Oncogene (2004) 23, 9247–9258 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc Nonsteroidal anti-inflammatory agents differ in their ability to suppress NF-jB activation, inhibition of expression of cyclooxygenase-2 and cyclin D1, and abrogation of tumor cell proliferation Yasunari Takada1, Anjana Bhardwaj1, Pravin Potdar1 and Bharat B Aggarwal*,1 1Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Box 143, 1515 Holcombe Boulevard, Houston, TX 77030, USA Nonsteroidal anti-inflammatory drugs (NSAIDs) such as Introduction aspirin have been shown to suppress transcription factor NF-jB, which controls the expression of genes such as Owing to its analgesic and anti-inflammatory effects, cyclooxygenase (COX)-2 and cyclin D1, leading to aspirin (acetylsalicylic acid), first synthesized in 1897, inhibition of proliferation of tumor cells. There is no was approved for the treatment of rheumatoid arthritis systematic study as to how these drugs differ in their ability in 1899 (Botting, 1999). Since then several other to suppress NF-jB activation and NF-jB-regulated gene nonsteroidal anti-inflammatory drugs (NSAIDs) have expression or cell proliferation. In the present study, we been synthesized and approved for human use. The anti- investigated the effect of almost a dozen different commonly inflammatory and analgesic effects of NSAIDs have used NSAIDs on tumor necrosis factor (TNF)-induced NF- been shown to be due to their ability to inhibit the jB activation and NF-jB-regulated gene products, and on enzymatic activity of cyclooxygenases (COXs), which cell proliferation. Dexamethasone, an anti-inflammatory convert arachidonic acid to prostaglandins (PGs) (Vane, steroid, was included for comparison with NSAIDs. As 1971). Aspirin is one of the most widely used drugs, with indicated by DNA binding, none of the drugs alone an average yearly consumption of 30 g/person in the activated NF-jB. All compounds inhibited TNF-induced industrialized countries (Roth and Calverley, 1994) and NF-jB activation, but with highly variable efficacy. The a daily consumption in the United States alone of 50% inhibitory concentration required was 5.67, 3.49, 3.03, 35 000 kg (Jack, 1997). 1.25, 0.94, 0.60, 0.38, 0.084, 0.043, 0.027, 0.024, and In most cases, aspirin or another NSAID either 0.010 mM for aspirin, ibuprofen, sulindac, phenylbutazone, prevents or delays the onset of the disease. Extensive naproxen, indomethacin, diclofenac, resveratrol, curcumin, research has shown that aspirin has a potential in the dexamethasone, celecoxib, and tamoxifen, respectively. All treatment of a wide variety of inflammatory diseases drugs inhibited IjBa kinase and suppressed IjBa degrada- including cancer, arthritis, cardiovascular diseases, and tion and NF-jB-regulated reporter gene expression. They Alzheimer’s disease (O’Neill, 1998). For instance, also suppressed NF-jB-regulated COX-2 and cyclin D1 aspirin has been found effective against colon cancer, protein expression in a dose-dependent manner. All especially familial adenomatous polyposis coli, a her- compounds inhibited the proliferation of tumor cells, with editary precancerous disease caused by the loss of the 50% inhibitory concentrations of 6.09, 1.12, 0.65, 0.49, tumor suppressor gene adenomatous polyposis coli 1.01, 0.19, 0.36, 0.012, 0.016, 0.047, 0.013, and 0.008 mM (Giercksky, 2001). Both preventive and therapeutic for aspirin, ibuprofen, sulindac, phenylbutazone, naproxen, affects of aspirin have been reported against colorectal indomethacin, diclofenac, resveratrol, curcumin, dexa- adenomas (Baron et al., 2003; Sandler et al., 2003). methasone, celecoxib, and tamoxifen, respectively. Overall Moysich et al. (2002) found that regular aspirin use may these results indicate that aspirin and ibuprofen are least be associated with reduced risk of lung cancer. When the potent, while resveratrol, curcumin, celecoxib, and tamox- analyses were restricted to former and current smokers, ifen are the most potent anti-inflammatory and antiproli- participants with the lowest cigarette exposure tended to ferative agents of those we studied. benefit most from the potential chemopreventive effect Oncogene (2004) 23, 9247–9258. doi:10.1038/sj.onc.1208169 of aspirin. Published online 18 October 2004 How aspirin and other NSAID mediate their effects is not fully understood. Whether inhibition of COX-1 Keywords: NSAID; NF-kB; IkBa; COX-2; cyclin D1; (constitutive) or COX-2 (inducible) activity is a major proliferation mechanism of its action has been questioned. For instance, Goel et al. (2003) showed that growth inhibition of human colon cancer cells by aspirin is *Correspondence: BB Aggarwal; E-mail: [email protected] through COX-independent mechanisms and is due to Received 21 April 2004; revised 1 September 2004; accepted 1 September the change in expression of DNA mismatch repair 2004; published online 18 October 2004 proteins (Goel et al., 2003). Similarly, Williams et al. Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9248 (2000) showed that the cytotoxic effects of celecoxib, were prepared and analysed for NF-kB activation by another NSAID, were independent of COX-2 inhibition electrophoretic mobility shift assay (EMSA). As shown because similar effects were observed in COX-2 ( þ / þ ), in Figure 2, none of the NSAIDs by themselves COX-2 ( þ /À) and COX-2 (À/À) fibroblasts. activated NF-kB. TNF-induced NF-kB activation, Generally, a strong correlation has been found however, was inhibited by all NSAID in a dose- between inflammation and cancer (Coussens and Werb, dependent manner. The 50% inhibitory concentration 2002), suggesting that cancer is an inflammatory disease. required was 5.67, 3.49, 3.03, 1.25, 0.94, 0.60, 0.38, Celecoxib, however, has been shown to reduce pulmon- 0.084, 0.043, 0.027, 0.024, and 0.01 mM for aspirin, ary inflammation but not lung tumorigenesis in mice ibuprofen, sulindac, phenylbutazone, naproxen, indo- (Kisley et al., 2002). Thus, novel targets to explain the methacin, diclofenac, resveratrol, curcumin, dexametha- effects of NSAID are being sought. Nuclear factor NF- sone, celecoxib, and tamoxifen, respectively (Table 1). kB is one such target that has been shown to mediate Based on IC50, aspirin was least potent and tamoxifen inflammation and suppress apoptosis, and it is com- was most potent (567-fold). The IC50 of celecoxib was monly overexpressed in cancer cells (Bharti and comparable with dexamethasone (236- vs 210-fold). Aggarwal, 2002). The expression of most genes that Since NF-kB is a family of proteins, various are involved in inflammation (e.g. COX-2) or in cellular combinations of Rel/NF-kB protein can constitute an proliferation (e.g. cyclin D1) are regulated by NF-kB. active NF-kB heterodimer that binds to a specific Thus, both antiproliferative and anti-inflammatory sequence in DNA (Ghosh and Karin, 2002). To show effects of NSAID can be explained through their ability that the retarded band visualized by EMSA in TNF- to suppress NF-kB. Although aspirin was the first treated cells was indeed NF-kB, we incubated nuclear NSAID shown to suppress NF-kB (Kopp and Ghosh, extracts from TNF-activated cells with antibodies to 1994), now several NSAID have been shown to suppress either the p50 (NF-kB1) or the p65 (RelA) subunit of NF-kB activation (Kazmi et al., 1995; Scheuren et al., NF-kB. Both shifted the band to a higher molecular 1998; Yamamoto et al., 1999; Chuang et al., 2002; mass (Figure 2b), thus suggesting that the TNF- Bryant et al., 2003). activated complex consisted of p50 and p65 subunits. How various NSAID differ in their ability to suppress Preimmune serum (PIS) had no effect on NF-kB NF-kB activation, NF-kB-regulated gene expression, complex. Excess unlabeled NF-kB oligonucleotide and proliferation has not been examined and is the focus (100-fold) caused complete disappearance of the band of the current study. We examined 11 different NSAID and no effect by using mutant NF-kB oligonucleotide. for inhibition of tumor necrosis factor (TNF)-induced To further determine the specificity of the effect of NF-kB activation, IkBa degradation, NF-kB reporter NSAIDs on NF-kB, we examined its effect on the gene expression, expression of cyclin D1 and COX-2, transcription factor Oct-1. For this, cells were treated and antiproliferative effects. with the indicated concentrations of aspirin or curcumin for 4 h, the nuclear extracts were prepared, and the extracts examined for binding to labeled oligonucleo- Results tides containing specific binding sites for Oct-1 by In the present study, we investigated the relative anti- inflammatory and antiproliferative effects of most commonly used NSAIDs by examining their effects on Table 1 NSAIDs inhibit TNF-induced NF-kB activation and on cell TNF-induced NF-kB activation, IkBa kinase, IkBa proliferation degradation, NF-kB-regulated reporter gene expression, NSAID 50% inhibitory concentration (IC50) NF-kB regulating proteins, and proliferation of tumor NF-kBa Cell viabilityb COX-1c COX-2c cells. We examined the effect of the oldest (aspirin) and the most recently identified (celecoxib) NSAIDs. In all, Aspirin 5.67 (1.0)d 6.09 (1.0) 1.7 >100 11 different NSAIDs, along with dexamethasone, were Ibuprofen 3.49 (1.6) 1.12 (5.4) 7.2 7.6 investigated. The similarities and dissimilarities in the Sulindac 3.03 (1.9) 0.65 (9.4) 1.9 55.0 Phenylbutazone 1.25 (4.5) 0.49 (12.4) N/A N/A chemical structure of these NSAIDs are shown in Naproxen 0.94 (6.0) 1.01 (6.0) 9.3 28.0 Figure 1. Indomethacin 0.60 (9.5) 0.19 (32.1) 0.013 1.0 Diclofenac 0.38 (14.9) 0.36 (16.9) 0.075 0.038 Resveratrol 0.084 (67.5) 0.012 (507.0) 30.0 39.0 NSAIDs inhibit TNF-induced NF-kB activation Curcumin 0.043 (131.0) 0.016 (380.0) N/A N/A Activation of NF-kB is one of the earliest events that Dexamethasone 0.027 (210.0) 0.047 (130.0) N/A N/A Celecoxib 0.024 (236.0) 0.013 (468.0) 1.2 0.83 mediate inflammation induced by various stimuli in Tamoxifen 0.010 (567.0) 0.008 (761.0) 15.0 95.0 most cells (Bharti and Aggarwal, 2002).