Non-steroidal anti-inflammatory drugs (NSAIDs) are a diverse class of drugs commonly used for the treatment of inflammatory conditions, analgesia, and fever. Specific indications include arthritis, headaches, menstrual cramps, and mild-to-moderate pain from injuries. Figure 1 shows some of the most commonly used NSAIDs and selective cyclooxygenase (COX)-2 inhibitors. The pharmacological basis for the anti-inflammatory properties of NSAIDs is attributed to inhibition of COX-1 and COX-2 enzymes that catalyze the conversion of arachidonic acid into prostaglandin H₂, a precursor for the synthesis of prostaglandins, prostacyclins, and thromboxanes. These eicosanoids are known to promote inflammation, pain, and fever (1). In addition, they provide protection for the lining of the stomach and intestines from the damaging effects of acid, promote blood clotting by activating blood platelets, and regulate kidney function. COX-1 is constitutively expressed in many tissues and plays an important role in tissue homeostasis, while COX-2 is induced by inflammatory stimuli and is generally believed to be more involved in pathological processes (2). The prostaglandin synthesis pathway and its relation to tumorigenesis are illustrated in Figure 2.

Epidemiological, clinical, and laboratory studies provide convincing evidence that NSAIDs, including aspirin, non-aspirin NSAIDs, and COX-2 selective inhibitors also have strong antineoplastic properties. The chemopreventive efficacy of NSAIDs against colorectal cancer (CRC) is particularly well-established. For example, numerous population-based studies have shown that regular, long-term users of NSAIDs have a significantly lower risk of colorectal adenomatous polyps and CRC than non-users (3–5). Clinical evidence of activity was first reported in case studies by Waddell and Loughry in 1983 in which administration of the NSAID sulindac (Clinoril^®) was found to be associated with the reduction of precancerous colorectal adenomas in patients with Garner’s syndrome (6). Later, several clinical trials, including a randomized trial conducted by Giardiello and colleagues reported that sulindac can strongly inhibit the formation of adenomatous polyps and cause regression of existing polyps in patients with familial adenomatous polyposis (FAP) (7–10). Subsequently, the COX-2 selective inhibitor, celecoxib (Celebrex^®), was reported by Steinbach and colleagues to inhibit adenoma formation in FAP patients (11). This study led to the FDA approval of celecoxib for the treatment of FAP in 1999, but was recently withdrawn for this indication upon request by the manufacturer, Pfizer.

Although there are fewer epidemiological studies on cancers other than CRC, multiple studies demonstrate an association between prolonged use of NSAIDs and lower incidence of or deaths from cancers arising from diverse tissues. These include tumors of breast (12–16), lung (12, 17), prostate (12, 18), bladder (12), ovaries (12, 19), esophagus (12), and stomach (12). Epidemiological and clinical studies are supported by evidence from numerous investigators reporting inhibitory effects of NSAIDs on tumorigenesis in various rodent models, including carcinogen-induced or transgenic models of colorectal, breast, and other types of carcinogenesis (20, 21). Among the earliest reports of the anticancer activity of NSAIDs were publications by Pollard and Luckert who described inhibitory effects of indomethacin on carcinogen-induced intestinal tumors (22, 23). Studies in cell culture models have been numerous as well and suggest that NSAIDs have direct inhibitory effects on tumor cell growth and have been especially useful for defining the underlying mechanism of action.

Unfortunately, suppression of prostaglandin synthesis from COX-1 or COX-2 inhibition is associated with gastrointestinal, renal, and cardiovascular toxicities that limit the dosage and long-term use of NSAIDs for cancer chemoprevention. In addition, currently available NSAIDs and COX-2 inhibitors do not completely protect all individuals from developing cancer. For example, there have been reports of resistant adenomas and breakthrough carcinomas during treatment with sulindac, which highlight the limitations of currently available NSAIDs and COX-2 inhibitors for chemoprevention (24). However, it is not clear if their efficacy limitations are attributable to inherent mechanisms of drug resistance or inability to achieve a sufficiently high dosage, given that the anticancer activity appears to require high dosages administered over extended periods of time, which increase the risk of toxicity.

Evidence for the involvement of COX-2 in colorectal carcinogenesis and its constitutive expression in multiple tumor types has led researchers to postulate that inhibition of COX enzymes, especially COX-2, is responsible for the chemopreventive efficacy of NSAIDs and selective COX-2 inhibitors. Detailed reviews on the role of COXs in tumorigenesis have been previously published (5, 24, 25). However, other studies suggest that COX-independent mechanisms may contribute to, or be fully responsible for their anticancer properties. Identification of alternative targets and additional biochemical processes involved in NSAID activity could lead to the development of safer and more efficacious drugs for cancer chemoprevention. This review examines the biochemical processes associated with the antineoplastic effects of NSAIDs and discusses their COX-independent mechanism of action. Detailed mechanistic analyses that link reported direct non-COX targets with the various cellular effects of this class of drugs are provided. In addition, current and potential future approaches to develop safer and more efficacious NSAID derivatives are outlined.

Numerous studies challenge the theory that COX inhibition is solely responsible for the chemopreventive action of NSAIDs by providing evidence that these effects can be exerted, at least partially, through COX-independent mechanisms. For example, in vitro studies have demonstrated that NSAIDs can inhibit proliferation and/or induce apoptosis in multiple tumor cell lines of different origins irrespective of their levels of COX-1 or COX-2 expression (26–29). In addition, the growth-inhibitory activity of NSAIDs cannot be reversed by supplementation with prostaglandins, pointing to a mechanism that is independent of suppressing prostaglandin production (30, 31). Furthermore, there is often a significant discrepancy between the potency of a particular NSAID to inhibit COX-1 and/or COX-2 and its potency to inhibit tumor cell growth in vitro and in vivo. This is highlighted in Table 1, which shows that various chemical families of NSAIDs display appreciably different potencies to inhibit tumor cell growth, yet there is no correlation between their potency to inhibit growth and potency to inhibit COX-1 or COX-2. For example, the non-selective COX inhibitor indomethacin has much lower antiproliferative activity compared with sulindac sulfide despite having a similar chemical scaffold and an approximately 10-fold lower IC₅₀ to inhibit both COX-1 and COX-2 in whole blood COX assays (32). Similarly, while selective COX-2 inhibitors celecoxib and rofecoxib inhibit COX-2 with similar IC₅₀ values, celecoxib has much higher antiproliferative activity in both COX-2-positive and COX-2-negative cell lines (33). Other studies confirm these findings through the use of genetic methods by showing that: (1) tumor cells in which expression of COX-2 has been knocked down by antisense cDNA do not display increased apoptosis but remain sensitive to COX-2 inhibitors, (2) the level of knockdown does not affect sensitivity to COX inhibitors, and (3) fibroblasts from COX-1^−/−, COX-2^−/−, or COX-1/2^−/− knockout mice retain sensitivity to various NSAIDs (34–36).

In general, the concentration of a given NSAID or selective COX-2 inhibitor required to inhibit tumor cell proliferation in vitro is much higher than that required to inhibit COX-1 and/or COX-2 activity (37). This is an important consideration since experimental studies in rodent models, as well as clinical studies typically demonstrate chemopreventive efficacy of NSAIDs only at doses higher than those necessary for anti-inflammatory effects. For example, Reddy et al. showed that doses of celecoxib required to decrease incidence and multiplicity of aberrant crypt foci (ACF) in the azoxymethane (AOM)-induced mouse carcinogenesis model reached plasma concentrations of approximately 9 μmol/L, while plasma concentrations of 1.3 μmol/L were sufficient to inhibit adjuvant-induced arthritis (38). Lower doses that reached around 1.8 μmol/L plasma concentrations did not have an effect on ACF development (39). In a randomized, placebo-controlled clinical trial, Steinbach et al. reported that celecoxib caused a significant reduction in colorectal polyp burden in FAP patients at a dose of 800 mg/day but not at the standard anti-inflammatory dose of 200 mg/day bid (11). Additional in vivo evidence for COX-independent mechanisms of NSAID chemoprevention is provided by a study in the APC^Min/+ mouse model of colorectal carcinogenesis. Administration of sulindac dramatically reduced the number of tumors in Min without altering eicosanoid formation (40). Also, increasing the levels of prostaglandin E₂ and leukotriene B₄ by dietary arachidonic acid supplementation did not affect tumor number or size. It has to be noted, however, that prostaglandin levels are decreased in the colorectal mucosa of FAP patients with adenoma regression on sulindac (41, 42). These results may explain the modest chemopreventive efficacy of currently available NSAIDs such as sulindac or celecoxib at the anti-inflammatory dosages and highlight the need for more potent and selective inhibitors.

Perhaps the most compelling evidence that COX-independent mechanisms exist comes from studies showing that NSAID metabolites or derivatives that lack COX-inhibitory activity can retain or have improved antitumor activity. Sulindac sulfone (exisulind) is a prototypical example of a non-COX-inhibitory NSAID derivative with in vitro and in vivo anticancer activity (43–49). As shown in Figure 3, sulindac is a prodrug that undergoes reversible reduction into the active sulfide form through the action of liver enzymes and colonic bacteria (50). Sulindac sulfide is a non-selective COX inhibitor and is responsible for the anti-inflammatory properties of sulindac. The sulfone metabolite is generated by irreversible oxidation of the sulfoxide in the liver, and does not have anti-inflammatory activity. Numerous studies have shown that sulindac sulfone can inhibit tumor cell growth and induce apoptosis in multiple tumor types despite lacking COX-1 and COX-2 inhibition. Furthermore, sulindac sulfone was shown to effectively inhibit carcinogen-induced tumorigenesis of the colon, mammary glands, lung, and bladder (43–46, 48, 51, 52). In studies involving the AOM model of rat colon tumorigenesis, sulindac sulfone did not reduce prostaglandin levels in the colon mucosa and was able to reach plasma concentrations above those required to inhibit tumor cell growth and induce apoptosis in vitro (Table 2). In clinical trials, sulindac sulfone (exisulind, Aptosyn^®) caused significant regression of polyps in patients with familial (53) or sporadic (54) adenomatous polyposis. Unfortunately, exisulind did not receive FDA approval because of hepatotoxicity, which also limited the dosage.

Additional evidence to suggest that COX inhibition is not required for the chemopreventive effects of NSAIDs is provided by studies of NSAIDs with a chiral center that exist as a racemic mixture of S- and R-enantiomers. The S-ibuprofen and S-flurbiprofen are non-selective COX inhibitors and have antiproliferative activity (55, 56). However, R-ibuprofen and R-flurbiprofen, which lack COX-1 or COX-2 enzyme activity, also inhibit tumor cell growth in vitro, in xenograft mouse models of human tumors, and in transgenic mouse models of tumorigenesis (56–58). Similarly, the R-enantiomer of etodolac, devoid of COX inhibition, has been shown to inhibit colorectal carcinogenesis and induce cytotoxicity in multiple myeloma cells (59, 60). It is important to note that about 50% of R-ibuprofen is converted to the S-enantiomer in vivo although the antiproliferative activity does not correlate with COX-2 expression. Synthetic NSAID analogs in which COX-inhibitory activity has been designed-out but retain anticancer activity provide further proof for the existence of COX-independent mechanisms. The sulindac derivatives, SSA and SBA, aspirin derivative, NCX-4016, and celecoxib derivatives, OSU-03012 and dimethyl-celecoxib (DMC), represent some of the non-COX-inhibitory NSAID analogs that display equal or higher antitumor efficacy compared to the parent drug (61–64).

Taken together, these studies provide a strong case that mechanisms independent of COX-1 and/or COX-2 inhibition fully or partially contribute to the chemopreventive activity of traditional NSAIDs and selective COX-2 inhibitors. The anticancer effects of this class of compounds have been proposed to consist of multiple cellular mechanisms, which include induction of apoptosis, inhibition of proliferation and angiogenesis, and more recently, induction of autophagy. A detailed mechanistic analysis of how currently known direct NSAID targets can lead to these biochemical effects is discussed below. These targets are summarized in Table 3.

It is now widely accepted that apoptosis is the primary mechanism responsible for the antineoplastic properties of NSAIDs, which was first reported to occur in cancer cells treated with sulindac sulfide by two different groups in 1995 (45, 65). The COX-independent activity of sulindac sulfide was evident by the ability of sulindac sulfone to also induce apoptosis (44, 45). The pharmacological relevance of this effect was demonstrated by studies reporting that treatment with sulindac can stimulate apoptosis in the normal rectal mucosa of FAP patients (66, 67), normal intestinal mucosa of APC^Min/+ mice (68), and in the colorectal carcinomas of carcinogen-treated rats (69, 70). Interestingly, the non-COX-inhibitory sulindac sulfone was found to induce apoptosis selectively in rectal polyps of FAP patients but not in normal rectal mucosa, which implies an aspect of selectivity not apparent with conventional chemotherapeutic drugs that also act by inducing apoptosis (53). Consistent evidence from in vitro studies also demonstrates that traditional NSAIDs and COX-2 selective inhibitors, as well as their non-COX-inhibitory derivatives can induce apoptosis in various cancer cell lines (2, 5, 45). Many mechanisms and targets have been proposed to mediate apoptosis induced by NSAID treatment. While a particular NSAID may have its own, more or less specific, COX-independent target, it is generally recognized that a combination of effects on multiple pathways through direct and indirect targets is responsible for the apoptosis-inducing properties of NSAIDs. Major direct cellular targets that have been shown to mediate apoptosis induction by NSAIDs are discussed below.

Unlike their well-defined ability to induce apoptosis, inhibition of proliferation by NSAID treatment is primarily observed in vitro or in experimental animal models. Notably, in clinical studies, sulindac caused adenoma regression in FAP patients without affecting proliferation in rectal mucosa (66). Although these findings do not exclude potential antiproliferative effects in adenomas, sulindac sulfone was also shown to lack antiproliferative activity in colorectal polyps of FAP patients (53). Conversely, other clinical studies indicate that celecoxib can decrease proliferation rates in adenomas from FAP patients (132) as well as in the bronchial epithelium of former smokers (133). Animal studies support these observations by providing evidence that various NSAIDs, including sulindac sulfide, can decrease proliferation rates in tumors from carcinogen-treated rats (69) and in APC^Δ716 mice (134). Indeed, aspirin, sodium salicylate, sulindac sulfide, sulindac sulfone, indomethacin, celecoxib, and piroxicam have all been reported to inhibit cell cycle progression in vitro by inducing a G₀/G₁ cell cycle arrest (2).

Transitions between different phases of the cell cycle are controlled by various cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors. Studies from our lab have shown that direct inhibition of PDE5 by sulindac sulfide can downregulate the expression level of G₁/S transition-specific cyclin D1 in breast cancer cell lines (61). More recent results extend these findings into colorectal adenoma and carcinoma cell lines suggesting that PDE5 inhibition can directly attenuate proliferation in tumor cell lines (135). Since cyclin D1 is a target gene for the β-catenin/TCF-Lef complex, the proposed mechanism of cyclin D1 downregulation was shown to involve elevation of cGMP, activation of PKG, and subsequent attenuation of nuclear β-catenin levels via proteasomal degradation and transcriptional inhibition (Figure 5). Previously, sulindac sulfone and its analogs with improved PDE5 inhibition were also shown to downregulate cyclin D1 levels (136), suggesting that the ability to block cell cycle progression by sulindac metabolites is COX-independent and at least partially mediated through PDE5 inhibition. Another target gene of the APC/β-catenin pathway important for cellular proliferation is the c-myc oncogene, which upregulates cyclins but downregulates p21. Therefore, it is logical to suggest that inhibition of c-myc expression by targeting PDE5 is an additional mechanism by which sulindac can reduce tumor cell proliferation. Importantly, upregulation of the CDK inhibitor p21^waf1, but not p27^kip1, was established to be required for the in vivo efficacy of sulindac by the finding that tumors in APC^1638N mice with p21 inactivation (APC^+/−, p21^{+/−or −/−} mice) were unresponsive to sulindac treatment (137–139). Later studies demonstrated that the induction of p21 and inhibition of proliferation after sulindac treatment, in vitro and in vivo, was dependent on the expression of JNK1 (84). These findings provide a potential link between the activation of PKG by sulindac metabolites and the induction of cell cycle arrest via consequent upregulation of JNK1 and p21. Other studies have shown that p21^waf1 upregulation by sulindac treatment is partially mediated through the COOH-terminal Src kinase (Csk), pointing to the involvement of Csk/Src pathway in the antiproliferative effects of sulindac (140).

The G₁-phase cell cycle arrest induced by celecoxib treatment in multiple tumor cell lines is accompanied by decreased expression of cyclins A, B, and D; and increased expression of cell cycle inhibitors p21^waf1 and p27^kip1 (141–143). One of the targets that can mediate these effects is proposed to be protein kinase B (PKB/Akt) or its upstream kinase PDK-1. As mentioned previously, celecoxib and its COX-sparing analog OSU-03012, can directly inhibit PDK-1 in an ATP-competitive manner thereby blocking signaling through the downstream Akt pathway (111, 113). Akt acts a positive regulator of cell cycle progression by phosphorylating and inactivating CDK inhibitors p21^waf1 and p27^kip1. This results in the activation of various cyclin-CDK complexes leading to DNA replication and proliferation (144–146). Therefore, inhibition of PDK-1/Akt signaling represents one mechanism by which celecoxib induces a cell cycle block. Although sulindac sulfide has been shown to inhibit Akt signaling by directly binding RXR, it remains unclear whether its effects on p21 levels and cell cycle progression are mediated through this pathway. However, p21 induction by sulindac treatment is likely not mediated by p53, the well-known positive regulator of p21, since p53 induction is not a determinant of sensitivity to sulindac metabolites in colon cancer cell lines (51).

It is tempting to suggest that inhibition of SERCA by celecoxib and sulindac sulfide is an important mechanism responsible for the antiproliferative properties of these compounds. As a result of ESR-mediated downregulation of general translation, short-lived proteins, such as various cyclins, quickly disappear and cannot be replenished. This leads to the loss of CDK activity for which they are essential subunits. Consequently, CDK target proteins, most notably the retinoblastoma (Rb) tumor-suppressor protein, cannot be phosphorylated allowing them to remain active and prevent cells from progressing toward the S phase. Although a functional link is difficult to establish due to the presence of multiple upstream events, the effects of celecoxib and sulindac sulfide on SERCA provide a plausible explanation for their effects on the cell cycle.

Lastly, direct inhibition of IKKβ by aspirin, salicylate, and sulindac may be another mechanism that could account for the antiproliferative effects of these NSAIDs via downregulation of NF-κB-mediated proliferative genes such as interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), c-myc, cyclin D1, and COX-2, among others.

It is now well-established through a large body of evidence that COX-2 derived eicosanoids (prostaglandins, thromboxanes, and leukotrienes) contribute to tumor development through their role in angiogenesis. COX-2 overexpression in tumor cells, surrounding stroma, and/or interstitial inflammatory cells (predominantly macrophages) promotes tumor vascularization by inducing the expression of vascular endothelial growth factor (VEGF) and increasing endothelial cell proliferation and migration (25, 147, 148). Consequently, traditional NSAIDs as well as selective COX-2 inhibitors have been shown to inhibit tumor growth through antiangiogenic mechanisms in experimental models (148). However, several studies provide evidence that COX-independent mechanisms may contribute to the antiangiogenic effects of NSAIDs. For example, sulindac sulfone was found to inhibit angiogenesis in intradermal lung tumor xenografts in mice (149), in the ex vivo chick embryo chorioallontoic membrane (CAM) assay (150, 151) and in an in vivo mouse corneal neovascularization assay (152). It is important to note that the CAM and corneal neovascularization assays represent non-inflammatory angiogenesis models and hence, the efficacy of sulindac metabolites points to an underlying mechanism unrelated to COX-2 inhibition.

As for potential alternative targets, it is plausible that cGMP phosphodiesterase inhibition by these compounds and subsequent activation of PKG is responsible for their antiangiogenic properties, given the ability of sulindac sulfone to inhibit angiogenesis. In support of this possibility, studies by Browning et al. have demonstrated that PKG overexpression in SW620 colon cancer cells significantly inhibits angiogenesis and reduces VEGF levels after subcutaneous implantation in mice (153). In addition, ectopic PKG expression was shown to block hypoxic adaptation in SW620 xenografts by inhibiting the transcriptional activity of Hif-1α, a critical driver of VEGF expression and angiogenesis (154). Although the mechanism of Hif-1α inhibition by PKG is not fully elucidated, an earlier study by Kaidi et al. provides evidence that β-catenin can enhance Hif-1-mediated transcription through a direct binding interaction (155). In addition to these findings, SW620 cells with PKG overexpression show reduced levels of β-catenin compared with parental cells. Therefore, cGMP PDEs represent potential targets that can mediate the antiangiogenic properties of NSAIDs through attenuation of β-catenin levels (Figure 5). It needs to be considered, however, that the PDE5 inhibitors, sildenafil, and tadalafil, can promote angiogenesis in various models of tissue damage and wound healing (156). Nonetheless, functional consequences of cGMP PDE inhibition on angiogenesis are yet to be tested in neoplastic models.

Studies using human PANC-1 pancreatic adenocarcinoma cell lines indicate that the transcription factor Sp1 may have a direct role in the inhibition of angiogenesis by celecoxib. Constitutive activation of Sp1 is a crucial determinant of VEGF overexpression in pancreatic cancers. Wei et al. demonstrate that celecoxib potently downregulates Sp1 protein levels and inhibits its transactivating activity in PANC-1 cells (157). By conducting deletion and mutational analyses of the VEGF promoter, Sp1 binding sites were shown to be required for celecoxib-mediated attenuation of VEGF expression. In cell-free assays, celecoxib can inhibit the DNA-binding ability of Sp1, suggesting a direct interaction may be responsible for the effects of celecoxib on Sp1 activity. In addition, celecoxib, and other COX-2 inhibitors such as nimesulide and NS-398 can induce proteasomal degradation of Sp1 and decrease Sp1 phosphorylation, in multiple colon cancer cell lines, which is required for its transcriptional activity (158). Although the precise mechanisms responsible for these effects remain unknown, comparable responses were observed in COX-2-expressing and -non-expressing cell lines, suggesting that mechanisms unrelated to COX may be involved.

Evidence implicating a direct NSAID target in the Hif-1α/VEGF-mediated angiogenic response in human tumors is thin. As such, it needs to be considered that the antiangiogenic properties of NSAIDs can be secondary to their effects on endothelial cell survival, proliferation, and/or migration. For example, inhibition of angiogenesis by sulindac sulfide and sulfone in the CAM model was paralleled by induction of apoptosis (150). Similarly, celecoxib and the COX-2-inactive analog, dimethyl-celecoxib (DMC), were shown to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) and display efficacy in the CAM model at concentrations lower than those required to induce apoptosis (159). These effects were associated with the induction of a G1-phase cell cycle arrest and inhibition of PDK-1/Akt signaling. More recent studies implicate SERCA inhibition as a potential contributor by showing that DMC causes ESR-mediated cell death in tumor-associated brain endothelial cells (TuBECs) (160). As support for this possibility, Neiderberger et al. reported that celecoxib-induced cell death in HUVEC cells is accompanied by elevation of intracellular Ca⁺² and activation of caspases (161). In addition, rofecoxib was unable to induce apoptosis in HUVECs indicating the involvement of a celecoxib-specific COX-independent target.

Matrix metalloproteases (MMPs) represent another class of major contributors to angiogenesis and invasion that are inhibited by NSAID treatment. MMPs 2 and 9 are the principal enzymes involved in degrading type IV collagen of the basement membrane allowing endothelial cells to reach incipient tumors or cancer cells to invade adjacent tissue leading to metastases (162). In U87 MG human glioblastoma cell lines, celecoxib (40 μM), sulindac (300 μM), sulindac sulfide (150 μM), and sulindac sulfone (400 μM) have been shown to inhibit invasion through downregulation of MMPs 2 and 9 (163). This anti-invasive activity could be abrogated by overexpression of phosphorylated Akt using a Myr-Akt vector or potentiated through the use of a dominant-negative Akt (DN-Akt) vector. Furthermore, downregulation of MMP levels was found to be due to the inhibition of Akt-mediated transcription in glioblastoma cell lines. Although these findings do not provide a definitive target, it can be postulated that celecoxib, through its direct inhibition of PDK-1, and sulindac, via its interaction with RXRα, are able to downregulate Akt-dependent tumor cell invasion. Intriguingly, NSAIDs that have not been previously reported to inhibit Akt signaling such as aspirin, ketoprofen, and naproxen were found to lack anti-invasive properties in these cell lines.

Finally, a recent report by Li et al. provides evidence that sulindac sulfide (50 μM) can inhibit tumor cell invasion by suppressing Nf-κB-mediated transcription of microRNAs in human colon and breast cancer cell lines (164). The majority of microRNAs modulated by sulindac sulfide treatment were found to contain Nf-κB binding sites in their promoter regions. In addition, Nf-κB could be isolated from the promoters of several microRNAs, such as miR-10b, downregulated by sulindac sulfide treatment and previously shown to have a role in tumor cell invasion. Furthermore, sulindac sulfide was able to significantly block the invasion stimulated by the overexpression of these microRNAs. Notably, anti-invasive activity was observed at concentrations lower than those required to significantly inhibit the growth of these cell lines, which suggests potential benefits for preventing metastasis in patients at risk of disease progression. These effects were associated with inhibition of IKKβ and IκB phosphorylation, and attenuation of nuclear Nf-κB levels. Although it is unclear whether these effects are mediated by direct IKKβ binding, this study establishes a mechanistic link between inhibition of Nf-κB signaling by sulindac and its anti-invasive effects via microRNA modulation. Future studies are warranted to test whether aspirin and/or salicylate can replicate these effects since IKKβ is a direct target of these compounds.

Autophagy is a catabolic process whereby cells degrade cytoplasmic components in lysosomes. This process is crucial for maintaining cellular homeostasis by removing damaged proteins and organelles, eliminating invading pathogens, and recycling cellular building blocks thereby providing substrates for energy generation. In the past decade, multiple studies have provided genetic and functional links between impaired autophagy and cancer suggesting that autophagy can serve as a tumor-suppressor mechanism. Conversely, consistent with its role in damage mitigation, autophagy has been demonstrated to promote growth of established tumors under conditions of hypoxia, and in response to chemo- or radio-therapy. In certain cases, autophagy has also been shown to mediate cell death either directly through excessive degradation of the cytoplasm or selective digestion of vital organelles such as mitochondria, or indirectly via triggering apoptosis. Excellent reviews on the role of autophagy in cancer have been previously published (165–167).

Regarding NSAID chemoprevention, a growing number of studies report that various NSAIDs and/or NSAID analogs can induce autophagy in tumor cell lines. An interesting connection between aspirin and autophagy was established in a recent report by Din et al. who showed that aspirin (5 mM) can inhibit Akt/mTOR signaling, activate AMP-activated kinase (AMPK), and induce autophagy in multiple colon tumor cell lines, in mice, and in the rectal mucosa of patients on a daily aspirin regimen. Activation of AMPK by aspirin was found to be comparable to that produced by known activators of this kinase, phenformin and metformin. An independent study by Hawley and colleagues concomitantly showed that AMPK is a direct molecular target of salicylate, the active metabolite of aspirin using purified kinase assays (168). These findings are important in the context of numerous epidemiologic, clinical, and experimental studies which indicate that use of metformin, a commonly prescribed antidiabetic drug, is associated with reduced cancer incidence and mortality (169, 170). Furthermore, its antineoplastic activity has been attributed to activation of AMPK and subsequent inhibition of mTOR activity, which among others, results in induction of autophagy (171, 172). Therefore, it is reasonable to suggest that activation of AMPK and subsequent autophagy induction by aspirin may contribute to its chemopreventive effects.

Another report demonstrates that sulindac sulfide can induce autophagy, followed by cell death, in gastric cancer cells at physiologically relevant concentrations (10 μM) (173). Tumor cell death was found to be dependent on downregulation of survivin, which could be abrogated by siRNA knockdown of the essential autophagy protein LC3. These results suggest that survivin may be selectively degraded by autophagic vacuoles after sulindac sulfide treatment. As such, sulindac sulfide-induced autophagy may function as an intermediate process that enables later apoptotic events by preventing the accumulation of an anti-apoptotic protein. As evidence for a COX-independent mechanism, we have recently reported that a novel non-COX-inhibitory sulindac derivative, sulindac sulfide amide (SSA), inhibits the growth of human lung adenocarcinoma cell lines primarily through the induction of autophagy (174). SSA also induced apoptosis, albeit at concentrations appreciably higher than its IC₅₀ value for growth inhibition. Nonetheless, treatment with pan-caspase inhibitor z-VAD-FMK could not prevent cell death, whereas suppressing autophagy by Atg7 siRNA could significantly increase cell viability after SSA treatment. Furthermore, the induction of autophagy and loss of cell viability was found to be mediated by Akt/mTOR inhibition and could be partially blocked by the overexpression of activated Akt via a Myr-Akt plasmid. On the other hand, sulindac sulfide only induced autophagy at concentrations higher than those required to inhibit tumor cell growth and apoptosis appeared to be the primary mechanism of cell death. These findings suggest that SSA and sulindac sulfide may share similar targets that ultimately lead to autophagy induction, but that SSA represents a much more potent inducer of autophagy than the COX-inhibitory sulfide metabolite of sulindac. The in vivo relevance of the induction of cell death through autophagy by SSA is still untested, but remains an interesting possibility to explain its tumor-selective effects and strong antitumor efficacy as reported in the human HT-29 xenograft mouse model (175). Together, these findings provide proof-of-concept evidence that COX-independent targets of sulindac and possibly other NSAIDs are relevant for the induction of autophagy. As such, celecoxib and its non-COX-inhibitory analog, OSU-03012, are the only NSAIDs known to directly target a negative regulator of autophagy signaling, PDK-1. Nonetheless, the relevance of PDK-1/Akt inhibition in autophagy induction by these compounds is unclear since OSU-03012 has previously been shown to induce autophagy and cell death without affecting Akt phosphorylation (176).

Altogether, the amount of data providing a mechanistic understanding of the precise role of autophagy after NSAID treatment is sparse. Studies described above suggest that induction of autophagy may contribute to the chemopreventive properties of NSAIDs. However, several other studies demonstrate that upregulation of autophagy by NSAID treatment can delay or inhibit apoptosis induction in tumor cell lines (177, 178). The most accurate approach for assessing the role of autophagy in NSAID chemoprevention would be a tumorigenesis model, whereas experiments in cell culture or on established xenograft tumors might reflect conditions in which autophagy will serve to counteract NSAID cytotoxicity. Functional evidence linking autophagy induction and inhibition of tumor formation by NSAIDs in genetic or carcinogen-induced animal models of tumorigenesis is lacking. In order to conclusively answer this question, new mouse strains that harbor mutations in both a tumor-suppressor gene and an autophagy gene such as APC^Min/+, ATG5^−/− or APC^Min/+, Beclin^−/+ mice need to be generated.

Studies of the pathways by which NSAIDs inhibit carcinogenesis have not provided conclusive evidence of the molecular targets that are clinically relevant to their chemopreventive activity. Evidence described above provides a strong case that inhibition of COX enzymes cannot explain the complex antineoplastic activity of NSAIDs. Identification of COX-independent targets and mechanisms most important for the antineoplastic properties of these drugs can be used to develop more efficacious chemopreventive drugs without the gastrointestinal, renal, and cardiovascular side effects associated with NSAIDs and COX-2 inhibitors. Currently, sulindac derivatives have been developed that inhibit PDE5 and have antitumor activity without inhibiting COX-1 or COX-2 (179). These experimental agents demonstrate the feasibility of developing safer and more efficacious drugs for chemoprevention by targeting PDE5. Furthermore, the sulindac derivative K-80003 that has been designed to selectively target RXRα and celecoxib derivatives developed to inhibit PDK-1 without COX-inhibition represent other examples of separating COX-inhibitory activity and antitumor efficacy. Previously published non-COX-inhibitory derivatives of sulindac and celecoxib are shown in Figure 5.

It needs to be stated that most of the studies described in this review were performed using human cancer cell lines and their in vivo significance is yet to be determined. The concentrations of NSAIDs used in cell culture experiments often significantly exceed maximum plasma levels that can be achieved in patients in clinical studies. However, despite relatively low blood levels, NSAIDs are able to achieve efficacy in vivo while these concentrations are not sufficient to inhibit tumor cell growth in vitro. This apparent discrepancy suggests that the in vivo response to NSAID therapy may reflect accumulative effects in tissues as a result of chronic administration to patients. Indeed, there is evidence that colonic epithelial cells are exposed to much higher concentrations of sulindac sulfide than those detected in plasma, owing to its unique metabolism by colonic bacteria (180–182). Other studies show that antiproliferative activity can be achieved in vitro at clinically relevant concentrations by increasing the duration of treatment. For example, Patel et al. have reported that low-dose (2.5–10 μM) celecoxib treatment can inhibit the growth of COX-2-negative PC-3 and LNCAP human prostate cancer cell lines when treatment period is extended to 96 h (183). Altogether, these findings highlight the need for more detailed studies on tissue pharmacokinetics and tumor uptake of various NSAIDs. In this respect, it is possible that dose-limiting toxicities from COX-inhibition prevent complete response during NSAID therapy and lead to a pharmacodynamic mechanism of resistance, which can be overcome by the optimization of non-COX targets.

Given the complexity and the multitude of pathways that mediate the biochemical effects of NSAIDs, it is highly challenging to identify relevant targets suitable for cancer chemoprevention. Treatment with a single drug can lead to pleiotropic effects by targeting multiple cellular molecules and pathways, while specific targeting of a single molecule can also induce a wide variety of changes in cellular functions. Therefore, it will be useful to consider targeting pathways that show a higher apoptosis-inducing or antiproliferative activity with the least potential for off-target effects. In addition to tissue culture studies, it is crucial to validate the in vivo roles of candidate targets of chemoprevention in experimental animal studies for an accurate assessment of efficacy and toxicity. Further elucidation of COX-independent NSAID targets has the potential to contribute to future chemopreventive strategies by enabling identification of novel agents and/or driving rational modification of existing chemopreventive drugs.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.