar- a molecule that selectively accumulates in tumors and macologic compounds [141]. Consequently, the chemother- is activated by light (600–850 nm). The photosensitizers apeutic agents are immediately extruded from the cells. P-gp may accumulate in different compartments of tumor cells (P-glycoprotein) is one of the best-understood mechanisms like mitochondria (i.e., porphycene monomer), nucleus, leading to multidrug resistance (MDR). Tremendous efforts lysosomes (i.e., lysyl chlorin p6), and plasma membrane have been made to find solutions to overcome MDR. (i.e., monocationic porphyrin like Photofrin). Then, the Recently, COX-2 inhibitors showed an ability to sensitize photosensitizer is excited with a laser from a single state to a tumor cells to chemotherapeutic agents in several models triplet state. The triplet-state photosensitizer is implicated in and also in clinical assays. Colorectal cancers are particularly two oxygen-dependent reactions. In the first one, the triplet affected by chemoresistance. One study revealed that the can react with cell membrane or molecules, leading to radical COXs inhibitors naproxen and indomethacin heptyl ester formation, which in combination with oxygen produce were able to downregulate P-glycoprotein in human colorec- oxygenated products, cytotoxic for the cells [146, 147]. In the talCACO-2cellline.[39]. Indomethacin inhibited the activ- second reaction, the triplet-state photosensitizer can transfer ity of the protein and affected COX-2 mRNA and protein its energy directly to oxygen in order to produce singlet level [39]. Another study showed that meloxicam was able oxygen ( 02), which is known to be a very highly reactive to downregulate MDR1 in HL60 (a human promyelocytic oxygen species and is implicated in cell damage. Therefore, leukemia) cell line as well as in acute myeloid leukemic blasts this therapy leads to tumor destruction due to cell death [142]. The regulation of MDR1 by COX-2 has been also occurring via apoptosis and necrosis. Vasculature damages suggested in another study [143] in which it was reported and activation of immune response are two important effects that transfection of COX-2 cDNA with adenovirus in renal implicated in tumor ablation. rat mesangial cells led to an upregulation of MDR1 gene. Some parameters affect PDT efficiency, such as the distri- bution of the photosensitizer, photobleaching, hypoxia/ano- The combination of COX-2 inhibitors with chemotherapy was also assayed in a study in which the sensitivity of a xia, and the vascularization of the tumor [146]. The main human gastric cancer cell line MKN45 to cisplatin (alkylating reason of failure of this therapeutic approach is linked to agent) resulted increased by COX-2 downregulation with an up-regulation of angiogenic and inflammatory factors siRNA [35], suggesting a possible therapeutic application of in the tumor microenvironment that strongly reduces the this combination. Similarly, the sensitivity to cisplatin was PTD efficiency with a consequent tumor relapse. The link increased by celecoxib in a human osteosarcoma cell line between inflammation and survival pathway activation, (MG-63) and this effect was linked to a down-regulation of cell proliferation, and angiogenesis is well known and anti-apoptotic proteins survivin, Bcl-2, and an inhibition of contributes to tumor progression [3, 6]. It has been shown the survival pathway PI3K/Akt [112]. It was also reported that PDT leads to an increase of TNFα,IL1β, PGE2, VEGF that B-CLL (B chronic lymphoid leukemia) overexpressed (vascular endothelial growth factor), and MMP9 (matrix COX-2 and the combination of NS398 with chlorambucil, metalloproteinase 9) [147]. These molecules can counteract an alkylating agent, increased the level of apoptosis in B- tumor responses to PTD by promoting cell proliferation and CLL blasts coming from patients [32]. Moreover, several cell survival [38]. Interestingly, it has been demonstrated that lymphoma cell lines overexpressed COX-2, such as RAJI, COX-2 is upregulated during PDT treatment in different BJAB, BL41 and treatment of these cells with celecoxib led to cancer models. As for radiotherapy and chemotherapy, International Journal of Cell Biology 13 this suggests COX-2 as a possible target to increase PDT case, this effect may be cell-type dependent as for chemother- efficiency. apy or radiotherapy. Indeed, celecoxib has been proved to affect the Photofrin- induced PDT in in vitro and in vivo studies performed 7. Inhibition of COX-2 Expression by on a mouse mammary carcinoma BA cell line [148]. In Natural Compounds vitro, celecoxib and NS-398 increase PDT-induced apoptosis. These results were correlated with caspase-3 and PARP Synthetic cyclooxygenase-2 inhibitors hold promise for can- cleavage and Bcl-2 degradation. In vivo, the photosensi- cer chemoprevention; however, recent toxicity problems sug- tization by COX-2 inhibitors was not due to apoptosis gest that new strategies are needed. Natural compounds with exacerbation. Interestingly, celecoxib and NS-398 decrease the potential to inhibit key cell signaling pathways including PDT-induced apoptosis but were also able to decrease the COX-2 gained much attention over the last regarding years level of angiogenic factors such as TNFα,IL1β, PGE2, VEGF, whether they are used alone or in combination with existing and MMP9 [148]. chemotherapeutic agents. Upon chlorin-induced PDT, COX-2 was found up-regu- Recently, Bhui et al. demonstrated that Bromelain, a lated 25-fold in mouse mammary carcinoma RIF cells pharmacologically active compound present in pineapple [149]. This up-regulation was associated with an increase (Ananas cosmosus), leads to a marked inhibition of COX-2 of PGE2 level in the tumor microenvironment. When expression and inactivation of NFκB. Bromelain treatment RIF cells were transplanted in CH3/HeJ mice, for in vivo induces up-regulation of p53 and Bax and subsequent studies, PDT similarly induced an increase of COX-2 and activation of caspase-3 and caspase-9 with a decrease in Bcl-2 PGE2. These effects were prevented by NS-398. Here, it was expression [154]. Furthermore bromelain induces apoptosis- demonstrated that PDT induced vascular endothelial growth related proteins along with inhibition of NFκB -driven COX- factor expression (VEGF) and this increase was attenuated by 2 expression by blocking the MAPK and Akt/protein kinase B treating mice with NS-398, meaning that COX-2 might play signaling in DMBA-TPA-induced mouse skin tumors [155]. a role also in angiogenesis. In consequence of these effects, Curcumin, a naturally occurring polyphenol from Cur- the combination of COX-2 inhibitors with PDT resulted in cuma longa, was described to act as an antiinflammatory an increased efficiency of tumor treatment. and antiproliferative agent by causing downregulation of Possible correlation between COX-2 level and resistance COX-2 in cervical cancer. Curcumin-mediated apoptosis in to PDT has been also investigated in Hela (human cervix these cells is initiated by up-regulation of pro-apoptotic carcinoma cells) and T24 (human transitional cell carcinoma Bax, AIF, release of cytochrome c, and downregulation of of the urinary bladder) cells [150]. It has been reported anti-apoptotic Bcl-2, Bcl-xL in HeLa and SiHa cell lines. that in PDT induced by hypericin, a natural photosensitizer This onset of apoptosis was accompanied by an increase in which accumulates in endoplasmic reticulum and Golgi caspase-3 and -9 activity, suggesting the role of mitochondria apparatus, an increase of PGE2 levels occurred. Hypericin in curcumin-mediated apoptotic cell death as described by induces apoptosis by triggering the release of cytochrome M. Singh and N. Singh [156]. Mar´ ın et al., furthermore, c after light excitation through a process requiring the concluded that curcumin inhibits NFκBactivityaswellas activation of p38 MAPK, which it is known to induce an the expression of its downstream target genes, and also selec- up-regulation of COX-2 [23, 151]. The increase in PGE2 tively induces apoptosis of melanoma cells but not normal levels was prevented by the use of a p38 MAPK inhibitor melanocytes [157]. In addition, curcumin-induced apoptosis (PD169316). Moreover, the impairment of p38 MAPK was was also associated with the activation of caspase-3 and associated with an increase in the susceptibility of tumor caspase-9, and the degradation of PARP. Curcumin decreased cells to PDT. However, COX-2 inhibitors did not lead to the the expression levels of COX-2 mRNA and protein without same effect, meaning that COX-2 was not involved in PDT causing significant changes in COX-1 levels, which was resistance in this model. correlated with the inhibition of prostaglandin E(2) synthesis In contrast to the study of Ferrario et al. [148, 149], [158]. In BV-2 microglial cells, curcumin and analogs were a report from Makowski et al. [152] has revealed that shown to inhibit LPS-induced COX-2 expression; analogs rofecoxib, NS-398, and nimesulide were unable to potentiate identified as more potent than curcumin in the screening PDT in C-26 cells (poorly differentiated colon adenocarci- assay were also more potent than curcumin in preventing noma cell line) in vitro. However, chronic exposition of mice COX-2 expression [159]. bearing C-26 cells to nimesulide potentiated PDT. These data suggest that COX-2 inhibitors may indirectly potentiate PDT. Coumarin (1,2-benzopyrone) is a naturally occurring It is known that vasculature damages are important fragrant compound found in numerous plants and spices. for PDT efficiency and that COX-2 inhibitors act as anti- Results obtained with human nonsmall cell lung cancer A549 angiogenic factors [153]. It has been hypothesized that these cells suggest that downregulation of Bcl-xL, COX-2, and antiangiogenic effects could be responsible for the anti- MAP kinase pathway and up-regulation of p53, Akt, and tumor effect. NFκB pathway are involved in the underlying molecular Currently, the link between COX-2 and PDT efficiency mechanism of apoptosis induction as suggested by Goel et al. is not well characterized. Some studies have revealed an [160]. improvement of efficiency with COX-2 inhibitors whereas Suh et al. concluded that the plant flavonoid fisetin indu- other reports have demonstrated no direct effects. In any ces apoptosis and suppresses the growth of colon cancer cells 14 International Journal of Cell Biology Radiotherapy - Nimesulide in nonsmall cell lung cancer in vivo and in vitro (A549 cell line) - Ns-398 in melanoma cell line (WM35 and LU1205) - siRNA COX-2 in human prostate carcinoma (PC3) and human cervical carcinoma COX-2 inhibitors Chemotherapy Photodynamic therapy - Naproxen/indomethacin downregulate - Celecoxib and NS-398 in photofrin-induced- P-glycoprotein in human colorectal cell line (CACO-2) photodynamic therapy in mouse mammary - COX-2 siRNA/cisplatin in human gastric cancer cell carcinoma cell line (BA) line (MKN 45) - NS-398 in mouse mammary carcinoma (RIF) - Celecoxib/cisplatin in human osteosarcoma cell line (MG-63) - NS-398/chlorambucil in B-CLL - NS-398/gemcitabine in nonsmall cell lung carcinoma cell line (A549) Figure 5: COX-2 inhibition in cancer therapy. by inhibition of COX-2- and Wnt/EGFR/NFκB-signaling effects of combinatorial chemopreventive agents and further pathways [161]. underscore the need for rational design of human clinical Sulforaphane (SFN) is a biologically active compound trials involving such natural compounds [165]. extracted from cruciferous vegetables, and presents potent Pandey et al. published that butein inhibited the expres- anti-cancer and anti-inflammatory activities by suppression sion of the NFκB-regulated gene products involved in anti- of NFkB-dependent genes involved in anti-apoptotic signal- apoptosis (IAP2, Bcl-2, and Bcl-xL), proliferation (cyclin D1 ing (IAP-1, IAP-2, XIAP, Bcl-2, and Bcl-xL), cell proliferation and c-Myc), and invasion (COX-2 and MMP-9). Suppression (c-Myc, COX-2, and cyclin D1), and metastasis (VEGF and of these gene products correlated with enhancement of the MMP-9) as published by Moon et al. [162]. apoptosis induced by TNF and chemotherapeutic agents, and Nontoxic apigenin can suppress anti-apoptotic pathways inhibition of cytokine-induced cellular invasion. This group involving NFκB activation including cFLIP and COX-2 exp- clearly demonstrates that antitumor and anti-inflammatory ression as demonstrated by Xu et al. [88]. According to Nam activities assigned to butein may be mediated in part through et al., DA-6034, a synthetic derivative of flavonoid Eupatilin, the direct inhibition of IKK, leading to the suppression of the strongly enhanced apoptosis and inhibited the expression NFκB activation pathway [166]. of COX-2 and phospho-IKKalpha in inflammation-related Hostanska et al. used human colon COX-2-positive HT colon cancer models [163]. 29 and COX-2-negative HCT 116 or lung COX-2 proficient EGCG from green tea was described to attenuate the A 549 and low COX-2 expressing SW2 cells and showed that AR, to downregulate IGF-1, to modulate COX-2 expression, willow bark extract BNO 1455 and its fractions inhibit the and to decrease MAPK signaling leading to the reduction cell growth and promote apoptosis in human colon and lung in cell proliferation and induction of apoptosis in prostate cancer cell lines irrespective of their COX selectivity [167]. cancer without toxicity [164]. Interestingly, combination of EGCG and COX-2 inhibitor NS-398 enhanced cell growth 8. COX-2 Independent Effects inhibition, apoptosis induction, expression of Bax, pro- caspase-6, and pro-caspase-9, and PARP cleavage, inhibition It is currently well known that several selective COX-2 of PPAR gamma, and inhibition of NFκB compared with the inhibitors inhibit cell proliferation and induce apoptosis additive effects of the two agents alone, suggesting a possible independently of COX-2. Celecoxib is particularly known to synergism. In vivo, combination treatment with green tea have these COX-2-independent effects, which were reviewed polyphenols and COX-2 inhibitor celecoxib resulted in by Grosch et al. [68]. Indeed, celecoxib was able to directly enhanced tumor growth inhibition, lowering of prostate- bind and inhibit PKB/Akt, which plays an important role specific antigen levels, lowering of IGF-I levels, and circu- in cell proliferation and in apoptosis. Concerning cell cycle, lating levels of serum IGF-1 binding protein-3 compared PKB is able to phosphorylate cdk inhibitors, such as p21 and with results of single-agent treatment. Accordingly, Adhami p27, leading to PCNA activation [168, 169]. Furthermore, et al. postulate the efficiency of synergistic and/or additive PKB can also activate several cyclin-cdk complexes and International Journal of Cell Biology 15 induce E2F factor in some cases [68], stimulating cell prolif- selective COX-2 inhibitors with radiotherapy or different eration. Besides, PKB inhibits apoptosis, by phosphorylating chemotherapeutics revealed a sensitization to apoptosis. the pro-apoptotic protein Bad and by inhibiting caspase-9 This effect was also observed with several agents inducing cleavage [51]. apoptosis in a physiological way, thus suggesting that COX-2 The COX-2-independent effects concern also the extrin- inhibitors used in combination with death receptors agonists sic apoptotic pathway. Indeed, we discussed that selective might be a novel approach to elicit apoptosis of cancer cells. COX-2 inhibitors, such as NS-398, celecoxib, and meloxi- However, the fact that COX-2 inhibitors can mediate their cam, are able to modulate the sensitivity of several tumor effects by COX-2-independent mechanisms suggests caution cells to Fas- and TRAIL-induced apoptosis. It has been in the interpretation of the data. discussed that this modulation could be due to COX-2- Nowadays, selective COX-2 inhibitors have been independent effects. In fact, NS-398 and nimesulide were included in several clinical assays. Some of them effectively able to promote TNF and TRAIL-induced apoptosis of D98 increase the efficiency of radiotherapy and chemotherapy and H21 Hela cell lines [170]. In D98, COX-2 is inactive. [172]. For example, celecoxib is in a clinical phase II assay in Moreover, prostaglandin E2 readdition was not able to revert combination with Paclitaxel, carboplatin, and radiotherapy the sensitization effect. In the same report, it has been shown for patients with inoperable stage IIIA/B nonsmall cell lung that NS-398 was able to promote apoptosis induced by TNF cancer [172]. These clinical assays confirm that COX-2 in MCF-7 cell line (human breast adenocarcinoma cells), inhibition may be a promising field in cancer treatment. which again does not express COX-2 [170]. However, the selective COX-2 inhibitors are responsible for A report from Ryan et al. [171] demonstrated that side effects, including an increasing risk of cardiovascular SC58125 and CAY10404, two selective COX-2 inhibitors, complications [67, 68]. It is hoped that other methods to were able to decrease intracellular content of GSH in inhibit COX-2 will be developed. To this purpose, RNA malignant human B-cells. This effect was accompanied by interference using vehicle (i.e., adenovirus) as well as natural an increase of reactive oxygen species production. Indeed, compounds were suggested by some studies [35, 76], as GSH is the most important intracellular nonprotein thiol alternative and promising strategy. antioxidant defense against free radicals, meaning that it pro- tects the cells from cellular damages. The GSH depletion was Conflicts of Interest correlated in this study with a reduced survival for these cells. The fact that many studies imply an association between The authors have no actual or potential conflict of interest. COX-2 inhibition and apoptosis induction or cell prolifera- tion inhibition, without assessing whether COX-2 activity is effectively decreased, suggests caution in the interpretation Acknowledgments of the data. This is confirmed by the observation that C. Sobolewski. and C. Cerella are recipients of doctoral different COX-2 inhibitors may trigger apoptosis in the same and postdoctoral Tel ´ ev ´ ie gtrants, respectively. Research at cancer cell model by modulating different mechanisms. For the Laboratoire de Biologie Moleculair ´ e et Cellulaire du example, celecoxib [113] induced apoptosis by an inhibition Cancer (LBMCC) is financially supported by “Recherche of Akt phosphorylation in prostate cancer cells COX-2- Cancer et Sang” foundation, by the “Recherches Scientifiques positive LNCaP without affecting Bcl-2 level. In contrast, a Luxembourg” association, by “Een Haerz fir kriibskrank study by Liu et al. [102] revealed that NS-398 in the same Kanner” association, by the Action Lions “Vaincre le Can- cell line was able to induce apoptosis but down-regulation of cer”association and by Tel ´ ev ´ ie Luxembourg. The authors are Bcl-2. 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The Role of Cyclooxygenase-2 in Cell Proliferation and Cell Death in Human Malignancies

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Hindawi Publishing Corporation
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Copyright © 2010 Cyril Sobolewski et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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1687-8884
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10.1155/2010/215158
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Hindawi Publishing Corporation International Journal of Cell Biology Volume 2010, Article ID 215158, 21 pages doi:10.1155/2010/215158 Review Article TheRoleofCyclooxygenase-2inCellProliferationand Cell Death in Human Malignancies 1 1 1 2 1 Cyril Sobolewski, Claudia Cerella, Mario Dicato, Lina Ghibelli, and Marc Diederich LaboratoiredeBiologieMol´eculaire et Cellulaire du Cancer, Hopital ˆ Kirchberg, 9 rue Edward Steichen, 2540 Luxembourg, Luxembourg Dipartimento di Biologia, Universita ` di Roma di Roma Tor Vergata, Via Ricerca Scientifica snc, 00133 Rome, Italy Correspondence should be addressed to Marc Diederich, marc.diederich@lbmcc.lu Received 16 July 2009; Accepted 18 December 2009 Academic Editor: Simone Fulda Copyright © 2010 Cyril Sobolewski et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. It is well admitted that the link between chronic inflammation and cancer involves cytokines and mediators of inflammatory pathways, which act during the different steps of tumorigenesis. The cyclooxygenases (COXs) are a family of enzymes, which catalyze the rate-limiting step of prostaglandin biosynthesis. This family contains three members: ubiquitously expressed COX- 1, which is involved in homeostasis; the inducible COX-2 isoform, which is upregulated during both inflammation and cancer; and COX-3, expressed in brain and spinal cord, whose functions remain to be elucidated. COX-2 was described to modulate cell proliferation and apoptosis mainly in solid tumors, that is, colorectal, breast, and prostate cancers, and, more recently, in hematological malignancies. These findings prompt us to analyze here the effects of a combination of COX-2 inhibitors together with different clinically used therapeutic strategies in order to further improve the efficiency of future anticancer treatments. COX- 2 modulation is a promising field investigated by many research groups. 1. Introduction: Inflammation and ago [3], only recently, this link has been further investigated, thus evidencing that the incidence of several cancers is Cancer are Linked tightly associated to inflammation such as colon, breast, and Inflammation is the major reaction of natural immunity with prostate cancers [4–6]. This hypothesis is supported by the the goal to defend the organism against pathogens. It can be findings that the tumor microenvironment is characterized induced upon bacterial infections by compounds including by the infiltration with different types of immune cells (i.e., lipopolysaccharides, as well as by viruses, which are detected dendritic cells, lymphocytes, and macrophages) responsible by Toll-like receptors (TLRs), expressed by immune cells for the release of cytokines [1]. The role of these cytokines like macrophages. Besides, inflammation can be triggered in tumor incidence has been established in many studies. by physical injuries (i.e., UV) or chemical compounds (i.e., For example, the overexpression of TNFα in transgenic reactive oxygen species) [1]. The activation of specific recep- mice bearing a lung tumor is associated with an increase tors triggers intracellular signals (i.e., NFκB, p38, or MAPKs- of the size of the tumor [7]. Moreover, a chronic intake mediated), which regulate pro-inflammatory cytokine expre- of nonsteroidal antiinflammatory drugs (NSAIDs) leads to ssion, such as interleukin 1 beta (IL1β), tumor necrosis factor a significant reduction in the incidence of such tumors. alpha (TNFα), interleukin 6 (Il6), together with chemokines Colorectal cancer (CRC), which remains an important cause and cell adhesion proteins [1], in turn, leading to the of death in the industrialized world, is one of the most recruitment and the activation of immune cells. characterized types of tumor that benefits from treatment by Several diseases are associated to chronic inflamma- NSAIDs [8]. Interestingly, chronic use of aspirin is reported tion, such as osteoarthritis, Crohn’s disease, and cancer to reduce the relative risk of CRC by about 50% [9]. Familial [2]. Although the first evidence of a connection between adenomatous polyposis, an inherited form of colon cancer, inflammation and cancer dates back to more than a century is characterized by the development of preneoplastic polyps. 2 International Journal of Cell Biology At the molecular level, this disease is caused with a mutation conversion of arachidonic acid released from the plasma of a tumor suppressor gene called Adenomatous polyposis membrane by phospholipase A2 to prostaglandin G2 by coli (APC). It has been shown that the use of NSAIDs, like the cyclooxygenase activity. The second reaction is mediated sulindac, as a chemopreventive treatment, is able to decrease by the peroxidase activity and leads to the conversion the incidence of polyp formation [10]. Similar results were of prostaglandin G2 to prostaglandin H2. Then, different obtained with celecoxib [11], which is now approved by the synthases convert prostaglandin H2 to prostaglandin D2, Food and Drug Administration’s Oncologic Drugs Advisory F2α, E2, I2, and thromboxane A2 (Figure 1). Committee as an adjuvant in FAP therapy. Prostanoids (prostaglandins and thromboxanes) are A body of evidence indicates a role for inflammation immediately released from the cells, where it is believed in the development/modulation of different steps of cancer that they act locally in an autocrine and paracrine manner progression. Inflammation may play a role in tumor initia- through different receptors activating different intracellular tion by triggering the production of reactive oxygen species pathways still to be completely elucidated (Figure 1)[22]. (ROS), responsible for DNA damage, thus increasing the Prostaglandins, specifically, are important for physiological rate of mutations [12]. It may also be implicated in tumor functions like vasodilatation (PGD2, PGE2, PGI2), gastric promotion, where inflammation triggers the secretion of cytoprotection (PGI2), maintenance of renal homeostasis, growth factors, such as the epithelial (EGF) and fibroblast and platelet aggregation. Besides, prostaglandins play a growth factors (FGF). These, in turn, favor the proliferation major role in mediating fever (PGE2), pain sensitivity, and of initiated tumor cells by determining an imbalance between inflammation [21]. cell proliferation and cell death stimuli [6], due to the So far, three isoforms of COXs have been identified. activation of different cell survival pathways [7]. Cyclooxygenase-1 (COX-1) is a glycoprotein of 71kDa, which Besides, the different cytokines produced during inflam- is constitutively expressed in different tissues. COX-1 is mation (i.e., TNFα,IL1β, IL6, and IL8) can also activate encoded by a gene on chromosome 9 and plays a role in tissue several survival pathways, thus leading to an escape of tumor homeostasis by modulating several cellular processes ranging cells from cell death. Well known is the case of TNFα, from cell proliferation to angiogenesis or platelet aggregation produced by tumor and immune cells, which leads to the due to thromboxane production [21]. survival of cancer cells by the upregulation of antiapoptotic Cyclooxygenase-2 (COX-2) is the inducible isoform, proteins, that is, Bcl-2 [13–15], via the activation of the which is regulated by growth factors and different cytokines nuclear factor kappa B (NFκB) [16]. The modulation of such as IL1β,IL6,orTNFα [23], therefore overexpressed pro-survival pathways or anti-apoptotic proteins makes the during inflammation. The COX-2 gene is located on chro- expression/activation of such proinflammatory mediators mosome 1 and its promoter displays an NFκBresponse also a determining factor in chemoresistance. A constitu- element as well as other cytokine-dependent (i.e., IL6) tive activation of such proinflammatory factors has been response elements [21]. The protein shows a 60% homology frequently found in many cancers, such as hepatocellular with COX-1 [24]; in addition, COX-2 presents a C-terminal carcinoma [17], prostate cancer [18], as well as chronic and extension and a different binding site for NSAIDs, which acute myeloid leukemia [19], where it is frequently associated makes COX-2 a preferential target compared to COX-1, thus with a bad prognosis. In these instances, the modulation being specifically inhibited at lower doses [25]. of Bcl-2 anti-apoptotic family members has been frequently Finally, COX-3 has been identified as a splice variant of shown [13–15, 20]. COX-1, and it is present mainly in brain and spinal cord Amongst the different mediators of inflammation, the [26, 27]. Currently, the role of COX-3 is not known. Some cyclooxygenases (COXs) clearly appear to be implicated in pieces of evidence suggest a possible role in pain sensitivity, cancer. This review focuses on COX-2, the inducible form, based on studies focused on the mechanism of action of normally induced and implicated in inflammation, and acetaminophen (paracetamol), recently evoked as a selective intends to analyze what is currently known about the link inhibitor of COX-3 [28]. However, this hypothesis is debated between COX-2 and cancer, in terms of effects on cell pro- because other findings argue that acetaminophen targets at liferation and cell death. In this view, we will focus our the same time COX-2 [29]. attention on studies analyzing the effects of COX-2 inhibitors on cancer cells, when used alone as well as in combination 3. COX-2 As a Tumor Promoter and a Good with therapeutic approaches, including radiotherapy, chem- Candidate for Cancer Therapy otherapeutic agents, and photodynamic therapy. Finally, we will consider the relevance of COX-2-independent effects. Overexpression of COX-2 has been detected in a number of tumors,suchascolorectalbreastaswellaspancreatic and lung cancers [2, 30–32], where it correlates with a 2. The Cyclooxygenase Enzyme Family poor prognosis. Moreover, overexpression of COX-2 has Cyclooxygenases (or prostaglandin H synthases), commonly been reported in hematological cancer models such as referred to as COXs, are a family of myeloperoxidases located RAJI (Burkitt’s lymphoma) and U937 (acute promonocytic at the luminal side of the endoplasmic reticulum and nuclear leukemia) [33, 34]aswellasinpatient’s blastcells [32, 34]. membrane [21], which catalyze the rate-limiting step of Data suggested that COX-2 may play a role in different steps prostaglandin biosynthesis from arachidonic acid [21]. These of cancer progression, by increasing proliferation of mutated enzymes act by two coupled reactions. The first one is the cells [30], thus favoring tumor promotion as well as by International Journal of Cell Biology 3 Arachidonic acid COX-2 NSAIDs, COX-2 selective PGG2 inhibitors COX-2 PGH2 Isomerases PGF2α PGD2 PGJ2 PGE2 PGI2 EP 1, 2, 3, 4 FP receptor DP receptor PPARγ IP receptor PPARδ Figure 1: Metabolism of arachidonic acid by COX-2 and receptors implicated in response to prostaglandins (according to Chandrasekharan et al. [21]). Prostaglandins act through different receptors to mediate their effects. PGE2 is able to bind four receptors (EP1, 2, 3, and 4). These receptors do not possess the same ligand affinity and their expression is tissuedependent. The different receptors are associated with different intracellular pathways. Most of these receptors are localized in the plasma membrane but nuclear receptors PPARγ can also bind PGJ2. Abbreviation: COX-2, cyclooxygenase-2; PG, prostaglandin; FP, prostaglandin F receptor; DP, prostaglandin D receptor; EP, prostaglandin E receptor; IP, prostaglandin I receptor; PPAR, peroxisome proliferator-activated receptor; NSAIDs, nonsteroidal anti-inflammatory drugs. affecting programmed cell death and affecting the efficacy are mostly described for adherent tumors while this link of anticancer therapies [35–39] to be, finally, implicated in is poorly understood for hematopoietic malignancies such metastasis formation, for example, by affecting apoptosis as leukemia or lymphoma. Indeed, several papers have induced by loss of cell anchorage (anoikis) [40]. reported that PGE2 is the most important prostaglandin COX-2 induction or overexpression is associated with an produced during colorectal carcinogenesis [44]. Moreover, it increased production of PGE2, one of the major products of is known that the level of PGE2 increases in a size-dependent COX-2 which is known to modulate cell proliferation, cell manner in Familial Adenomatous Polyposis (FAP) patients death, and tumor invasion in many types of cancer including [45], suggesting a correlation between tumor growth and colon, breast, and lung. Prostaglandin E2 acts through prostaglandin biosynthesis. Tumorigenesis is characterized different membrane receptors called EP receptors (EP1, EP2, by a disequilibrium between cell proliferation and cell death. EP3, and EP4) [41]. Thesereceptors areall locatedon PGE2 is able to inhibit apoptosis in human colon cancer the cell surface and characterized by seven-transmembrane cells. It has been demonstrated that PGE2 can upregulate domains, and rhodopsin-type G protein-coupled receptors, the level of the anti-apoptotic protein Bcl-2 in HCA-7 cells but trigger different signaling pathways. Thus, it is known (adenocarcinoma), which produce significant amounts of that EP1 signaling acts through phospholipase C/inositol PGE2. This paper described a modulation of the MAPK triphosphate signaling, leading to intracellular mobilization pathway that precedes the upregulation of Bcl-2 [46]. PGE2 of calcium. EP2 and EP4 receptors are coupled with G can mediate its effect through EGF receptor, leading to proteins which activate adenylate cyclase, leading to an MAPK activation. The ability of PGE2 to modulate tumor increase of intracellular cAMP [41]. cAMP is then able to progression in colorectal cell has been shown in other activate kinases such as protein kinase A (PKA) or PI3K for models of colon cancer such as HT-29 cells that express example, and also GSK3 leading to an activation of β-catenin, EP receptors. In this cell type, PGE2 is associated with an a pathway regulating cell proliferation [42, 43]. In contrary increase of cAMP through EP4 receptor. The effect can be to EP2 and EP4, EP3 is coupled with Gi protein, leading reversed by L-161982, an antagonist of EP4 [47]. Moreover, to an inhibition of adenylate cyclase, and thus a decrease PGE2 transactivates EGFR by triggering the release of of cAMP inside the cells [41]. The differential expression amphiregulin, a well-known EGFR ligand [48]. SC-236, an of these different receptors according to the cell type may inhibitor of COX-2, is able to inhibit cell proliferation of explain the diverse and antagonist effects of PGE2 described HT-29 cells and this effect is greater in combination with an in literature. amphiregulin neutralizing antibody [47]. In this cell line, the Until now, there are multiple evidences about the role expression of amphiregulin is correlated to the expression of of PGE2 in tumorigenesis in some cancers. These evidences COX-2. 4 International Journal of Cell Biology The transactivation of EGFR by PGE2 can lead also to AKT Alltogether these data together suggest that PGE2 and, activation, which is a well-known survival pathway [49]. thus, COX-2 play an important role in tumor progression This effect was well described in a study by Tessner et al. by enhancing cell proliferation, cell survival, and tumor [50] demonstrating that 16,16-dimethyl PGE2 (dmPGE2) invasion. The diversity of PGE2 receptors and their different inhibits radiation-induced apoptosis in the mouse intestinal signaling pathways suggest that the protumorigenic effect epithelium. Using HCT-116 cell line as a model to reflect of PGE2 depends on the cell type and the type of receptor the effect on mouse small intestine, it has been shown expressed. Until now, many signaling pathways associated that the anti-apoptotic effect of dmPGE2, which is known with tumor progression are linked to PGE2 and this could to bind EP2, was tightly related to AKT phosphorylation explain why the use of COX-2 inhibitors is a good strategy through activation of EGFR and leads to an inhibition of in cancer therapy. However, the signaling pathways of EP Bax translocation in mitochondria, an important step for receptors are not completely characterized and their precise apoptosis [51]. roles in the different cancers remain to be elucidated before a PGE2 modulates also tumor growth of lung cancer. This clinical application. effect has been described by Yamaki et al. [52] showing COXs may be targets of several compounds that may that PGE2 activates Src kinase in A549 cells, leading to inhibit their functions. Combination of such preferential or an induction of cell growth. These cells express EP3 that selective COX-2 inhibitors with anti-cancer agents already activates Src (sarcoma) kinase. This study has demonstrated used in clinics were tested with the goal to improve the that the activation of Src leads to an activating phosphory- efficiency of anti-cancer protocols. lation of STAT3, a transcription factor known to regulate COX-2 is the preferential target of several NSAIDs cyclin D1 transcription, an important positive regulator (Figure 2)[64, 65]. Historically, NSAIDs used for clinical and of cell proliferation. Apoptosis can be inhibited because anti-inflammatory purposes were represented by the nonse- STAT3 regulates the transcription of Bcl-XL, a well-known lective COX-2 inhibitors, to which belong aspirin, sulindac anti-apoptotic protein [53]. Moreover, Src phosphorylates acid and, more recently, agents such as nimesulide, ibuprofen p27, a protein known to inhibit cell cycle progression and naproxen. As their definition well reflects, this first especially at the G1/S transition [54]. However, it has been generation of NSAIDs may affectbothmainCOXsisoforms, recently shown that this protein plays a dual role as the even if preferentially COX-2 (see above). Their mechanisms unphosphorylated form of p27 inhibits the cell cycle, and of action are not all completely elucidated, complicated by thus cell proliferation. If phosphorylation occurs on T157 the fact that different agents seem to act in different ways. and T198 by PI3K (phosphoinositide 3-kinase), it triggers For example, different NSAIDs bind the active site of COX- cell cycle transition by stabilizing the cyclin D1/cdk4 complex 2. Commonly, binding occurs by a reversible competitive [55]. Thus phosphorylation of S10 appears to be important inhibition (i.e., ibuprofen, naproxen, and indomethacin). for other phosphorylation steps and it has been hypothesized In contrast, aspirin is able to acetylate the active site of that Src kinase can play this role [55]. Moreover, it is COX at a serine residue, leading to an irreversible inhibition known that phosphorylation of p27 is responsible also for (see Figure 2, summarizing the classification of COX-2 its degradation by the proteasome [56]. All together these inhibitors mentioned in this review). Considerable side data suggest that PGE2 increases cell proliferation via p27 effects generated by the interference with homeostatic func- phosphorylation through EP4 receptors. tions modulated by COX-1 include increased incidence of Nonsmall lung cancer is characterized by a Ras mutation gastrointestinal hemorrhage and ulceration upon chronic or correlated with a poor prognosis [57]. Activation of Ras leads long-time intake [66]. A novel generation of COX-2-selective to an upregulation of COX-2 resulting in increased PGE2 inhibitors NSAIDs termed “Coxibs” was then developed. production [58]. PGE2 increases cell proliferation of A549 These compounds promised to be much less gastrotoxic. cells (adenocarcinoma) and this effect is associated with an They act as competitive inhibitors of the active site of activation of Ras pathway via EP4 receptor. In this case, PGE2 COX-2 and present indeed a higher specificity. However, mediates its effect by the release of amphiregulin, the most concerns related to a long-time/chronic intake of these abundant ligand in A549 cells [59]. EGFR activation leads to drugs raised quite soon, following some clinical reports, activation of MAPK pathway that regulates cell proliferation suggest a correlation between an increased risk of myocardial by transactivating several oncogenes such as c-myc [60]. infarction and their consumption [67]. This has lead to PGE2 is also important for tumor invasion. A study by the voluntary withdrawal of some of these agents, that is, Ma et al. [61] described that PGE2 can increase the number rofecoxib and valdecoxib [68], and drastic regulatory advices of metastasis. This effect has been demonstrated in a model regarding the use of the other ones, thus opening a discussion in which murine mammary tumor cells 66.1 were injected on the real benefitsversussideeffects of their use in clinics. in syngenic immune competent BALB/CByJ mice. All these Consequently, studies focused on the use of traditional cell lines express EP1, 2, 3, and 4. The use of EP4 antagonists versus COX-2-selective NSAIDs, frequently associated to the (AH23848 and AH6809) decreased surface tumor colonies elaboration of economical models, have been performed in and reduced tumor invasion. Another study has revealed that these latest years, with the aim to evaluate the real risks PGE2 increases the level of VEGF in granuloma [62]. VEGF together with the costeffectiveness and, possibly, identify is an important factor of angiogenesis, and thus of tumor classes of users/patients where regular NSAIDs intake may be progression by enhancing the vascularization of the tumors beneficial. Although, further analyses need to be performed, [63]. a number of reports suggest that Coxibs may really increase International Journal of Cell Biology 5 O O HO H C CH CH Aspirin COOH Ibuprofen COOH Sulindac sulphone Cl Sulindac sulphide OH NH Cl OH Nabumetone Naproxen Cl Diclofenac OH N N H CO CH 3 S Indomethacine Piroxicam O O CH COOH NH 2 O O S O NO H C CH CF H C S 3 NH H N S Valdecoxib NS-398 CF Br H C 3 Celecoxib Rofecoxib F C 3 SC-236 O O S DUP697 O S CH Cay10404 NH 2 O OH NO O 2 CH NH CH O NH Meloxicam Nimesulide O Figure 2: COX-2 inhibitor classification. COX-2 is the target of many compounds. COX-2 inhibitors described in this review are classified according to their ability to inhibit COX-2: nonselective (green), selective (pale blue), and preferential (grey). O 6 International Journal of Cell Biology cardiovascular risks only in patients presenting a positivity transition, which requires activation of the cyclin B-cdk2 to other cardiovascular factor risks, as high blood pressure complex. This complex accumulates during the previous step and altered lipid metabolism [69–73]. These results suggest of the cell cycle but is inactivated by a phosphorylation that their use should be limited to patients with a low risk at tyrosine 15 and threonine 14 by Wee 1 and Myt 1. of cardiovascular complications after analysis of multiple These phosphate groups are removed by the phosphatase biomarkers [Chaiamnuay et al., 2006, clinical reviews]. CDC25A when cells enter mitosis. In the case of DNA Therefore, the future perspective in the pharmacological damages, p53 is activated and increases the level of p21 use of preferential versus selective COX-2 inhibitors is the that is directly inhibiting cdk2. Moreover, 14-3-3 protein, identification of a panel of interesting biomarkers, helping a transcriptional target of p53, leads to a sequestration of in defining individual biological risk factors and limiting the cdk2 in the cytoplasm [83]. Other mechanisms involved in use of a specific class of COX-2 inhibitors to the appropriate the regulation of the G2 checkpoint or the mitotic spindle responders [74, 75]. This approach will have a considerable checkpoint are reviewed by Stewart et al. [54]. implication in therapy as well as in chemoprevention of Cancer cells are characterized by deregulation of the cell inherited forms of colon cancer. cycle via alteration of cell cycle controllers (cyclins) and cell It is interesting to mention that recent alternative cycle regulators (p53) [54], resulting in a perturbation of cell approaches have been considered. Strillacci et al. [76]and cycle checkpoints. Chan et al. suggested RNA interference using adenoviral Currently, there is evidence that prostaglandins produced vehicles. Moreover, other selective COX-2 inhibitors have by COX-2 intervene in tumor cell proliferation as NSAIDs been developed and experimentally used: SC-558 [35], and selective COX-2 inhibitors inhibit proliferation of DUP-697 [77], SC-58125 [78], and NS-398 [8]. Some of different cancer cell types expressing COX-2 [30]. NS-398, them induce an irreversible inhibition. This is the case for a COX-2 specific inhibitor, was described to reduce cell NS-398, which acts by inducing a conformational change proliferation of MC-26 cell line, a highly invasive mouse of COX-2 [25](Figure 2). Another strategy discussed in CRC cell model expressing constitutively COX-2 [8]. This literature could be the use of EP receptor antagonists. Indeed, effect was associated with a reduction of cyclin D level, a it has been demonstrated that EP antagonists can decrease key protein involved in G1-S transition [54], and PCNA, cell proliferation and cell invasion [47, 61, 79]. This could be thus increasing the processivity of DNA polymerase [82]. a more specific strategy that could limit the other side effects NS-398 and COX-2 specific inhibitor nabumetone reduced of classic COX-2 inhibitors. cell proliferation of U937 (acute promonocytic leukemia) and ML1 (human myeloblastic leukemia), thus leading to an accumulation in G0/G1 phase [33]. Interestingly, meloxicam 4. COX-2 As a Regulator of Cell Proliferation was also able to downregulate PCNA and cyclin A in HepG2 Cell cycle is regulated by different serine-threonine kinase cell line (hepatocellular carcinoma cells), leading to an proteins called cyclin-dependent kinase (Cdk). These pro- inhibition of the cell proliferation and an accumulation of teins regulate the different steps of cell cycle progression the cells in G0/G1 phase of cell cycle [84]. Alternatively, by phosphorylating many substrates (i.e., nuclear lamins) the link between COX-2 and CRC has been demonstrated by the fact that prostaglandin E2 (PGE2) derivating from [54]. These proteins are regulated by phosphorylation and dephosphorylation. Thus, Cdks can be activated by phos- COX-2-mediated arachidonic acid metabolism increased the phatases such as CDC25C (cell division cycle 25 homolog proliferation of colorectal cancer cells [85]. The inhibitory effect of NSAIDs on cell proliferation C) for CDK1 or kinase like CAK (Cdk activating kinase). The activity of cdks is also regulated by cyclins, which form of CRC has been also observed in ovarian cancer. Indeed, heterodimers with cdks leading to an activation of Cdks by treatment of OVCAR-3 tumors xenotransplanted in nu/nu conformational change [54, 80]. mice (nude mice) with aspirin and piroxicam (NSAIDs) and Cell cycle is under the control of other factors, implicated the selective COX-2 inhibitor meloxicam led to a reduction in the regulation of cell cycle transition. These regulatory of tumor growth [86]. mechanisms form checkpoints where the cell cycle can be It has been estimated that 40% of breast cancers show stopped after cellular damage in order to allow repair and an overexpression of COX-2, which is associated with a bad prognosis [5]. Indomethacin (NSAIDs), celecoxib, rofecoxib to maintain cellular integrity or, alternatively, to eliminate mutated and potentially dangerous cells. The INK4 family and nimesulide have been shown to able to inhibit cell prolif- (p16, p15, p18, and p19) and the Cip/Kip family (p21, p27, eration of these cells [5]. Moreover, prostaglandins were able and p57) [54, 80, 81] are key regulators of G1/S transition. to increase cell proliferation of hormonal-dependent breast For example, after DNA damage, p53, a tumor suppres- cancer by increasing transcription of CYP19 aromatase sor gene, activates transcription of p21, which inhibits implicated in estrogen biosynthesis [87]. cyclin E phosphorylation leading to hypophosphorylation Several studies revealed that inhibition of COX-2 by of retinoblastoma protein (pRb) [81]. INK4 family inhibits celecoxib in Burkitt’s lymphoma cell lines RAJI, BjAB, (Epstein-Barr virusnegative), and BL41 led to a reduction Cdk4 and Cdk6, whereas Cip/Kip family inhibits all Cdks. Retinoblastoma protein needs to be phosphorylated in order of cell proliferation [34]. NS-398 and celecoxib were able to to release transcription factor E2F activating genes involved reduce proliferation of pancreatic cancer cell line, Panc-1 in a dose-dependent manner [88]. Treatment with celecoxib of in the S phase-like PCNA (proliferating cell nuclear antigen) [82]. p53 is also important for the regulation of the G2/M these cells implanted into nude mice led to a reduction of International Journal of Cell Biology 7 Cip1 p21 kip1 p27 CDC25 NS-398 (MC26) Growth factors Celecoxib (K562) Cyclin B E2F 1-3 DUP-697 (K562) Genotoxic CDK1 stress Cyclin B Cyclin D pRB CDK1 CDK 4/6 Wee1 G2 E2F 1-3 Celecoxib(K562) DUP-697(K562) G1 p15, p16, p18, p19 p53 NS-398 (MC-26) meloxicam (HepG2) Cyclin E CDK2 PCNA S Cip1 Meloxicam p21 PP (HepG2) Cyclin A p21,p27 CDK2 E2F1/2, pRB PCNA cyclin A/E Pol α ··· P P p21 E2F AAAAA p27 Figure 3: Effects of COX-2 inhibitors on cell proliferation. Cell cycle is divided into different steps: G1, S, G2, and M (mitosis). This process is regulated by cyclin proteins, which activate cyclin-dependent kinase (cdk) and phosphatase (i.e., CDC25) or kinase like cyclin-dependent kinase inhibitors such as p16, p15, p18, p19, p21, and p27 [54]. Selective COX-2 inhibitors are able to modulate some cell cycle checkpoints. In this picture, some examples of this link have been shown for different cell types: MC26, colorectal cancer; HepG2, hepatocellular carcinoma; K562, chronic myeloid leukemia. Cdk; cyclin-dependent kinase; pRb, retinoblastoma protein; PCNA, proliferating cell nuclear antigen. the volume of the tumor [88]. Other studies have shown that pathway, also called the mitochondrial or stress-induced celecoxib is able to reduce cell proliferation of the chronic apoptotic pathway, is activated in response to damaging myeloid leukemia (CML) cell line K562, which expresses stresses, such as DNA damage. Typical hallmarks of this path- COX-2 at the mRNA and protein level [89]. This effect way are mitochondrial outer membrane permeabilization was accompanied by an accumulation of cells in G0/G1. (MOMP), accompanied by a collapse of the mitochondrial Moreover, the inhibition of cell proliferation was correlated membrane potential [51]. These events lead to the release to a downregulation of cyclin D1, cyclin E, and pRb and the of cytochrome c into the cytosol, which is an indispensable upregulation of p16 and p27 [89]. Similar results were found component of the apoptosome, the death complex formed on this cell type with the other selective COX-2 inhibitor also by APAF-1, and procaspase-9. Once recruited, this DUP-697 [77]. Different effects are recapitulated in Figure 3. protease is cleaved to its activated form (caspase-9) to further activate the executor caspase-3 and, finally, to finalize the apoptotic program. 5. Implication of COX-2 in Cell Death Alternatively, the extrinsic, or physiological, apoptotic pathway (Figure 4) can be triggered upon binding of specific 5.1. Apoptosis. Apoptosis (type I cell death) is important for the development and maintenance of tissue homeostasis ligands to death receptors characterized by the presence of of multicellular organisms [90, 91]. This active form of a death effector domain [94]. Ligands include cytokines, such as TNFα, tumor necrosis factor-related apoptosis- cell death is characterized by the occurrence of typical cell alterations including plasma membrane blebbing, cell inducing ligand-induced apoptosis (TRAIL), or FAS. After shrinkage, chromatin condensation and nuclear fragmen- binding, death inducing silencing complex (DISC) is formed. tation, and, finally, formation of apoptotic bodies, which The DISC is composed by the adaptors proteins TRADD can be phagocyted by macrophages [92]. Deregulation of (TNF receptor-associated death domain) and FADD (Fas- associated death domain) and is able to recruit and activate apoptosis is linked to several pathophysiological disorders, including autoimmune disorders, Alzheimer’s disease, and pro-caspase-8. Finally, caspase-8 activates caspase-3 in order cancer [93]. to trigger the final steps of apoptosis (Figure 4). Two major cascades of intracellular events are commonly Cross-talks between the two pathways take place. The involved in mediating apoptosis (Figure 4). The intrinsic extrinsic apoptotic pathway can activate the intrinsic 8 International Journal of Cell Biology Increase of TRAIL receptor Sulindac TNFα, indomethacine by NS-398 and CAY10404 in TRAIL, SC-236 (HT29) SK-Hep 1 and HLE cells DR5 clustering in FasL celecoxib (MG-63) cholesterol-rich domain by DUP-697 in HT29 cells 14-3-3 PI3K/PKB GSH depletion Bad Bax Bad DD Bak Bax Bcl-2 Cyt Bax Bid Cyt Cyt Cyt Diablo Bak Cyt Pro Bak Bak Bcl-2 Pro-casp-8 Casp9 Bcl-x DUP-697 Bax Cyt Cyt (K562) Diclofenac Bak (HT29; HCT-15) Cyt Cyt Bcl-x L Casp-8 Cyt Cyt Cyt Bcl-2 APAF Cyt NS-398 IAPs CAY10404 Apoptosome (SK-Hep; HLE) NS-398 Diablo LAP Casp-9 Casp-3 CAY10404 (SK-Hep1; HLE) Celecoxib DNA damage (neuroblastoma) Activation Gelsolin Tap 73 transcription Bax, Bid, Lamin A NFkB casp-9 β-catenin Figure 4: Effects of COX-2 inhibitors on apoptosis. Apoptosis can be mainly mediated by two pathways: the mitochondrial, intrinsic, or stress-induced apoptosis, which is activated in response to damaging stresses and the extrinsic pathway, triggered by the binding of ligands to specific death receptors [51]. COX-2 inhibitors are able to modulate stress-induced apoptosis as well as extrinsic apoptosis in several cell types. In this picture, some examples of these interaction discussed in the text are presented for different cell types: LNCaP, prostate cancer; K562, chronic myeloid leukemia; HT29, colorectal cancer; SK-Hep 1 and HLE, human hepatocarcinoma cells; HepG2, hepatocarcinoma; Be17402, hepatocarcinoma; SMMC-7402, hepatocarcinoma; MG-63, osteosarcoma. Abbreviation: AIF: apoptosis-inducing factor; Bcl-2, B cell lymphoma 2; Bid, Bcl-2 interacting domain; Casp, caspase; Cyt, cytochrome C; DD, death domain; DED, death effector domain; DISC, death-inducing silencing complex; PI3K/PKB, phosphatidyl inositol-3 kinase/protein kinase B; FADD, Fas-associated death domain; GSH, glutathione; PTP, transition permeability pore; TNF, tumor necrosis factor; TRAIL, TNF-related-inducing-apoptosis-ligand. pathway via truncation of the BH3-only protein Bid (t- same role [99]. In contrast to Bax, Bak is already present Bid) by caspase-8. t-Bid interacts with mitochondria, by at the surface of mitochondria, normally sequestered in its favoring the activation of the proapoptotic Bcl-2 family active monomeric form by the Bcl-2 anti-apoptotic members members Bak and Bax, thus leading to MOMP and caspase- Bcl-xL and Mcl-1 (see Burlacu for a general overview of 9activation[51, 95](Figure 4). The intrinsic apoptotic the Bcl-2 family members modulation involved in Bax/Bak pathway may, in turn, activate caspase-8, downstream to activation [51]). caspase-3 [96](Figure 4). Cross-talks represent an important Apoptosis is regulated in order to maintain tissue home- strategy of amplification loops carried out by dying cells to ostasis. This regulation implicates protein-protein inter- ensure/potentiate cell death. actions, with some of them counteracting apoptosis. In this view, the interaction between Bcl-2 family pro- and antiapoptotic members represents a crucial and delicate step. 5.1.1. Involvement of COX-2 in Intrinsic Apoptosis. When cells are damaged by a variety of chemicals or physical stress Bcl-2 is the best described member of this family preventing (i.e., reactive oxygen species, UV, and ionizing radiation), Bax activation [51]. Bax can form also a complex with the anti-apoptotic protein Bcl-xL [53] and Mcl-1 [14]. Similarly, they undergo apoptosis by triggering the intrinsic apoptotic pathway (Figure 4). This pathway may be associated with a Bak activity is monitored by the anti-apoptotic members Bcl- xL and Mcl-1 [51]. The interaction between Bax/Bak and the redox disequilibrium, mediated by depletion of glutathione (GSH) [94, 97, 98], required for the activation and transloca- Bcl-2 family anti-apoptotic members is carefully regulated by tion to mitochondria of the Bcl-2 pro-apoptotic member Bax the BH-3-only proteins. Another carefully regulated down- stream checkpoint of the apoptotic pathway is the activation [98], which, in turn, forms complexes (oligomers) mediating MOMP and cytochrome c release. As Bax, Bak may play the of caspases. Inhibitor of apoptosis (IAP) family, by directly PTP DED FADD DED FADD DISC International Journal of Cell Biology 9 interacting with caspases (i.e., XIAP, survivin [100]) controls is anti-apoptotic and lacks the transactivation domain. and prevents their activity once cleaved. IAPs monitoring DeltaNp73 is overexpressed in neuroblastoma, leading to function can be, in turn, counteracted by the pro-apoptotic chemotherapy resistance [109]. It has been shown that SMAC/DIABLO, a mitochondrial heterodimer, which is celecoxib was able to upregulate Tap73 and downregulate released from mitochondria when MOMP is affected [101]. DeltaNp73. These data suggest the use of COX-2 inhibitors as This interaction favors the induction of apoptosis. p73 modulators in order to improve efficiency of chemother- Imbalance between cell proliferation and apoptosis apy [110]. observed in cancer can be tightly related to an altered The apoptotic effect of COX-2 inhibitors has been also function of pro-apoptotic proteins as well as to an up- observed for other tumor cell types, such as in the chronic regulation of anti-apoptotic proteins (i.e., Bcl-2 or IAPs) myeloid leukemia model K562 where DUP-697 induced or a downregulation of tumor suppressor genes (i.e., p53). apoptosis by cell cycle arrest and caspase-8 activation [77]. In addition, the activation of prosurvival pathways (i.e., COX-2 inhibitors can also activate prosurvival pathways. PI3K/Akt) may be implicated upstream. Inflammation can The PI3K/Akt pathway is a survival pathway, frequently contribute to this imbalance via cytokines secreted in the activated in cancer cells [49]. PI3K produces PIP3 (phos- tumor microenvironment able to activate survival path- phatidylinositol 3,4,5 triphosphate) that activates PDK1 ways. For example, TNFα can induce NFκB, leading to (pyruvate deshydrogenase kinase). This protein phosphory- an inhibition of apoptosis [38]. COX-2 seems also to play lates and activates PKB (protein kinase B), which, in turn, is a role in this process because it is known that COX-2 responsible for the phosphorylation of several targets playing inhibition is correlated to an increase of apoptosis in several a modulator function in apoptosis. An anti-cancer effect of cancer models. NS-398 downregulated Bcl-2 expression in an celecoxib due to the inhibition of Akt signaling [111]was androgen-sensitive human prostate cancer cell line LNCaP observed in a gastric cancer model. Celecoxib triggered also that exhibited a high constitutive level of COX-2 [102]. apoptosis in osteosarcoma cells (MG-63) through down- Similar results have been observed in human colorectal regulation of Bcl-2, survivin and PI3K (phosphoinositide 3- cancer cells (HCA-7 cell line which expresses COX-2) where kinase) pathway [112]. Similarly, Hsu et al. [113] found that PGE2 was able to inhibit apoptosis induced by SC58125, inhibition of Akt phosphorylation by celecoxib in prostate a selective COX-2 inhibitor, and increase Bcl-2 expression cancer models (LNCaP and PC3 cell lines which express [46]. Different mechanisms are supposed to explain how constitutively COX-2) led to apoptosis, but in this case COX-2 inhibitors may trigger apoptosis. In a number of without affecting Bcl-2 level. studies, COX-2 inhibition was linked to a concomitant The PI3K pathway is negatively regulated by PTEN increase of intracellular arachidonic acid. In HT-29 human (phosphatase and TENsin homolog), which converts PIP3 colon adenocarcinoma cell this accumulation led to the in PIP2, preventing PKB activation and Bad phosphory- induction of apoptosis [103]. The arachidonic acid-induced lation/sequestration. Thus PTEN is considered as a tumor apoptosis was inhibited by Bcl-2 transfection, indicating suppressor gene. It has been shown that NS-398 was able to a role of arachidonic acid in affecting Bcl-2 intracellular increase the level of PTEN in human gastric carcinoma cell levels [103]. Accumulation of arachidonic acid can affect line MKN45 [114]. apoptosis by mediating an increase of pro-apoptotic intra- One of the PKB targets is Bad, a BH3-only member [51]. cellular ceramides caused by activation of sphingomyelinase The nonphosphorylated form of Bad plays a pro-apoptotic [104, 105]. Sulindac sulphide, a metabolite of sulindac, also role, by binding Bcl-xL or Bcl-2 and, thus, preventing their activates sphingomyelinase and enhances the ceramide level interactions with Bak and Bax. The activation of PI3K/Akt in the two human colorectal carcinoma cell lines HCT116 pathway may lead to the phosphorylation of Bad, which and SW480 [106]. is consequently sequestrated in the cytoplasm by 14-3- COX-2 reduces pro-apoptotic nitric oxide (NO) levels in 3 protein and, in this way, inhibited in its pro-apoptotic cancer cells downstream of prostaglandin production [30] function [51]. It has been reported that sulindac sulphone, (see Brune ¨ et al. [107] for an overview on nitric oxide role indomethacine, and SC-236 were able to induce apoptosis in apoptosis). Chang et al. reported that PGE2 prevented via Bad activation, by inhibiting 14-3-3 expression in a apoptosis induced by NGF (nerve growth factor) withdrawal dose- and time-dependent manner in HT-29 cells [115]. by increasing the level of dynein light chain, an inhibitor This effect was tightly related to PPARδ. It is known, of neuronal NO synthase in pheochromocytoma of the indeed, that 14-3-3 protein contains PPRE recognized and rat adrenal medulla PC12 cells, thus leading to decreased bound by PPARδ [115]. COX-2 can mediate the synthesis of intracellular NO levels [108]. prostaglandin I2, which can bind and activate PPARδ [41]. More recently, connections between COX-2 inhibitors Thus, it has been suggested that the inhibition of COX-2, and p53 family members have been described. For example, leading to a decrease of PGI2, impaired PPARδ activation, celecoxib was shown to able to modulate different isoforms of which, in turn, was responsible for a downregulation of p73, a p53 family member in neuroblastoma cell lines [109]. 14-3-3 protein, thus allowing Bad to play its pro-apoptotic p73 encodes many isoforms with different roles. Tap73 is functions [115]. pro-apoptotic and contains a transactivation domain. This NFκB is a most important transcription factor involved isoform is considered as a tumor suppressor gene because in survival by enhancing transcription of anti-apoptotic it seemstobeinvolvedincellcycle regulationaswellas proteins such as Bcl-2 [14, 15]. Sulindac inhibits NFκBin in apoptosis induction [109, 110]. In contrast, DeltaNp73 two colon cancer cell lines (human colon adenocarcinoma 10 International Journal of Cell Biology HCT-15 and HT29 cell lines) [116]. Similar results were Altogether, these results encourage the perspective that obtained with diclofenac, which was able to inhibit nuclear COX-2 inhibitors could be used in future as a therapeutic accumulation of NFκB[117]. In the same study, PGE2 was strategy to sensitize tumor cells to apoptosis induced by demonstrated to increase the transcriptional activity of NFκB physiological stimuli. p65/p50 dimer in CACO-2 cells (human epithelial colorectal adenocarcinoma cells), transfected with a luciferase con- 5.2. Involvement of COX-2 in Other Types of Cell Death struct containing NFκB response elements [117]. It is known that activation of prostaglandin receptors 5.2.1. Anoikis. Anoikis is a form of apoptosis mediated by the induces an increased cAMP level which in turn can activate loss of cell anchorage. This pathway plays a fundamental role protein kinase A (PKA) [58]. Studies have suggested that during development and maintenance of tissue homeostasis PKA, like PKB, phosphorylates Bad [118], leading to its by killing damaged cells or detached cells in order to main- sequestration and inhibition of apoptosis. Some of the pro- tain tissue architecture. For example, the inner endodermal or anti-apoptotic mediators affected by COX-2 inhibitors are cells undergo anoikis after the loss of anchorage to the matrix schematized in Figure 4. during development [124]. It is known also that intestinal epithelial cells loose anchorage when located at the luminal surface, leading to anoikis [42]. As a form of apoptosis, 5.1.2. Implication of COX-2 in Extrinsic Apoptotic Cell Signal- anoikis is dependent on caspase activation and cytochrome ing Mechanisms. Studies reported that COX-2 inhibitors are c release by mitochondria and is regulated by Bcl-2 family also associated with a sensitization of tumor cells to extrinsic members [42]. apoptosis. Thus, DUP-697 sensitized HT29 colon cancer It has been shown that anoikis is prevented in cancer cell line to TRAIL-induced apoptosis. This effect was due cells, thus favoring tumor progression with the formation to an accumulation of arachidonic acid inside the cells, of metastasis [42]. Accordingly, modulation of anoikis is which activates sphingomyelinase, triggering a clustering of considered a promising target for anti-cancer strategies. deathreceptor(DR)5receptorsinceramideand cholesterol- Cell anchorage is due to cell-cell and cell-matrix interac- rich domains [119]. Alternatively, the expression of COX- tions. Cell-cell interactions are mainly mediated by integrins 2 has been frequently associated with a modulation of the which are transmembrane receptors located at the cell expression of death receptors, thus leading to an upstream surface and composed of alpha and beta chains [125]. Many control of the extrinsic apoptotic pathway. Tang et al. [120] intracellular signals can act downstream to integrins, which, showed that COX-2 overexpression in human colon cancers correctly switched on, can ensure cell survival. Some of them cells led to an inhibition of DR5 expression and a resistance are mediated by kinases such as Focal-adhesion-kinase (Fak) to TRAIL-induced apoptosis. Accordingly, COX-2 specific or integrin-linked kinase (ILK) [42]. Fak is phosphorylated inhibitors, NS-398 and CAY10404, are sensitizing human upon integrin adhesion, leading to activation of other sig- hepatocarcinoma cells (SK-Hep1 and HLE) to TRAIL- naling pathways like PI3K, MAPK. ILK is a serine/threonine induced apoptosis. This effectwas duetoanupregulation kinase that directly phosphorylates PKB. of TRAIL receptors (TRAIL R2/DR5 and TRAIL-R1/DR4), Together with cell-cell and cell-matrix interactions, para- together with an ability of the compounds to induce a down- crine factors could be important for the regulation of anoikis. regulation of the anti-apoptotic proteins survivin (IAP) and It has been shown that E-cadherin (epithelial cadherin) Bcl-xL [121]. In hepatocellular carcinoma models (HepG2, can activate COX-2 [23]. It is possible that prostaglandins Bel7402, and SMMC-7402), Li et al. [84] showed that COX- produced by COX-2, which act in an autocrine and a 2 inhibition with meloxicam led to an upregulation of Fas- paracrine manner, favor cell survival. A study from Joseph et mediated apoptosis. In vivo studies performed on transgenic al. [126] showed that PGE2 inhibited anoikis in IEC-18 cells mice constitutively expressing human COX-2 confirmed an (rat intestine ileum cells). This effect was suggested to be due increased resistance to Fas-induced apoptosis in liver, as to cAMP signaling because prostaglandin E2 receptors are coupled to adenylate cyclase, which converts AMP to cAMP shown by the preservation of liver architecture in COX- 2-expressing mice compared to wild type [122]. Similarly, [126]. another study performed on human extrahepatic bile duct Other studies demonstrated that COX-2 inhibits anoikis carcinoma cell line showed that COX-2 induction led to the via activation of PI3K/Akt pathway, as the case of a human inhibition of Fas-induced apoptosis, whereas the inhibition bladder cancer cell line expressing COX-2 [40]. A link of COX-2 with NS-398 in cytokine-treated cells exacerbated between COX-2 and anoikis has been described, further- apoptosis induced by CH-11, an agonist of Fas receptor more, in uterine endometrial carcinoma [127]. COX-2 is [123]. over-expressed in this type of cancer and this is associated AKT pro-survival pathway may play a role also in with tumor aggressiveness. In addition, a recent report based the modulation of extrinsic apoptosis. The human gastric on HEC-1B and RL95-2 (two human endometrial cancer carcinoma cell line MKN45, which expresses COX-2, was cell lines) showed that the treatment of these cells with sensitized to Fas-induced apoptosis by NS-398. The COX- hepatocyte growth factor (HGF) led to an up-regulation of 2 inhibitor, indeed, was able to increase the level of PTEN, COX-2. Hepatocyte growth factor interacts with its tyrosine leading to a decrease of Akt phosphorylation and activation kinase receptor c-Met. This interaction is responsible for of Bad [114]. Some effects of COX-2 inhibitors on extrinsic tumor progression. Overexpression of HGF/c-Met has been apoptosis are summarized in Figure 4. described in different tumors such as breast cancer [128] International Journal of Cell Biology 11 as well as head and neck cancer [129], also in endometrial Polyposis (for celecoxib) [134, 135] and also in breast cancer carcinoma [130]. It has been demonstrated that HGF [136]. This property of COX-2 inhibitors could be useful for inhibited anoikis and treatment of HEC-1B and RL95-2 patients with a high risk to develop cancer such as people cells with the COX-2 selective inhibitor meloxicam prevented with Li-Fraumeni syndrome, for example [137]. The fact HGF-mediated anoikis resistance [127]. Similar results were that many reports in literature suggest that COX-2 inhibitors obtained in head and neck squamous cell carcinoma [131]. are responsible for an inhibition of cell proliferation and Altogether these data suggest that COX-2 may be impli- apoptosis induction in a number of different cancer cell cated in the inhibition of anoikis and that COX-2 inhibitors models prompts to consider a possible use of COX-2 may play a role in inhibiting tumor progression (metastasis), inhibitors in future therapeutical protocols, administered by sensitizing tumor cell to anoikis. alone as well as in combination with anti-cancer clinical protocols in order to improve tumor cell death. 5.2.2. Autophagy. Autophagy is a process triggering cells 6.1. COX-2 Inhibitors in Combination with Radiotherapy. to degrade intracellular constituents, ranging from proteins Radiation therapy is a common treatment used for the up to entire organelles. It represents an important process treatment of solid tumors, such as breast, prostate, colorectal, ensuring the turnover of long-lived cellular components, and lung cancers. It is known that the anti-cancer properties which can be activated also by stress conditions like nutrient of ionizing radiation are due to pleiotropic mechanisms. starvation in order to avoid cell death. The process starts Radiation leads to the formation of DNA doubled-strand with the formation of doubled membrane-bound vacuoles breaks in proliferating cells, which triggers the activation of corresponding to autophagosomes that entrap parts of the DNA damage pathways (i.e., p53), followed by the induction cytoplasm or organelles (i.e., mitochondria). Then, these of apoptosis [36]. The importance of Bcl-2 family members structures are fused with lysosomes (autolysosomes), thus during apoptosis [51] suggests that prosurvival proteins (i.e., leading to the degradation of the intracellular parts previ- Bcl-2, Bcl-xL) play an important role in radioprotection of ously enclosed. Together with apoptosis, when exacerbated, tumor cells. The NFκB pathway seems to be implicated, autophagy contributes to the modulation of homeostasis, by being required in regulating expression of the anti-apoptotic eliminating damaged and potentially dangerous cells (type II Bcl-2 family members like Bcl-xL [36]. Moreover, it is well cell death) [132]. However, the relationship between apopto- established that NFκB regulates the level of COX-2, suggest- sis and autophagy is currently still poorly understood [132] ing that COX-2 may play a role in radiotherapy resistance because in some cases autophagy permits an adaptation of [21]. Similarly, nimesulide could increase radiation efficiency the cells to stress (i.e., nutrient starvation), thus counteract- in nonsmall cell lung cancer in vivo (nude mice) and in ing apoptosis, whereas, in other cases, autophagy is a process vitro (A549 cell line) as shown by Grimes et al. [138]. This triggering downstream apoptosis [132]. Indeed, similar effect was due to a down-regulation of MnSOD (superoxide stimuli can induce both apoptosis or autophagy [132]. dismutase containing manganese (Mn) and localized in This process is implicated in pathologies such as Alz- mitochondria), a primary antioxidant protein and survivin, heimer’s disease and cancer, suggesting a promising field in an anti-apoptotic protein (IAPS family member). These two therapy. By considering that COX-2 is supposed to play a proteins are regulated by NFκB. It is well known that during role in apoptosis and a link between apoptosis and autophagy radiation therapy NFκB can be upregulated due to reactive exists, it is conceivable that COX-2 plays a role also in this oxygen species release and inflammation (i.e., PGE2). This process. Currently, not many studies aimed at investigating report suggests that nimesulide may act on NFκB to inhibit a possible link between COX-2 and autophagy have been MnSOD and survivin. published. Nevertheless, one study revealed that sulindac Melanoma is known to be very resistant to conven- sulphide (NSAIDs) induced apoptosis of the colon cancer tional radiotherapy and chemotherapy. Irradiation of two HT29 cell line. This effect was increased by treatment of melanoma cell lines WM35 and LU1205 in the presence of the cells with 3 methyl-adenine, a well-known inhibitor NS-398, a selective COX-2 inhibitor, strongly exacerbated the of autophagy [133]. Moreover, the extent of apoptosis in G2/M arrest as well as the induction in apoptosis. Accord- Q204L cells (a clone of HT-29 cells in which 3 methyl- ingly, the down-regulation of COX-2 by RNA interference in adenine-sensitive autophagic sequestration is impaired) was these cell lines was followed by an upregulation of p53 and less than in HT29. These data suggest that autophagy can G2/M arrest [36], thus confirming that the effect of NS-398 delay sulindac sulphide-induced apoptosis [133]. is due to its role on COX-2 inhibition. Other studies have shown that the radiosensitivity of PC3 6. COX-2 Inhibitors in Cancer Therapy (human prostate carcinoma cells) and Hela (human cervical Despite the latest progress in cancer research and the differ- carcinoma cells) was enhanced after silencing of COX-2 by ent strategies to kill cancer cells, several tumors are resistant siRNA. NS-398 was able to increase radiosensitivity of PC3 to conventional therapeutics treatment (i.e., radiotherapy, cells expressing COX-2, but not in PC3 silenced for COX-2. chemotherapy, and photodynamic therapy). In contrast, NS-398 enhanced radiosensitivity of Hela cells, COX-2 inhibitors play an important role in cancer irrespective to the level of COX-2 [37]. prevention. Indeed, the chronic intake of NSAIDs is able However, combination of COX-2 inhibitors with radi- to consistently reduce the appearance and incidence of ation therapy can also lead to a reduction of efficiency of many types of cancer as described in Familial Adenomatous the radiotherapy. In one report, it has been shown that the 12 International Journal of Cell Biology selective COX-2 inhibitor nimesulide decreased radiation a decrease of cell proliferation in a dose-dependent manner efficiency of two head-and-neck cancer cells lines (SCC9 and [34]. SCC25) which are COX-2 positive [139]. This suggests that NS-398 was able to increase the cytotoxicity of gemc- the sensitization of tumor cells to radiation might be strongly itabine, an analog of the antimetabolite nucleoside deoxycy- dependent on tumor cell type. tidine, used for treatment of nonsmall cell lung carcinoma, in A549ACA cell line (lung adenocarcinoma cell line) by 6.2. COX-2 Inhibitors in Combination with Chemotherapy. enhancing apoptosis [144]. The combination of NS-398 and Many types of cancer are treated with chemotherapeutic gemcitabine is also associated with an inhibition of cell proliferation with an accumulation of the cells in G0/G1 agents leading to inhibition of cell proliferation or induction of apoptosis [140]. phase of cell cycle and an increase of p21 [144]. One of the major causes of chemotherapy failure is the All of these data suggest that COX-2 is implicated in survival and/or development of multidrug resistant cancer anti-apoptotic and MDR pathways and that selective COX-2 cells. This resistance is mediated by many mechanisms inhibitors could be used to improve chemotherapy efficiency. including over-expression of proteins involved in inhibition of apoptosis (i.e, Bcl-2), leading to insensitivity of tumor cells to apoptotic stimuli; an up-regulation of DNA repair; 6.3. COX-2 Inhibitors in Combination with Photodynamic alteration of the target; up-regulation of detoxification Therapy. An alternative therapeutic approach to treat can- enzymes (i.e., Glutathione S-transferases); and extrusion of cers is photodynamic therapy. This procedure is particularly chemotherapeutic drugs by overexpression of ATP-binding used for such solid tumors including skin, bladder, and cassette family proteins, such as MRP (multidrug resistant- head and neck cancers in addition to other diseases like associated protein) BCRP (breast cancer resistance protein or age-related macular degeneration and psoriasis [145]. The mitoxantrone resistance protein) because these proteins reg- treatment consists in the administration of a photosensitizer, ulate absorption, distribution, and excretion of various phar- a molecule that selectively accumulates in tumors and macologic compounds [141]. Consequently, the chemother- is activated by light (600–850 nm). The photosensitizers apeutic agents are immediately extruded from the cells. P-gp may accumulate in different compartments of tumor cells (P-glycoprotein) is one of the best-understood mechanisms like mitochondria (i.e., porphycene monomer), nucleus, leading to multidrug resistance (MDR). Tremendous efforts lysosomes (i.e., lysyl chlorin p6), and plasma membrane have been made to find solutions to overcome MDR. (i.e., monocationic porphyrin like Photofrin). Then, the Recently, COX-2 inhibitors showed an ability to sensitize photosensitizer is excited with a laser from a single state to a tumor cells to chemotherapeutic agents in several models triplet state. The triplet-state photosensitizer is implicated in and also in clinical assays. Colorectal cancers are particularly two oxygen-dependent reactions. In the first one, the triplet affected by chemoresistance. One study revealed that the can react with cell membrane or molecules, leading to radical COXs inhibitors naproxen and indomethacin heptyl ester formation, which in combination with oxygen produce were able to downregulate P-glycoprotein in human colorec- oxygenated products, cytotoxic for the cells [146, 147]. In the talCACO-2cellline.[39]. Indomethacin inhibited the activ- second reaction, the triplet-state photosensitizer can transfer ity of the protein and affected COX-2 mRNA and protein its energy directly to oxygen in order to produce singlet level [39]. Another study showed that meloxicam was able oxygen ( 02), which is known to be a very highly reactive to downregulate MDR1 in HL60 (a human promyelocytic oxygen species and is implicated in cell damage. Therefore, leukemia) cell line as well as in acute myeloid leukemic blasts this therapy leads to tumor destruction due to cell death [142]. The regulation of MDR1 by COX-2 has been also occurring via apoptosis and necrosis. Vasculature damages suggested in another study [143] in which it was reported and activation of immune response are two important effects that transfection of COX-2 cDNA with adenovirus in renal implicated in tumor ablation. rat mesangial cells led to an upregulation of MDR1 gene. Some parameters affect PDT efficiency, such as the distri- bution of the photosensitizer, photobleaching, hypoxia/ano- The combination of COX-2 inhibitors with chemotherapy was also assayed in a study in which the sensitivity of a xia, and the vascularization of the tumor [146]. The main human gastric cancer cell line MKN45 to cisplatin (alkylating reason of failure of this therapeutic approach is linked to agent) resulted increased by COX-2 downregulation with an up-regulation of angiogenic and inflammatory factors siRNA [35], suggesting a possible therapeutic application of in the tumor microenvironment that strongly reduces the this combination. Similarly, the sensitivity to cisplatin was PTD efficiency with a consequent tumor relapse. The link increased by celecoxib in a human osteosarcoma cell line between inflammation and survival pathway activation, (MG-63) and this effect was linked to a down-regulation of cell proliferation, and angiogenesis is well known and anti-apoptotic proteins survivin, Bcl-2, and an inhibition of contributes to tumor progression [3, 6]. It has been shown the survival pathway PI3K/Akt [112]. It was also reported that PDT leads to an increase of TNFα,IL1β, PGE2, VEGF that B-CLL (B chronic lymphoid leukemia) overexpressed (vascular endothelial growth factor), and MMP9 (matrix COX-2 and the combination of NS398 with chlorambucil, metalloproteinase 9) [147]. These molecules can counteract an alkylating agent, increased the level of apoptosis in B- tumor responses to PTD by promoting cell proliferation and CLL blasts coming from patients [32]. Moreover, several cell survival [38]. Interestingly, it has been demonstrated that lymphoma cell lines overexpressed COX-2, such as RAJI, COX-2 is upregulated during PDT treatment in different BJAB, BL41 and treatment of these cells with celecoxib led to cancer models. As for radiotherapy and chemotherapy, International Journal of Cell Biology 13 this suggests COX-2 as a possible target to increase PDT case, this effect may be cell-type dependent as for chemother- efficiency. apy or radiotherapy. Indeed, celecoxib has been proved to affect the Photofrin- induced PDT in in vitro and in vivo studies performed 7. Inhibition of COX-2 Expression by on a mouse mammary carcinoma BA cell line [148]. In Natural Compounds vitro, celecoxib and NS-398 increase PDT-induced apoptosis. These results were correlated with caspase-3 and PARP Synthetic cyclooxygenase-2 inhibitors hold promise for can- cleavage and Bcl-2 degradation. In vivo, the photosensi- cer chemoprevention; however, recent toxicity problems sug- tization by COX-2 inhibitors was not due to apoptosis gest that new strategies are needed. Natural compounds with exacerbation. Interestingly, celecoxib and NS-398 decrease the potential to inhibit key cell signaling pathways including PDT-induced apoptosis but were also able to decrease the COX-2 gained much attention over the last regarding years level of angiogenic factors such as TNFα,IL1β, PGE2, VEGF, whether they are used alone or in combination with existing and MMP9 [148]. chemotherapeutic agents. Upon chlorin-induced PDT, COX-2 was found up-regu- Recently, Bhui et al. demonstrated that Bromelain, a lated 25-fold in mouse mammary carcinoma RIF cells pharmacologically active compound present in pineapple [149]. This up-regulation was associated with an increase (Ananas cosmosus), leads to a marked inhibition of COX-2 of PGE2 level in the tumor microenvironment. When expression and inactivation of NFκB. Bromelain treatment RIF cells were transplanted in CH3/HeJ mice, for in vivo induces up-regulation of p53 and Bax and subsequent studies, PDT similarly induced an increase of COX-2 and activation of caspase-3 and caspase-9 with a decrease in Bcl-2 PGE2. These effects were prevented by NS-398. Here, it was expression [154]. Furthermore bromelain induces apoptosis- demonstrated that PDT induced vascular endothelial growth related proteins along with inhibition of NFκB -driven COX- factor expression (VEGF) and this increase was attenuated by 2 expression by blocking the MAPK and Akt/protein kinase B treating mice with NS-398, meaning that COX-2 might play signaling in DMBA-TPA-induced mouse skin tumors [155]. a role also in angiogenesis. In consequence of these effects, Curcumin, a naturally occurring polyphenol from Cur- the combination of COX-2 inhibitors with PDT resulted in cuma longa, was described to act as an antiinflammatory an increased efficiency of tumor treatment. and antiproliferative agent by causing downregulation of Possible correlation between COX-2 level and resistance COX-2 in cervical cancer. Curcumin-mediated apoptosis in to PDT has been also investigated in Hela (human cervix these cells is initiated by up-regulation of pro-apoptotic carcinoma cells) and T24 (human transitional cell carcinoma Bax, AIF, release of cytochrome c, and downregulation of of the urinary bladder) cells [150]. It has been reported anti-apoptotic Bcl-2, Bcl-xL in HeLa and SiHa cell lines. that in PDT induced by hypericin, a natural photosensitizer This onset of apoptosis was accompanied by an increase in which accumulates in endoplasmic reticulum and Golgi caspase-3 and -9 activity, suggesting the role of mitochondria apparatus, an increase of PGE2 levels occurred. Hypericin in curcumin-mediated apoptotic cell death as described by induces apoptosis by triggering the release of cytochrome M. Singh and N. Singh [156]. Mar´ ın et al., furthermore, c after light excitation through a process requiring the concluded that curcumin inhibits NFκBactivityaswellas activation of p38 MAPK, which it is known to induce an the expression of its downstream target genes, and also selec- up-regulation of COX-2 [23, 151]. The increase in PGE2 tively induces apoptosis of melanoma cells but not normal levels was prevented by the use of a p38 MAPK inhibitor melanocytes [157]. In addition, curcumin-induced apoptosis (PD169316). Moreover, the impairment of p38 MAPK was was also associated with the activation of caspase-3 and associated with an increase in the susceptibility of tumor caspase-9, and the degradation of PARP. Curcumin decreased cells to PDT. However, COX-2 inhibitors did not lead to the the expression levels of COX-2 mRNA and protein without same effect, meaning that COX-2 was not involved in PDT causing significant changes in COX-1 levels, which was resistance in this model. correlated with the inhibition of prostaglandin E(2) synthesis In contrast to the study of Ferrario et al. [148, 149], [158]. In BV-2 microglial cells, curcumin and analogs were a report from Makowski et al. [152] has revealed that shown to inhibit LPS-induced COX-2 expression; analogs rofecoxib, NS-398, and nimesulide were unable to potentiate identified as more potent than curcumin in the screening PDT in C-26 cells (poorly differentiated colon adenocarci- assay were also more potent than curcumin in preventing noma cell line) in vitro. However, chronic exposition of mice COX-2 expression [159]. bearing C-26 cells to nimesulide potentiated PDT. These data suggest that COX-2 inhibitors may indirectly potentiate PDT. Coumarin (1,2-benzopyrone) is a naturally occurring It is known that vasculature damages are important fragrant compound found in numerous plants and spices. for PDT efficiency and that COX-2 inhibitors act as anti- Results obtained with human nonsmall cell lung cancer A549 angiogenic factors [153]. It has been hypothesized that these cells suggest that downregulation of Bcl-xL, COX-2, and antiangiogenic effects could be responsible for the anti- MAP kinase pathway and up-regulation of p53, Akt, and tumor effect. NFκB pathway are involved in the underlying molecular Currently, the link between COX-2 and PDT efficiency mechanism of apoptosis induction as suggested by Goel et al. is not well characterized. Some studies have revealed an [160]. improvement of efficiency with COX-2 inhibitors whereas Suh et al. concluded that the plant flavonoid fisetin indu- other reports have demonstrated no direct effects. In any ces apoptosis and suppresses the growth of colon cancer cells 14 International Journal of Cell Biology Radiotherapy - Nimesulide in nonsmall cell lung cancer in vivo and in vitro (A549 cell line) - Ns-398 in melanoma cell line (WM35 and LU1205) - siRNA COX-2 in human prostate carcinoma (PC3) and human cervical carcinoma COX-2 inhibitors Chemotherapy Photodynamic therapy - Naproxen/indomethacin downregulate - Celecoxib and NS-398 in photofrin-induced- P-glycoprotein in human colorectal cell line (CACO-2) photodynamic therapy in mouse mammary - COX-2 siRNA/cisplatin in human gastric cancer cell carcinoma cell line (BA) line (MKN 45) - NS-398 in mouse mammary carcinoma (RIF) - Celecoxib/cisplatin in human osteosarcoma cell line (MG-63) - NS-398/chlorambucil in B-CLL - NS-398/gemcitabine in nonsmall cell lung carcinoma cell line (A549) Figure 5: COX-2 inhibition in cancer therapy. by inhibition of COX-2- and Wnt/EGFR/NFκB-signaling effects of combinatorial chemopreventive agents and further pathways [161]. underscore the need for rational design of human clinical Sulforaphane (SFN) is a biologically active compound trials involving such natural compounds [165]. extracted from cruciferous vegetables, and presents potent Pandey et al. published that butein inhibited the expres- anti-cancer and anti-inflammatory activities by suppression sion of the NFκB-regulated gene products involved in anti- of NFkB-dependent genes involved in anti-apoptotic signal- apoptosis (IAP2, Bcl-2, and Bcl-xL), proliferation (cyclin D1 ing (IAP-1, IAP-2, XIAP, Bcl-2, and Bcl-xL), cell proliferation and c-Myc), and invasion (COX-2 and MMP-9). Suppression (c-Myc, COX-2, and cyclin D1), and metastasis (VEGF and of these gene products correlated with enhancement of the MMP-9) as published by Moon et al. [162]. apoptosis induced by TNF and chemotherapeutic agents, and Nontoxic apigenin can suppress anti-apoptotic pathways inhibition of cytokine-induced cellular invasion. This group involving NFκB activation including cFLIP and COX-2 exp- clearly demonstrates that antitumor and anti-inflammatory ression as demonstrated by Xu et al. [88]. According to Nam activities assigned to butein may be mediated in part through et al., DA-6034, a synthetic derivative of flavonoid Eupatilin, the direct inhibition of IKK, leading to the suppression of the strongly enhanced apoptosis and inhibited the expression NFκB activation pathway [166]. of COX-2 and phospho-IKKalpha in inflammation-related Hostanska et al. used human colon COX-2-positive HT colon cancer models [163]. 29 and COX-2-negative HCT 116 or lung COX-2 proficient EGCG from green tea was described to attenuate the A 549 and low COX-2 expressing SW2 cells and showed that AR, to downregulate IGF-1, to modulate COX-2 expression, willow bark extract BNO 1455 and its fractions inhibit the and to decrease MAPK signaling leading to the reduction cell growth and promote apoptosis in human colon and lung in cell proliferation and induction of apoptosis in prostate cancer cell lines irrespective of their COX selectivity [167]. cancer without toxicity [164]. Interestingly, combination of EGCG and COX-2 inhibitor NS-398 enhanced cell growth 8. COX-2 Independent Effects inhibition, apoptosis induction, expression of Bax, pro- caspase-6, and pro-caspase-9, and PARP cleavage, inhibition It is currently well known that several selective COX-2 of PPAR gamma, and inhibition of NFκB compared with the inhibitors inhibit cell proliferation and induce apoptosis additive effects of the two agents alone, suggesting a possible independently of COX-2. Celecoxib is particularly known to synergism. In vivo, combination treatment with green tea have these COX-2-independent effects, which were reviewed polyphenols and COX-2 inhibitor celecoxib resulted in by Grosch et al. [68]. Indeed, celecoxib was able to directly enhanced tumor growth inhibition, lowering of prostate- bind and inhibit PKB/Akt, which plays an important role specific antigen levels, lowering of IGF-I levels, and circu- in cell proliferation and in apoptosis. Concerning cell cycle, lating levels of serum IGF-1 binding protein-3 compared PKB is able to phosphorylate cdk inhibitors, such as p21 and with results of single-agent treatment. Accordingly, Adhami p27, leading to PCNA activation [168, 169]. Furthermore, et al. postulate the efficiency of synergistic and/or additive PKB can also activate several cyclin-cdk complexes and International Journal of Cell Biology 15 induce E2F factor in some cases [68], stimulating cell prolif- selective COX-2 inhibitors with radiotherapy or different eration. Besides, PKB inhibits apoptosis, by phosphorylating chemotherapeutics revealed a sensitization to apoptosis. the pro-apoptotic protein Bad and by inhibiting caspase-9 This effect was also observed with several agents inducing cleavage [51]. apoptosis in a physiological way, thus suggesting that COX-2 The COX-2-independent effects concern also the extrin- inhibitors used in combination with death receptors agonists sic apoptotic pathway. Indeed, we discussed that selective might be a novel approach to elicit apoptosis of cancer cells. COX-2 inhibitors, such as NS-398, celecoxib, and meloxi- However, the fact that COX-2 inhibitors can mediate their cam, are able to modulate the sensitivity of several tumor effects by COX-2-independent mechanisms suggests caution cells to Fas- and TRAIL-induced apoptosis. It has been in the interpretation of the data. discussed that this modulation could be due to COX-2- Nowadays, selective COX-2 inhibitors have been independent effects. In fact, NS-398 and nimesulide were included in several clinical assays. Some of them effectively able to promote TNF and TRAIL-induced apoptosis of D98 increase the efficiency of radiotherapy and chemotherapy and H21 Hela cell lines [170]. In D98, COX-2 is inactive. [172]. For example, celecoxib is in a clinical phase II assay in Moreover, prostaglandin E2 readdition was not able to revert combination with Paclitaxel, carboplatin, and radiotherapy the sensitization effect. In the same report, it has been shown for patients with inoperable stage IIIA/B nonsmall cell lung that NS-398 was able to promote apoptosis induced by TNF cancer [172]. These clinical assays confirm that COX-2 in MCF-7 cell line (human breast adenocarcinoma cells), inhibition may be a promising field in cancer treatment. which again does not express COX-2 [170]. However, the selective COX-2 inhibitors are responsible for A report from Ryan et al. [171] demonstrated that side effects, including an increasing risk of cardiovascular SC58125 and CAY10404, two selective COX-2 inhibitors, complications [67, 68]. It is hoped that other methods to were able to decrease intracellular content of GSH in inhibit COX-2 will be developed. To this purpose, RNA malignant human B-cells. This effect was accompanied by interference using vehicle (i.e., adenovirus) as well as natural an increase of reactive oxygen species production. Indeed, compounds were suggested by some studies [35, 76], as GSH is the most important intracellular nonprotein thiol alternative and promising strategy. antioxidant defense against free radicals, meaning that it pro- tects the cells from cellular damages. The GSH depletion was Conflicts of Interest correlated in this study with a reduced survival for these cells. The fact that many studies imply an association between The authors have no actual or potential conflict of interest. COX-2 inhibition and apoptosis induction or cell prolifera- tion inhibition, without assessing whether COX-2 activity is effectively decreased, suggests caution in the interpretation Acknowledgments of the data. This is confirmed by the observation that C. Sobolewski. and C. Cerella are recipients of doctoral different COX-2 inhibitors may trigger apoptosis in the same and postdoctoral Tel ´ ev ´ ie gtrants, respectively. Research at cancer cell model by modulating different mechanisms. For the Laboratoire de Biologie Moleculair ´ e et Cellulaire du example, celecoxib [113] induced apoptosis by an inhibition Cancer (LBMCC) is financially supported by “Recherche of Akt phosphorylation in prostate cancer cells COX-2- Cancer et Sang” foundation, by the “Recherches Scientifiques positive LNCaP without affecting Bcl-2 level. In contrast, a Luxembourg” association, by “Een Haerz fir kriibskrank study by Liu et al. [102] revealed that NS-398 in the same Kanner” association, by the Action Lions “Vaincre le Can- cell line was able to induce apoptosis but down-regulation of cer”association and by Tel ´ ev ´ ie Luxembourg. The authors are Bcl-2. 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