Cancer Letters 346 (2014) 217–224

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Cancer Letters

journal homepage: www.elsevier .com/locate /canlet

Mini-review

Anti-tumor activity of non-steroidal anti-inflammatorydrugs: Cyclooxygenase-independent targets

0304-3835/$ - see front matter � 2014 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.canlet.2014.01.021

Abbreviations: AA, arachidonic acid; NSAID, non-steroidal anti-inflammatorydrug; FAP, familial adenomatous polyposis; TA, tolfenamic acid; SS, sulindacsulfide; EGR-1, early growth response-1; NAG-1, NSAID-activated gene-1; EMT,epithelial-mesenchymal transition; bZIP, basic leucine zipper domain; EGFR,epidermal growth factor receptor; PDE5, phosphodiesterase type 5; TGF-b, trans-forming growth factor-beta; CRC, colorectal cancer; CHOP, C/EBP homologoustranscription factor; SP, specific protein.⇑ Corresponding author. Address: Department of Biomedical and Diagnostic

Sciences, College of Veterinary Medicine, University of Tennessee, 2407 River Dr.,Knoxville, TN 37996, USA. Tel.: +1 865 974 8216; fax: +1 865 974 5616.

E-mail address: sbaek2@utk.edu (S.J. Baek).

Jason L. Liggett a, Xiaobo Zhang b, Thomas E. Eling c, Seung Joon Baek a,⇑a Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 USAb College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, Chinac Laboratory of Molecular Carcinogenesis, National Institutes of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 9 December 2013Received in revised form 21 January 2014Accepted 22 January 2014

Keywords:NSAIDsCOXSulindac sulfideTolfenamic acidNAG-1

Non-steroidal anti-inflammatory drugs (NSAIDs) are used extensively for analgesic and antipyretic treat-ments. In addition, NSAIDs reduce the risk and mortality to several cancers. Their mechanisms in anti-tumorigenesis are not fully understood, but both cyclooxygenase (COX)-dependent and -independentpathways play a role. We and others have been interested in elucidating molecular targets of NSAID-induced apoptosis. In this review, we summarize updated literature regarding cellular and molecular tar-gets modulated by NSAIDs. Among those NSAIDs, sulindac sulfide and tolfenamic acid are emphasized inthis review because these two drugs have been well investigated for their anti-tumorigenic activity inmany different types of cancer.

� 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction tissues, whereas COX-2 expression is usually transient and can

Despite advancements in modern medicine, cancer is a majorcause of death worldwide, with a death toll of 7.6 million or about13% of all deaths per year [1]. This along with increasing life expec-tancies in the developed world produces more chances of acquiringcancer and makes research into the mechanisms of cancer preven-tion a high priority.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a class ofdrugs that have been utilized for their analgesic and antipyretic ef-fects for centuries. In more recent history, NSAIDs have also beenwidely used and studied for their anti-tumorigenic and chemopre-ventive properties. The classical pathway of action for NSAIDs isblocking the generation of prostaglandins from arachidonic acid(AA) via inhibition of cyclooxygenase-1 and -2 (COX-1 and -2)activities. COX-1 is present and expressed constitutively in most

be rapidly induced by cytokines, lipopolysaccharide (LPS), phorbolmyristate acetate (PMA), or growth factors [2–4]. The inhibition ofboth COX-1 and CO-2 by conventional NSAIDs such as aspirin, ibu-profen, and naproxen, could have negative consequences due toinhibition of prostaglandin (PG) synthesis. Particularly, PGE2 hasa cytoprotective effects on the gastric mucosa and renal epithelialcells [5]. Therefore, the prolonged use of conventional NSAIDs thatinhibit both COX-1 and COX-2 can lead to side effects includinggastrointestinal tract bleeding and kidney failure, which may beattributable to COX-1 inhibition. In addition, a recent clinical trialof familial adenomatous polyposis (FAP) patients show a three-foldincrease in the risk of cardio-toxicity from treatment with theCOX-2 inhibitor celecoxib [6,7]. These side effects are part of themotivation that drives research into the COX-independent mecha-nisms of NSAIDs. Among NSAIDs, tolfenamic acid (TA) and sulindacsulfide (SS) have been extensively investigated for their anti-tumorigenic properties because these two drugs exhibit betteranti-cancer activity than many other NSAIDs [8–10]. There is stillmuch work to be done elucidating the pathways of COX-indepen-dent activity of NSAIDs, and this review primarily focuses on intra-cellular molecular targets that are affected by NSAIDs.

2. Transcription factors

NSAIDs have been shown to affect a wide variety of transcrip-tion factors, including EGR-1, ATF3, ATF4, Sp1, CHOP, NF-jB,

218 J.L. Liggett et al. / Cancer Letters 346 (2014) 217–224

b-catenin/TCF, and Smad. These transcription factors have beenidentified as either pro-tumorigenic or anti-tumorigenic, depend-ing on cell context and types. In this section, we summarize howthese transcription factors are altered by NSAIDs as related totumorigenesis (Fig. 1).

2.1. EGR-1

The zinc-finger DNA binding protein early growth respose-1(EGR-1) is involved in many activities including differentiationand tumorigenesis. EGR-1 acts as a tumor suppressor in mousetwo-step skin carcinogenesis using EGR-1 knock-out mice, andbinds to the p53 promoter region both in vitro and in vivo [11]. Fur-thermore, EGR-1 plays an important role in up-regulating theexpression of the anti-tumorigenic/pro-apoptotic protein NSAIDactivated gene-1 (NAG-1) and the anti-angiogenic protein throm-bospondin-1 (TSP-1) in colorectal and lung cancer cells, respec-tively, during sulindac sulfide (SS) treatment [12,13]. NSAIDs up-regulate EGR-1, which binds to the promoter regions of NAG-1and TSP-1. This up-regulation has been confirmed in vivo, using aSprague–Dawley rat model for early colorectal tumorigenesis,which uses the colon-specific pro-carcinogen 1,2-dimethylhydra-zine dihydrochloride (DMH) [14]. EGR-1 protein expression is in-duced in rats treated with sulindac or celecoxib, along with lesstumor formation compared to control rats [14]. Another conven-tional NSAID, TA, also induces EGR-1 at the transcription level viaenhancement of epithelium-specific ETS transcription factor-1(ESE-1) nuclear translocation, which leads to activation of apopto-sis in colorectal cancer cells [15]. In addition, natural anti-cancercompounds such as resveratrol can induce apoptosis via EGR-1induction [16]. However, depending on the cell type and condi-tions, EGR-1 can also play a negative role in tumorigenesis. Inandrogen-independent prostate cancer, EGR-1 is required formediating CXCL5/ENA78’s oncogenic activity in cell growth, migra-tion, and epithelial-to-mesenchymal transition (EMT) [17]. Thus,EGR-1 plays a complex role, acting through many pathways includ-

Fig. 1. NSAIDs’ effects on transcriptional factors. NSAIDs induce apoptosis through a vcatenin, IjBa, and Sp1 are degraded by NSAIDs through ubiquitination pathways. NSAIDsup-regulates pro-apoptotic NAG-1 protein expression. ER stress is induced by NSAID treaThe phospho-eIF2a protein activates ATF4, which translocates to the nucleus where it biup-regulation of CHOP and NAG-1. NSAIDs also induce apoptosis via unknown mechani

ing anti- and/or pro-tumorigenic activity. Therefore, although NSA-IDs increase EGR-1 expression, further study of EGR-1 function intumorigenesis is needed.

2.2. ATF3

ATF3 is a cyclic adenosine monophosphate (cAMP)-dependenttranscription factor that is a member of the basic region leucinezipper (bZIP) family [18]. ATF3 is induced by the conventional NSA-IDs SS and TA in human colorectal cancer cells [19]. Treatment ofcolorectal cancer cells (CRC) with TA increases ATF3 proteinexpression, which is highly associated with apoptosis induction.Furthermore, ATF2 phosphorylation by TA increases ATF3 pro-moter activity via direct binding of phospho-ATF2 in the ATF3 pro-moter [19].

Both ATF3 over-expression and SS treatment down-regulatemetastasis-associated protein (MTA1) and b-catenin in HCT-116cells [20]. Thus, ATF3 has been implicated in several anti-tumori-genic pathways and represents one explanation for NSAIDs’COX-independent activity.

2.3. ATF4

ATF4 is another bZIP transcriptional factor that is usually up-regulated under cellular stress by a translational control mecha-nism [21]. In addition, hypoxia can induce ATF4 up-regulation byincreasing its stability [22]. Various NSAIDs have been shown totrigger endoplasmic reticulum (ER) stress response, resulting inthe induction of ATF4, which is involved in NSAID-induced apopto-sis in cultured guinea-pig gastric mucosal cells [23]. Moreover,celecoxib activates the Ca2+-dependent PERK–eIF2a–ATF4 path-way, which causes apoptosis through up-regulation of PUMAexpression in human gastric carcinoma cell lines [24]. On the otherhand, celecoxib-induced ATF4 expression could contribute to theup-regulation of glucose-regulated protein 78 (GRP78), an ERchaperone, and S100P, a member of the S100 family of EF-hand

ariety of transcription factors and pathways. Pro-tumorigenic proteins such as b-can also induce ESE-1 nuclear translocation, where it binds to EGR-1’s promoter and

tment as well, which causes the ER membrane protein PERK to phosphorylate eIF2a.nds to the promoter regions of the transcription factors CHOP or ATF3. This leads tosms. All of these pathways funnel into induction of apoptosis.

J.L. Liggett et al. / Cancer Letters 346 (2014) 217–224 219

Ca2+-binding proteins, both of which may partially decrease thepotential capability of celecoxib against cancer [25,26]. More re-cently, we also showed evidence that ATF4 could play a role inapoptosis induced by TA in CRC cells [9]. Collectively, NSAIDs in-crease ATF4 expression via inducing ER stress, which may playcomplicated roles in gastric tumor suppression, and possibly drugresistance as well.

2.4. CHOP

The C/EBP-homologous protein (CHOP) is an ER stress responseprotein and member of the bZIP transcription factor family; CHOPinhibits CCAAT/enhancer-binding protein (C/EBP) and liver-en-riched transcriptional activator protein (LAP) DNA-binding activity[27]. CHOP mRNA and protein levels are both elevated by SS treat-ment in CRC cells [28]. Celecoxib also induces CHOP proteinexpression in malignant glioma cells, whereas SS does not unlessco-treated with carbonyl cyanide 4-(trifluoromethoxy)-phenylhy-razone (FCCP) [29]. FCCP is a protonophore which uncouples oxida-tive phosphorylation causing the loss of mitochondrial membranepotential; FCCP does not induce CHOP protein by itself in malig-nant glioma cells [29,30]. These results suggest that mitochondrialdisruption accompanied by ER stress response is needed to induceCHOP expression in malignant glioma cells. Subsequently, recentstudy from our group suggests that TA affects ER stress, therebyenhancing apoptosis via CHOP activity in human CRC cells [9].Interestingly, pranoprofen suppresses ER stress-induced CHOPexpression, likely through the inhibition of XBP-1 splicing in glialcells [31]. Taken together, these results suggest that there are com-plicated mechanisms involved in NSAID-induced CHOP expression,and that these mechanisms are dependent on cell context andchemical structure.

2.5. Sp

Specificity proteins (Sp) are zinc-finger transcription factorsthat are members of the Sp/KLF sub-family [32]. Sp proteins bindto the GC and/or GT boxes of many promoters. There are severalSp proteins, including Sp1, Sp2, Sp3, Sp4, Sp5, Sp6/KLF14, Sp7,and Sp8. This section focuses on Sp1, Sp3, and Sp4, which are phy-logenetically more similar to one another than to the other Sp pro-teins [33,34]. Sp1 is normally responsible for controlling varioushousekeeping genes; however, Sp1 has been linked to the regula-tion of tumorigenesis [35]. Sp proteins contribute to the prolifera-tion of metastatic tumor phenotypes, and thus overexpression ofthese transcription factors is a negative survival prognostic factorin many human cancers [35].

TA has been shown to decrease Sp1, Sp3, and Sp4 proteinexpression as well as vascular endothelial growth factor recep-tor-1 (VEGFR-1) protein expression in pancreatic cancer cells[36]. Interestingly, small interfering RNA (siRNA) for Sp1, Sp3,and Sp4 also lead to decreased VEGFR-1 [36]. Thus, TA decreasesthis important angiogenic factor via regulation of Sp proteins.These results are supported by nude mouse data that show thatTA reduces Sp1, Sp3, and Sp4 protein expression in vivo as well[8]. TA has also been shown to decrease Sp1, Sp3, and Sp4 in rhab-domyosarcoma (RMS) cells and in lung cancer cells both in vitro aswell as in mouse models [37,38]. COX-2-preferential NSAIDs,including celecoxib, also down-regulate Sp1 and Sp4 activity in hu-man CRC cells; however, Sp3 protein expression is unchanged inthis case [39]. This action was shown to be through COX-2-inde-pendent activation of Sp1 and Sp4 proteasomal degradation [39].Recently, Dr. Safe’s group has further shown that TA and the novelnitric oxide (NO) chimera containing NSAID ethyl 2-((2,3-bis(nitro-oxy)propyl) disulfanyl) benzoate (GT-094) down-regulate Sp1, Sp3,and Sp4 in human CRC cells both in vitro and in a mouse xenograft

model, leading to decreased expression of VEGF/VEGFR-1, cyclinD1, hepatocyte growth factor receptor, survivin, Bcl-2, and NF-jB[40,41]. Aspirin and sodium salicylate have also recently beenshown to decrease Sp1, Sp3, and Sp4 in a caspase-dependent man-ner in human CRC cells in vitro as well as in a mouse model [42].Taken together, these data show that NSAIDs down-regulate Spproteins in several different cancers including pancreas, lung, rhab-domyosarcoma, and colon cancers. The consistency of these resultsmakes NSAID action through Sp protein regulation a promisingpathway.

2.6. NF-jB

The roles of NF-jB in cancer progression and anti-cancer thera-peutics are complex. There is evidence to suggest that NF-jB acti-vation is associated with increased survival of cancer cells andresistance to chemotherapy; thus inhibition of NF-jB activity is re-garded as a target for anti-cancer therapy [43,44]. Aspirin inhibitsthe NF-jB pathway by interaction with IKKb [45]. In an in vivomodel using male Sprague–Dawley rats given DMH to produceearly stages of CRC, SS and celecoxib inhibited DMH-mediated IjBbdown-regulation and DMH-mediated IKKb up-regulation, followedby interfering nuclear localization of NF-jB [46]. Through in silico3D crystal structure modeling, this study also suggests that both SSand celecoxib directly bind to NF-jB docking sites [46]. However,NF-jB activation is capable of promoting a pro-apoptotic responseunder different circumstances [47,48]. SS and TA induce NF-jBactivity and apoptosis in human CRC cells [49,50]. Thus, furtherstudy is needed to elucidate NF-jB’s role in tumorigenesis,although NSAIDs could activate or inhibit NF-jB activity.

2.7. b-Catenin/TCF

The b-catenin/TCF signaling pathway plays a key role in regula-tion of tumorigenesis. In colorectal tumor cells, loss of APC func-tion results in increased levels of b-catenin. The elevated levelsof b-catenin promote its interaction with transcription factors ofthe TCF/Lef family and translocation of the b-catenin/TCF-complexto the nucleus, where it modulates transcription of downstreamgenes involved in pro-tumorigenesis. Both aspirin and indometha-cin suppress b-catenin target genes by modulating TCF activity[51]. Nuclear b-catenin content is also decreased by treatment withseveral other NSAIDs [52,53]. However, this effect of NSAIDs on b-catenin suppression is dependent on PPARc and RXRa expression[54]. Thus, NSAIDs down-regulate b-catenin expression at the tran-scriptional and/or translational levels, leading to its reduced nucle-ar translocation. Furthermore, it is speculated that NSAIDs may beuseful as anti-metastatic drugs by inhibiting b-catenin activity,leading to inhibition of metastatic protein S100A4 transcription[55].

2.8. Smad

Smad transcription factors serve as vital mediators in TGF-b sig-naling; these transcription factors normally form heteromericcomplexes and then translocate into the nucleus where they acti-vate target gene expression together with co-activators and/orsuppressors [56]. Smad signaling has tumor-suppressive effectsin the early stages of tumorigenesis; however, paradoxically, Smadsignaling contributes to metastasis in advanced stage tumors.There are a large number of proteins targeted by the Smad path-way, over half of which may be involved in cancer cell invasionand migration [57]. Therefore, disruption of Smad signaling inlate-stage tumorigenesis could be a potential cancer therapeuticstrategy. The NSAID, 5-aminosalicylic acid (5-ASA) has been re-ported to suppress TGF-b-driven Smad2/3 phosphorylation and

220 J.L. Liggett et al. / Cancer Letters 346 (2014) 217–224

subsequently inhibit translocation of Smad2/3 into nucleus in CRCcells [58]. Moreover, the intake of aspirin seems to modulate Smadgene variation in colorectal tumors [59]. Our recent finding alsoshows that TA inhibits TGF-b-induced Smad phosphorylation viathe ERK MAP kinase pathway in various cancer cells [60]. Moreimportantly, TA had the best inhibitory activity of Smad phosphor-ylation compared with other tested NSAIDs. These data suggestthat the interference of Smad signaling could be a novel mecha-nism of NSAID anti-tumor activity.

3. Cell signaling proteins

Cell signaling proteins are a diverse group of proteins that per-form a wide variety of functions. Some are adhesion modulesresponsible for cell–cell binding while others are responsible foranchoring the plasma membrane to the actin cytoskeleton. Cellsurface proteins can act as receptors for paracrine and autocrinecell signaling and are responsible for signal transduction to cyto-plasmic signaling proteins and even translocation into the nucleusto affect transcription. These proteins are highly studied in cancerresearch due to their potential prognostic value as cancer markersand the promising chemotherapy potential of cancer cell-specificdrug delivery (Fig. 2).

3.1. EGFR

Epidermal growth factor receptor (EGFR) is a receptor tyrosinekinase that is mutated and/or over-active in a high percentage ofcancers. EGFR signaling can lead to cell proliferation through theErk1/2 pathway and cell survival, invasion, and angiogenesisthrough the AKT pathway. Sulindac metabolites, SS and sulindacsulfone, down-regulate EGFR signaling by inhibiting activationand/or expression of EGFR in CRC cell lines [61]. The dual NSAIDlicofelone, which inhibits both COX and 5-lipoxygenase (5-LOX),has also been shown to decrease CRC membrane fluidity leadingto the inhibition of EGFR signaling, which contributes to apoptosis[62]. Thus, NSAIDs could be another attractive agent to target EGFRby co-treatment with other EGFR target drugs.

3.2. E-cadherin

E-cadherin is a calcium-dependent type-1 transmembrane pro-tein. It is important for tight cell–cell junctions and anchors to thecytoskeleton. E-cadherin can play an important role in tumor pro-gression through its sequestration of b-catenin to the plasmamembrane, which prevents b-catenin from acting in the Wnt sig-naling pathway and from performing its job as a cell adhesion mol-

Fig. 2. NSAIDs’ effects on cell signaling proteins. EGFR signaling and PDE5’s effecton cAMP and cGMP drive increased cell proliferation. NSAIDs inhibit cell prolifer-ation through down-regulation of EGFR protein expression and inhibition of PDE5activity. E-cadherin is an adhesion molecule that is important for maintaining tightcell junctions and is lost or reduced during EMT. TGF-b signaling also drives EMT.NSAIDs block EMT through reduction of Smad activity or serum levels of TGF-b1ligand, and through protection of E-cadherin protein expression.

ecule. Loss of E-cadherin function and/or expression is animportant step in epithelial-mechenchymal transition (EMT) lead-ing to invasion and migration. In an animal model, sulindac treat-ment protected APCMin/+ mice from E-cadherin loss andaccumulation of nuclear b-catenin in colon tumors [63]. On thecontrary, the non-selective NSAID indomethacin reduced proteinlevels of E-cadherin and collagen IV, while increasing the activityof matrix metalloprotease-9 (MMP-9) leading to enhanced motilityin vitro in lung cancer cell lines [64]. Furthermore, low doses ofcelecoxib treatment resulted in loss of E-cadherin, followed byinduction of EMT [65]. Thus, further study may need to clarifythe role of E-cadherin in NSAID-induced anti-tumorigenesis.

3.3. Phosphodiesterase-5 (PDE5)

Accumulating evidence suggests that modulating the intracellu-lar secondary messengers, cAMP and cGMP may be a promising ap-proach in the field of anti-cancer therapy. Inhibiting PDE5 leads toincreased intracellular cGMP levels and activated cGMP-dependentprotein kinase (PKG). PDE5 was found to be overexpressed in colo-rectal cancer cells as well as in colon cancer tissues compared toadjacent normal tissues [66]. Indeed, Dr. Piazza’s group has re-ported that SS and other NSAIDs inhibit PDE5 activity, and thisleads to induction of apoptosis in cancer cells [67]. Therefore, inhi-bition of PDE5 may contribute to the anti-cancer properties ofNSAIDs. Since PDE5 inhibitors have been previously reported tohave chemo-sensitizing effects [68], NSAIDs like SS may be usefulchemo-sensitizers along with other cancer drugs.

4. Cytoskeletal proteins

Cytoskeletal proteins are critical for maintaining normal cellu-lar form and function. They represent the highways for vesicle traf-ficking, which is a key element of autocrine and paracrine cellularsignaling. Cytoskeletal protein rearrangement is also a critical partof cell migration in cancer. All of these aspects are areas that wouldneed to be disregulated to allow a pre-cancerous cell to go throughEMT and metastasize to new sites.

4.1. Nesprin-2

Nesprin-2 is a 796 kDa protein containing an N-terminal actin-binding domain consisting of two calponin homology domains, acentral coiled-coil rod domain, and a C-terminal transmembraneKASH domain responsible for targeting to the nuclear envelopand interaction with inner nuclear membrane SUN proteins [69–71]. Also, the Nesprin-2/SUN complex is important for the propernuclear positioning that is necessary for fibroblast migration, sug-gesting that Nesprin-2 may have an important role in cell motility[72]. Recently, a link between Nesprin-2 and cancer has been sug-gested because it was observed in tumor tissues more frequentlyand at higher expression levels than normal tissues [73]. SS andother NSAIDs down-regulate Nesprin-2 expression in human colo-rectal cancer cells and its down-regulation is highly associatedwith NSAIDs’ effect on anti-tumorigenesis as assessed by cellimpedance experiments [73]. Therefore, Nesprin-2 is likely amolecular target of NSAIDs in a group of cell assembly proteins.

5. TGF-b signaling

The mixtures of cytokines that are produced in the tumormicroenvironment have important roles in cancer. TGF-b is a mul-tifunctional regulatory polypeptide that is part of a large family ofcytokines that control many aspects of cellular function, includingcellular proliferation, differentiation, migration, apoptosis,

J.L. Liggett et al. / Cancer Letters 346 (2014) 217–224 221

adhesion, angiogenesis, immune surveillance, and survival. TGF-bsignaling can drive either tumor suppression or tumor promotion.The TGF-b superfamily protein NAG-1 has been extensively studiedin NSAID-induced anti-tumorigenesis.

5.1. NAG-1

NAG-1, also known as GDF15, MIC-1, and PDF, is a divergentmember of the TGF-b superfamily. NAG-1 is one of the few TGF-bsuperfamily proteins whose receptors remain unknown. NAG-1 ishighly induced by most conventional NSAIDs [74] and its inductionby NSAIDs is mediated in a COX-independent manner [75]. NAG-1induction by NSAIDs not only plays a role in colorectal anti-tumor-igenesis, but also in others including head and neck cancer [76],prostate cancer [77], ovarian cancer [78], and thyroid cancer [79].Mechanistically, it has been reported that NSAIDs up-regulateEGR-1 in vitro and in vivo, and HER-1 then binds to the promoterregion of NAG-1 [14,80]. SS also induces NAG-1 expression, andits induction can be blocked by dominate negative PERK and bydominant negative CHOP [28], indicating that NAG-1 is down-stream of PERK and CHOP (Fig. 1). Since NAG-1 transgenic mice ex-hibit anti-cancer activity and anti-inflammation activity [81,82] inthe colon and lung, NAG-1 must be a key target protein to controlanti-inflammation and anti-cancer activity, mediated by NSAIDs.

6. Cell cycle regulators

Several studies have shown that NSAIDs can modulate cell cycleprogression. Expression and activity of cyclins and CDKs are mod-ulated by several NSAIDs in a COX-independent manner. Twoimportant cell cycle regulators, cyclin D1 and p21 are discussed.

6.1. Cyclin D1

Cyclin D1 serves as a key regulator in cell cycle progression byactivating cyclin dependent kinase (CDK) 4/6 and is frequentlyover-expressed in tumor cells. There is a substantial body of exper-imental evidence showing that cyclin D1 could be a potential can-cer therapeutic target [83]. NSAIDs, as potential anti-cancer agents,have been well documented to down-regulate cyclin D1 accompa-

Table 1Molecular targets of sulindac sulfide and tolfenamic acid in different cancer types.

NSAIDs Up-regulated targets Down-regulated targets Cancer types [

Sulindac sulfide EGR-1 Colon [80], LuATF3 Colon [20]

NF-jB Colon [46], BrNF-jB Colon [50]

b-catenin Colon [53], LuEGFR Colon [61]

E-cadherin Colon [63]PDE5 Colon [67]

NAG-1 Colon [74], OvNesprin-2 Colon [73]

p21 Breast [92], OCHOP Colon [28]

Cyclin D1 Breast [92], Pa

Tolfenamic acid EGR-1 Colon [15]ATF3 Colon [19]ATF4 Colon [9]CHOP Colon [9]

Sp1 Pancreas [36],NF-jB Colon [49]

Smad2/3 Lung [60]NAG-1 Colon [15], Orp21 Breast [104]

Cyclin D1 Colon [9]

nied by G1 cell cycle arrest; however, the underlying mechanismscould be diverse. Various NSAIDs can down-regulate cyclin D1expression by suppressing NF-jB activation in the tumor necrosisfactor (TNF)-induced KMB-5 cell model [84]. Also, aspirin cause cy-clin D1 degradation in SW480 cells through the p38 MAP kinasepathway, which, in turn, results in NF-jB pathway activation andsubsequently apoptosis [85]. On the other hand, the suppressionof Wnt/b-catenin signaling by NSAIDs could also contribute to cy-clin D1 down-regulation in colorectal cancer cells [86–90].Although TA-mediated cyclin D1 decreases have been proposedto result from degradation of Sp transcription factor protein [91],we recently showed that TA can inhibit cyclin D1 translation viaPERK/eIF2a pathway activation in colorectal cancer cells [9].Apparently, NSAIDs alter cyclin D1 expression by various transcrip-tional and post-transcriptional mechanisms.

6.2. p21

SS increases p21 mRNA expression in the immortalized humanbreast epithelial cell line MCF-10F [92] and increases p21 proteinin ovarian cancer cells [78]. Celecoxib also increases p21 proteinexpression in the human CRC cell lines HCT-15, Caco-2, and HT-29 [93]. COX-2-preferential NSAIDs NS398 and nimesulide also in-crease p21 expression at the transcriptional level [94]. Collectively,a decrease in p21 expression may be one of the main oncogenicevents in the development of cancer, and thus p21 induction byNSAIDs could be the molecular link between NSAIDs and chemo-preventive activity in several cancers.

7. Conclusion

NSAIDs interact with many pathways in cancer cells. There aremany promising cyclooxygenase-independent mechanisms uti-lized by NSAIDs that provide potential avenues for developingnew and better drugs that minimize or eliminate the undesirableside effects of cyclooxygenase inhibition such as gastric bleedingand cardiovascular risks. NSAIDs up-regulate a number of tran-scription factors such as the tumor suppressors EGR-1, ATF3, andCHOP and down-regulate oncogenic transcription factors Sp1 andb-catenin (Fig. 1). The oncogenic cell membrane protein EGFR is

References]

ng [13], Brain [95]

east [96]

ng and breast [97]

arian [78], Gastric [98], Prostate [99]

varian [78], Head/Neck [100], Pancreas [101]

ncreas [101], Colon [102]

Rhabdomyosarcoma [37], Lung [38], Esophageal [91]. Ovarian [103], Colon [40]

al [76], Thyroid [79]

222 J.L. Liggett et al. / Cancer Letters 346 (2014) 217–224

down-regulated, and the activity of the metastatic protein complexSmad2/3 is decreased by NSAID treatment in CRC cell lines, whilethe cell adhesion protein E-cadherin is protected in APCMin/+ micefrom loss of expression by NSAID treatment (Fig. 2). Some NSAIDsalso affect cytoskeletal reorganization and loss of actin stress fi-bers. The secreted tumor suppressor protein NAG-1 is induced byNSAIDs both in vitro and in vivo via p53, EGR-1, CHOP, and otherpathways. Thus, NSAIDs and improved derivatives of NSAIDs havegreat potential as both chemotherapeutic and chemopreventiveagents in cancer. Molecular targets of two NSAIDs, SS and TA, aresummarized in Table 1, and more targets could be identified inthe future.

Conflict of interest

No conflict of interest exists in the submission of thismanuscript.

Acknowledgements

We apologize to all colleagues whose important work we couldnot cite due to space restrictions. The authors thank Misty Baileyfor her critical review. This work was supported by grants fromthe American Cancer Society (CNE-111611), National Institutes ofHealth (R01CA108975), and the University of Tennessee, Centerof Excellence in Livestock Diseases and Human Health.

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