Nonsteroidal Anti-Inflammatory Agents Differ in Their Ability to Suppress

Home , Phenylbutazone, Sulindac

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). Whether all a NSAIDs inhibit NF-kB activation induced by TNF, a A dose required for 50% inhibition (IC50) by NSAIDs in mM on most potent inflammatory stimulus, was investigated. TNF-induced NF-kB activity was estimated by EMSA in KBM-5 cells. b IC50 of NSAIDs in mM on cell viability of KBM-5 cells was estimated Cells were pretreated with various concentrations of c by MTT method. IC50 in mM of NSAIDs on COX-1 and COX-2 NSAID either for 4 or 8 h (phenylbutazone, resveratrol activity is based on ref Warner et al. (1999). dNumbers within and diclofenac), and then activated for NF-kBby parentheses indicate fold activity normalized against aspirin set at 1. treatment with 0.1 nM TNF for 30 min. Nuclear extracts N/A is not available.

Oncogene Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9249

Figure 1 Chemical structure of NSAIDs. The molecular weight of each NSAID is indicated within parentheses

EMSA. Oct-1 bound to their speciﬁc DNA sequence, failed to induce the degradation of IkBa (Figure 3a). and the treatment of cells with neither aspirin (Figure 2c) The effect was dose-dependent. Based on IC50, like NF- nor curcumin (Figure 2d) affected the activity of Oct-1. kB activation, aspirin again was least potent and These results indicate that the effects of NSAIDs on tamoxifen was most potent in suppressing IkBa NF-kB activation are speciﬁc. degradation. Since IkBa degradation requires IkBa kinase (IKK) activation, we also examined the effect of various NSAIDs inhibit TNF-dependent IkBa degradation and NSAIDs on TNF-induced IKK activity. Cells were IkB kinase activity treated with NSAIDs as described above and then The activation of NF-kB is known to require the treated with 1 nM TNF for 5 min. Whole-cell extracts phosphorylation, ubquitination, and degradation of were prepared, immunoprecipitated with anti-IKK-a IkBa, the natural inhibitor of NF-kB (Ghosh antibody, and then subjected to IKK kinase assay. The and Karin, 2002). To determine whether inhibition of results indicate that TNF activated IKK and all TNF-induced NF-kB activation was due to the inhibi- NSAIDs inhibited TNF-induced IKK kinase activity tion of IkBa degradation, we pretreated cells with (Figure 3b). various concentrations of NSAIDs for either 4 or 8 h (phenylbutazone, resveratrol, and diclofenac), and NSAIDs inhibit TNF-induced NF-kB-dependent reporter then exposed them to 0.1 nM TNF for 30 min. The gene expression cell extracts were then examined for IkBa status in the cytoplasm by Western blot analysis. TNF induced Although we have shown that NSAIDs block the DNA- IkBa degradation, but in NSAID-pretreated cells, TNF binding step in NF-kB activation, DNA binding alone

Oncogene Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9250 does not always correlate with NF-kB-dependent NF-kB-dependent reporter gene expression, we tran- gene transcription, suggesting that there are additional siently transfected A293 cells with the NF-kB-regulated regulatory steps (Nasuhara et al., 1999). To secretory alkaline phosphatase (SEAP) reporter determine the effect of NSAIDs on TNF-induced construct, incubated them with NSAIDs, and then

Figure 2 Effect of NSAIDs on TNF-induced NF-kB activation. (a) KBM-5 cells were pretreated with various concentrations of NSAIDs either for 4 or 8 h (phenylbutazone, resveratrol, and diclofenac), and then treated with 0.1 nM TNF for 30 min. Nuclear extracts were prepared and analysed for NF-kB activation by EMSA as described in Materials and methods. (b) NF-kB induced by TNF is composed of p65 and p50 subunits. Nuclear extracts from untreated or 0.1 nM TNF-treated KBM-5 cells were incubated with different antibodies, unlabeled NF-kB oligo-probe or mutant oligo-probe, and then assayed for NF-kB activation by EMSA. Aspirin (c) and curcumin (d) had no effect on Oct-1 in KBM-5 cells. Cells were preincubated with the indicated concentrations of aspirin or curcumin for 4 h, and then treated with 0.1 nM TNF for 30 min. Nuclear extracts were prepared and analysed for Oct-1 activation by EMSA. Result shown are representative of two or three separate experiments

Figure 3 Effect of NSAIDs on TNF-induced IkBa degradation and IKK activation. KBM-5 cells were pretreated with various concentrations of NSAIDs either for 4 or 8 h (phenylbutazone, resveratrol, and diclofenac), and then treated with 0.1 nM TNF for 15 min (a)or1nM TNF for 5 min (b). Cytoplasmic extracts were prepared, resolved on 10% SDS–PAGE, and then electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-IkBa and anti-b-actin antibodies as described in Materials and methods (a). Whole-cell extracts were prepared, immunoprecipitated with anti-IKK-a antibody, and then subjected to IKK kinase assay as described in Materials and methods (b). For loading control, whole-cell extracts were also resolved on 7.5% SDS– PAGE, and performed Western blot analysis using anti-IKK-a antibody. Results shown are representative of two or three separate experiments

Oncogene Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9251

Oncogene Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9252 stimulated the cells with TNF. An almost eight-fold NSAIDs inhibit TNF-induced and NF-kB-regulated increase in SEAP activity over the vector control protein expression was observed upon stimulation with 1 nM TNF (Figure 4). NSAIDs repressed NF-kB-dependent Through activation of NF-kB, TNF induces cyclin D1 reporter gene expression induced by TNF in a dose- and COX-2, both of which have NF-kB-binding sites in dependent manner. In most cases, the dose required to their promoters (Yamamoto et al., 1995; Guttridge et al., suppress NF-kB-dependent reporter activity was similar 1999; Hinz et al., 1999). The suppression of COX-2 is to that needed for inhibition of NF-kB DNA-binding essential for the anti-inﬂammatory effects of NSAIDs, activity. Thus, these results suggest that NSAIDs whereas the suppression of cyclin D1 explains also suppressed TNF-induced NF-kB-dependent gene their antiproliferative effects. To determine whether expression. NSAIDs inhibit TNF-induced cyclin D1 and COX-2, we

Figure 4 Effect of NSAIDs on TNF-induced gene expression. A293 cells were plated in 12-well plates and transiently transfected with NF-kB-SEAP plasmid along with b-actin-Renilla plasmid (transfection control) by the FuGENE 6 as described in Materials and methods. Then cells were pretreated with various concentrations of NSAIDs either for 4 or 8 h (phenylbutazone, resveratrol, and diclofenac) and exposed to 1 nM TNF. The cell culture medium was harvested after 24 h and analysed for SEAP activity as described in Materials and methods. The cells were also lysed and analysed for luciferase activity as described in Materials and methods. The results are expressed as fold with basal NF-kB activity in the untreated samples as one. The activity was normalized against the b-actin-Renilla for transfection control. The results shown are the average7s.d. of two separate experiments. The signiﬁcance of results when compared with the TNF-treated cells are *P>0.1, **Po0.1, ***Po0.01, ****Po0.001

Oncogene Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9253 pretreated the cells with NSAIDs, and then exposed NSAIDs inhibit the proliferation of tumor cells them to TNF. TNF induced cyclin D1 expressions, and As NSAIDs inhibited the expression of cyclin D1, NSAIDs blocked TNF-induced expression of this gene which is a cell cycle regulator, we also examined the product in a dose-dependent manner (Figure 5). Simi- effect of NSAIDs on the proliferation of human lung larly, TNF also induced the expression of COX-2 adenocarcinoma H1299 cells and on human myeloid protein, and NSAIDs blocked TNF-induced gene KBM-5cells. KBM-5 cells were incubated for 72 h in the expression in a dose-dependent manner (Figure 6). presence of different concentrations of NSAIDs, These results further support the conclusion that and then cell viability was assayed. All NSAIDs NSAIDs block NF-kB activation and NF-kB-dependent decreased cell viability in a dose-dependent manner inﬂammatory gene expression. (Figure 7a). The 50% inhibitory concentration required in KBM-5 cells was 6.09, 1.12, 0.65, 0.49, 1.01, 0.19, M NSAIDs inhibit TNF-induced prostaglandin E2 (PGE2) 0.36, 0.012, 0.016, 0.047, 0.013, and 0.008 m synthesis for aspirin, ibuprofen, sulindac, phenylbutazone, naproxen, indomethacin, diclofenac, resveratrol, curcu- PGE2 is formed through COX-mediated conversion of min, dexamethasone, celecoxib, and tamoxifen, arachidonic acid (Lawrence et al., 2002). Whether respectively (Table 1). Based on the IC50, aspirin was suppression of COX-2 expression by NSAIDs correlates least potent and tamoxifen was the most potent with suppression of PGE2 synthesis was examined. To (761-fold) antiproliferative agent. H1299 cells were also determine this, we pretreated mouse macrophage with incubated for the indicated days in the presence of the indicated concentrations of aspirin or curcumin for different concentrations of NSAIDs, and then cell 4 h, and then cells were stimulated with 1 nM TNF for viability was assayed by the 3-(4, 5-dihydro-6-(4-(3, 4- 12 h, collected the culture media, and analysed for PGE2 dimethoxy benzoyl)-1-piperazinyl)-2(1H)-quinolinone secretion. Figure 6b shows that TNF induced PGE2 (MTT) uptake method. All NSAIDs also decreased secretion and aspirin suppressed it in a dose-dependent H1299 cell viability in a time- and dose-dependent manner (left panel). Similar results were observed with manner (Figure 7b). curcumin (right panel). These results thus suggest that To determine whether the effect of NSAIDs on cell NSAIDs inhibit both COX-2 protein expression as well viability is due to a decrease in cell proliferation or to an as its enzymatic activity. increase in apoptosis, we performed [3H]thymidine incorporation assay and PARP-cleavage assay as an indication of caspase activity. KBM-5 cells were treated with the indicated concentrations of aspirin or curcumin for 18 h, labeled them with [3H]thymidine for further 6 h, and analysed [3H]thymidine uptake (Figure 7c). We found that both curcumin and aspirin inhibited thymidine incorporation in a dose-dependent manner. Whether NSAIDs induce apoptosis was also examined. For this, KBM-5 cells were incubated with the indicated concentrations of aspirin or curcumin for 24 h, prepared whole-cell extract, and performed Western blot analysis using anti-PARP antibody (Figure 7d). These results indicated that NSAIDs induced PARP cleavage in a dose-dependent manner, thus suggesting an induction of apoptosis. Thus, NSAIDs can both inhibit cell proliferation and induce apoptosis.

Discussion

The aim of the current study was to investigate the relative efficacy of classical NSAIDs and those which are COX-2 specific (such as celebrex) in suppressing NF- kB activation and NF-kB-regulated protein expression, and cell proliferation. Our results clearly demonstrate Figure 5 Effect of NSAIDs on TNF-induced cyclin D1 expression. KBM-5 cells were pretreated with various concentrations of that all the commonly used NSAIDs can suppress TNF- NSAIDs either for 4 or 8 h (phenylbutazone, resveratrol, and induced DNA binding of NF-kB by inhibiting IKK diclofenac), and then treated with 1 nM TNF for 24 h. Whole-cell activation and IkBa degradation. They also inhibited extracts were prepared, resolved on 10% SDS–PAGE, and then NF-kB-dependent reporter gene expression and NF-kB- electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-cyclin D1 and anti-b-actin regulated protein expression. Both COX-2, which antibodies as described in Materials and methods. Results shown regulates inflammation, and cyclin D1, which regulates are representative of two or three independent experiments proliferation, were downregulated by all NSAIDs. All

Oncogene Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9254

Figure 6 Effect of NSAIDs on TNF-induced COX-2 expression and PGE2 secretion. (a) KBM-5 cells were pretreated with various concentrations of NSAIDs either for 4 or 8 h (phenylbutazone, resveratrol, and diclofenac), and then treated with 1 nM TNF for 24 h. Whole-cell extracts were prepared, resolved on 7.5% SDS–PAGE, and then electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-COX-2 and anti-b-actin antibodies as described in Materials and methods. Results shown are representative of two or three independent experiments. (b) Macrophages were pretreated with various concentrations of NSAID for 4 h, and then treated with 1 nM TNF for 12 h. Cell culture media was collected and analysed for PGE2 levels using with PGE2 ELISA kit. The results expressed are percentage of untreated control (100%). Results shown are representative of two independent experiments. The signiﬁcance of results when compared with the TNF-treated cells are *P>0.1, **Po0.1, ***Po0.01, ****Po0.001

the NSAIDs inhibited cell proliferation in a dose- 1994; Yamamoto et al., 1999), sulindac (Hass et al., dependent manner as well. 1992; Kopp and Ghosh, 1994; Yamamoto et al., 1999), That both TNF and NF-kB play a major role in curcumin (Singh and Aggarwal, 1995; Manna et al., inﬂammation is well established (Bharti and Aggarwal, 2000), and resveratrol (Singh and Aggarwal, 1995; 2002; Aggarwal, 2003). Our results indicate that all Manna et al., 2000) block NF-kB activation. Very little NSAIDs can block TNF-induced NF-kB activation, in is known, however, about the effect of ibuprofen, agreement with reports that aspirin (Hass et al., 1992; phenylbutazone, naproxen, indomethacin, diclofenac, Kopp and Ghosh, 1994; Yamamoto et al., 1999), celecoxib, and tamoxifen on the NF-kB activity (Kazmi dexamethasone (Hass et al., 1992; Kopp and Ghosh, et al., 1995; Scheuren et al., 1998; Chuang et al., 2002;

Oncogene Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9255

Figure 7 Effect of NSAIDs on cell viability, cell proliferation, and apoptosis. (a) In all, 5000 cells of KBM-5 were treated with various concentrations of NSAIDs for 72 h. Then, cell viability was determined by MTT incorporation as described in Materials and methods. (b) A total of 5000 H1299 cells were treated with various concentrations of NSAIDs for the indicated days. Then, cell viability was determined by MTT incorporation. (c) Effect of NSAIDs on [3H]thymidine incorporation. In total, 10 000 KBM-5 cells were treated with various concentrations of aspirin or curcumin for 18 h, and then labeled them with [3H]thymidine for further 6 h, and analysed [3H]thymidine uptake as described in Materials and methods. The results expressed are percentage of untreated control (100%). Results shown are representative of two independent experiments. The signiﬁcance of results when compared with the each untreated control are *P>0.1, **Po0.1, ***Po0.01, ****Po0.001. (d) KBM-5 cells were incubated with various concentrations of aspirin or curcumin for 24 h. Whole-cell extracts were prepared, resolved on 7.5% SDS–PAGE, and then electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-PARP and anti-b-actin antibodies as described in Materials and methods. Results shown are representative of two independent experiments

Bryant et al., 2003). Scheuren et al. (1998) showed that whereas in our system, celecoxib alone did not activate R(À)-ibuprofen as well as the S( þ )-enantiomer inhib- NF-kB. Another recent report showed that aspirin at 5– ited the activation of NF-kB in response to T-cell 10 mM activated NF-kB in colorectal cancer HRT 18 stimulation. One recent report showed that low doses of cells but not in other colorectal cell lines (Stark et al., celecoxib inhibit IL-1-induced NF-kB activation and 2001), suggesting a cell type speciﬁcity. Callejas et al. high doses stimulate it (Niederberger et al., 2001), (2002) also showed that salicylate, aspirin, indomethacin,

Oncogene Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9256 ibuprofen, and 5,5-dimethyl-3(3-fluorophenyl)-4-(4- possible that downregulation of NF-kB, COX-2, and methylsulfonyl)phenyl-2(5H)-furanone, a fluorinated cyclin D1 mediate the antiproliferative effects of derivative of rofecoxib, had no effect on IKK activity, NSAID. We found that NSAIDs (e.g. aspirin and the processing of NF-kB, or the expression of NF-kB- curcumin) can inhibit both DNA synthesis and induce dependent genes, such as NOS-2 in hepatocytes. Tegeder apoptosis as indicated by caspase-induced PARP et al. (2001) also showed that indomethacin failed to cleavage. inhibit NF-kB activation. Our results differ from those There are reports that suggest COX-independent reported in these studies. These differences could be actions of COX inhibitors (Tegeder et al., 2001). linked with cell type used, type of NF-kB inducer NSAIDs such as sodium salicylate, sulindac, and employed, NF-kB assay used (DNA binding vs reporter) ibuprofen can induce anti-inflammatory and antiproli- and its dose, or the dose of NSAID examined. ferative effects independent of COX activity. These Our results also indicate that various NSAID effects were, however, found to be mediated through suppress NF-kB activation through inhibition of IKK inhibition of NF-kB. Cyclin D1 is required for progres- activity, leading to suppression of IkBa degradation. sion of cells from G1 to S phase of the cell cycle and thus These results are in agreement with reports that aspirin, is critical for cell proliferation. The downregulation of sulindac, and sodium salicylate can inhibit the activity of cyclin D1 by NSAIDs may explain their antiprolifera- IKK (Yin et al., 1998; Yamamoto et al., 1999). tive effects against various tumor cells. Among all the Curcumin and resveratrol have also been shown to NSAID, once again tamoxifen was most potent (761- inhibit IKK (Singh and Aggarwal, 1995; Manna et al., fold) in its antiproliferative effects, and this effect must 2000). Our finding that indomethacin can suppress be ER-independent. Celecoxib was almost 468 times TNF-induced IKK activity, however, differ from that of more potent than aspirin in suppressing cell prolifera- Yamamoto et al. (1999), who reported that indometha- tion. cin had no effect on IKK. The difference again may be When compared with the IC50 of NSAIDs against due to differences in cell type or activator. COX-1 and COX-2 (Warner et al., 1999), the IC50 of Our results also indicate that among all the NSAID NSAIDs for NF-kB inhibition or for antiproliferative used, tamoxifen is the most potent inhibitor of NF-kB effects suggest that the NSAID effects described here are activation. Based on IC50, it is 567-fold more potent COX-independent. For instance, tamoxifen is a than aspirin. There is no prior report on the suppression weak inhibitor of COX-1 and COX-2 (see Table 1), of NF-kB activation by tamoxifen. Tamoxifen is a yet it is most potent in suppression of NF-kB and nonsteroidal antiestrogen that is widely used for cell proliferation. Similarly, celecoxib, which is 100 times chemoprevention. Since NF-kB suppression has been more potent than tamoxifen in inhibiting closely linked with chemoprevention (Bharti and Ag- COX-2 activity (Warner et al., 1999), is equally potent garwal, 2002), it is possible that chemopreventive effects for suppression of NF-kB and cell growth (see Table 1). of tamoxifen are due to NF-kB inhibition as described Overall our results suggest that all NSAID can here. Interestingly, the effects of tamoxifen do not inhibit NF-kB activation and NF-kB-regulated gene appear to be mediated through the estrogen receptor expression, and this may contribute to their (ER), as neither KBM-5 nor A293 cells used here are anti-inflammatory and antiproliferative effects. Since known to express ER. These results are in agreement NF-kB has been implicated in chemoresistance and with reports that tamoxifen can induce apoptosis in ER- radioresistance (Jung and Dritschilo, 2001; Bharti and negative human cancer cell lines (namely T-leukemic Aggarwal, 2002), NF-kB inhibitory activity of NSAIDs Jurkat and ovarian A2780 cancer cells) (Ferlini et al., can be exploited to overcome chemoresistance and 1999). radioresistance. We found that celecoxib, a specific COX-2 inhibitor, is 236 times more potent than aspirin in inhibiting NF- kB activation. This inhibition of NF-kB was again Materials and methods mediated through suppression of IKK and IkBa degradation. Materials We found that all NSAID downregulated NF-kB- Sulindac, indomethacin, aspirin, naproxin, ibuprofen, tamox- mediated cyclin D1 and COX-2 protein expression. ifen, dexamethasone, phenylbutazone, diclofenac, MTT and Aspirin and curcumin also suppressed TNF-induced anti-b-actin antibody were purchased from Sigma Chemical PGE2 secretion. However, the dose needed to suppress (St Louis, MO, USA). Curcumin, with purity greater than PGE2 secretion was lower than that for COX-2 protein 98%, and celecoxib were purchased from LKT laboratory (St expression. These results are in agreement with a Paul, MN, USA). Resveratrol, with purity greater than 95%, previous report (Fernandez de Arriba et al., 1999; was purchased from Alexis (San Diego, CA, USA). All Callejas et al., 2002). These results also point to a more NSAIDs were dissolved in 100% DMSO at different complex regulation of COX-2 expression besides NF-kB concentrations. Penicillin, streptomycin, Iscove’s modified Dulbecco’s medium, minimum essential medium, RPMI (Inoue et al., 1995; Miller et al., 1998). 1640, and FBS were obtained from Invitrogen (Grand Island, We found that all the NSAIDs were quite effective in NY, USA). Antibodies to IkBa, COX-2, and cyclin D1 were suppressing the proliferation of tumor cells. Both NF- purchased from Santa Cruz Biotechnology (Santa Cruz, CA, kB and NF-kB-regulated COX-2 and cyclinD1 have USA). Antibody to IKK-a was a kind gift from Imgenex (San been associated with cell proliferation. Therefore, it is Diego, CA, USA).

Oncogene Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9257 Cell lines and culture total amounts of IKK-a and IKK-b in each sample, 50 mg of the whole-cell protein was resolved on 7.5% We used the human leukemic cell line KBM-5, which is SDS–PAGE, electrotransferred to a nitrocellulose membrane, phenotypically myeloid with monocytic differentiation, A293, and then blotted with either anti-IKK-a or anti-IKK-b a human embryonic kidney cell line, and H1299, a human lung antibodies. cancer cell line. KBM-5 cells were maintained in Iscove’s modified Dulbecco’s medium supplemented with 15% FBS, A293 cells were maintained in minimum essential medium NF-kB-dependent reporter gene transcription assay supplemented with 10% FBS, and H1299 cells were main- The effect of NSAIDs on TNF-induced NF-kB-dependent tained in RPMI 1640 supplemented with 10% FBS. Culture reporter gene transcription was analysed by SEAP assay as media contained 100 U/ml penicillin and 100 mg/ml streptomy- previously described (Takada and Aggarwal, 2003a). To cin. examine TNF-induced reporter gene expression, A293 cells (5 Â 105 cells/well) were plated in 12-well plates and transiently EMSA transfected by the FuGENE 6 with pNF-kB-SEAP (0.5 mg) and b-actin-Renilla (12 ng). After 24 h, cells were washed and NF-kB activation was analysed by EMSA as described then treated with NSAIDs: aspirin, ibuprofen, sulindac, previously (Takada et al., 2003). In brief, 15 mg of nuclear naproxen, indomethacin, curcumin, dexamethasone, celecoxib, extracts prepared from TNF-treated or -untreated cells was and tamoxifen for 4 h or phenylbutazone, resveratrol, and 32 incubated with P end-labeled 45-mer double-stranded NF-kB diclofenac for 8 h. The medium was then changed, and exposed oligonucleotide from the human immunodeficiency virus-1 them to 1 nM TNF. The cell culture medium was harvested long terminal repeat (50-TTGTTACAA GGGACTTTCC 0 after 24 h and analysed for SEAP activity according to the GCT GGGGACTTTCC AGGGAGGCGTGG-3 ; underlined protocol essentially as described by the manufacturer (Clon- sequence is binding site) for 30 min at 371C, and the DNA– tech, Palo Alto, CA, USA) using a 96-well plate reader (Vecter protein complex resolved in a 6.6% native polyacrylamide gel. 3, Perkin–Elmer Life & Analytical Sciences, Boston, MA, For supershift assays, nuclear extracts prepared from TNF- USA) with excitation set at 360 nm and emission at 460 nm. treated cells were incubated with antibodies against either p50 The cells were also lysed and analysed for Renilla activity or p65 of NF-kB for 15 min at 371C before the complex was according to the protocol (Promega, Madison, WI, USA) analysed by EMSA. The specificity of binding was also using a 96-well plate reader (Vecter 3). examined by competition with the unlabeled and mutant oligonucleotide. PIS was included as negative controls. The PGE secretion effect of aspirin and curcumin on the binding of Oct-1 to DNA 2 was determined by incubating 15 mg of nuclear extracts with The effect of NSAIDs on the PGE2 secretion was studied. One 16 fmol of 32P end labeled with the octamer-binding protein million mouse macrophage were seeded into six-well plate, (Oct-1) consensus oligonucleotide 50-TGTCGAATGCAAAT- pretreated with the indicated concentrations of NSAID for 4 h, CACTAGAA-30 (underline indicates Oct-1 binding site) for then stimulated with 1 nM TNF for 12 h and collected culture 30 min at 371C, and then analysed using 5% native poly- media. The concentration of PGE2 was determined using acrylamide gel. The radioactive bands from the dried gels were PGE2 ELISA kit purchased from R&D Systems (Minneapolis, visualized and quantitated by PhosphorImager (Molecular MN, USA). Dynamics, Sunnyvale, CA, USA) using ImageQuant software. Cytotoxicity assay (MTT assay) Western blot analysis The cytotoxic effects of NSAIDs were determined by the MTT In all, 30–50 mg of cytoplasmic protein or whole-cell extract uptake method as described (Takada and Aggarwal, 2003b). was prepared as described (Ashikawa et al., 2002) and resolved Briefly, 5000 cells were incubated in duplicate in 96-well plates by SDS–polyacrylamide gel electrophoresis (PAGE). Then, the in the presence of NSAIDs at 371C. Thereafter, MTT solution proteins were electrotransferred to a nitrocellulose membrane, was added to each well. After 2 h incubation at 371C, blocked with 5% nonfat dry milk, and probed with primary extraction buffer (20% SDS, 50% dimethylformamide) was antibodies against either IkBa, cyclin D1, COX-2, or PARP added, the cells were incubated overnight at 371C, and then the for 2 h at 41C. The blotting membrane was washed, exposed to optical density was measured at 570 nm using a 96-well horse radish peroxidase-conjugated secondary antibodies for multiscanner (Dynex Tech., MRX Revelation; Chantilly, 1 h, and the blots finally detected by chemiluminescence (ECL, VA, USA). Amersham Pharmacia Biotech. Arlington Heights, IL, USA). [3H]thymidine incorporation assay IKK assay The effect of NSAIDs on DNA synthesis was monitored by 3 The IKK assay was performed by a method described the [ H]thymidine incorporation method (Bharti et al., previously (Takada et al., 2004). Briefly, IKK complex from 2004). In all, 10 000 KBM-5 cells in 100 ml of medium were whole-cell extracts were precipitated with antibody against cultured in triplicate in 96-well plates in the presence or IKK-a, followed by treatment with protein A/G–Sepharose absence of the indicated concentrations of NSAID for 18 h. 3 beads (Pierce, Rockford, IL, USA). After a 2 h incubation, the Then, cells were pulse treated with 0.5 mCi [ H]thymidine for 3 beads were washed with lysis buffer and then assayed in kinase 6 h, and the uptake of [ H]thymidine was monitored using a assay mixture containing 50 mM HEPES (pH 7.4), 20 mM Matrix-9600 b-counter (Packard Instruments, Downers 32 Grove, IL, USA). MgCl2,2mM DTT, 20 mCi [g- P]ATP, 10 mM unlabeled ATP, and 2 mg of substrate GST-IkBa (aa 1–54). After incubation at 301C for 30 min, the reaction was terminated by boiling with SDS sample buffer for 5 min. Finally, the protein was resolved Abbreviations on 10% SDS–PAGE, the gel was dried, and the radioactive NSAIDs, nonsteroidal anti-inflammatory drugs; TNF, tumor bands were visualized by PhosphorImager. To determine the necrosis factor; IkB, inhibitory subunit of NF-kB; IKK, IkBa

Oncogene Downregulation of NF-jB, COX-2, cyclin D1, and proliferation by NSAID Y Takada et al 9258 kinase; EMSA, electrophoretic mobility shift assays; SEAP, Research. This work was supported partially by the Clayton secretory alkaline phosphatase; COX, cyclooxygenase. Foundation for Research (to BBA), Department of Defense US Army Breast Cancer Research Program Grant BC010610 Acknowledgements (to BBA), a PO1 Grant (CA91844) from the National We thank Mr Walter Pagel for carefully proof-reading the Institutes of Health on Lung Cancer Chemoprevention manuscript and providing valuable comments. Dr Aggarwal is (to BBA), and a P50 Head and Neck SPORE Grant from a Ransom Horne Jr, Distinguished Professor of Cancer the National Institutes of Health (to BBA).

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