Salicylates Inhibit NF-κB Activation and Enhance TNF-α-Induced Apoptosis in Human Pancreatic Cancer Cells

Journal of Surgical Research 83, 56 – 61 (1999) Article ID jsre.1998.5560, available online at http://www.idealibrary.com on

Salicylates Inhibit NF-kB Activation and Enhance TNF-a-Induced Apoptosis in Human Pancreatic Cancer Cells Theodore P. McDade, M.D., Richard A. Perugini, M.D., Frank J. Vittimberga, Jr., M.D., Rebecca C. Carrigan, B.S., and Mark P. Callery, M.D., FACS 1 Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts 01655 Presented at the Annual Meeting of the Association for Academic Surgery, Seattle, Washington, November 18 –22, 1998

degradation in BxPC-3 human pancreatic cancer cells. © 1999 Academic Press Key Words: sodium salicylate; apoptosis; pancreatic cancer; nuclear factor kappa B (NF-kB); tumor necrosis factor alpha (TNF-a).

Introduction. Tumor necrosis factor (TNF-a)induced apoptosis is limited by its coactivation of nuclear factor kappa B (NF-kB)-dependent antiapoptotic genes. Sodium salicylate (NaSal) inhibits NF-kB activation by limiting phosphorylation and degradation of its bound inhibitor protein, IkB-a. We examined whether NaSal enhances TNF-a-induced apoptosis in cultured human pancreatic cancer cell lines. Methods. Two cultured human pancreatic cancer cell lines were studied. PANC-1 and BxPC-3 cells were serum-starved for 12 h, pretreated or not for 1 h with NaSal (5–20 mM), and then stimulated with recombinant human TNF-a (400 units/ml). Western blots of cytoplasmic lysates were performed to demonstrate IkB-a phosphorylation and degradation. Western blots of nuclear extracts were performed to assess nuclear translocation of NF-kB. In separate cultures, apoptosis was measured 4.5 h after TNF-a stimulation by both ELISA detection of interhistone DNA fragments and flow cytometry with propidium iodide staining. Results. TNF-a induced IkB-a phosphorylation and degradation, which was inhibited by NaSal in both cell lines. TNF-a-induced apoptosis (DNA fragmentation) increased significantly when BxPC-3 cells were pretreated with NaSal. Flow cytometry confirmed this, demonstrating increases in apoptotic cell fractions: 8.5% (untreated), 9.3% (TNF-a alone), 14.9% (15 mM NaSal), and 22.9% (NaSal and TNF-a). In contrast, no increases in apoptosis were measured in the PANC-1 cell line among the various treatment groups. Conclusions. NaSal enhances TNF-a-induced apoptosis while inhibiting IkB-a phosphorylation and

BACKGROUND

Pancreatic cancer is the fifth most common cause of cancer death in the Western world, affecting nearly 27,000 Americans in 1997 [1]. Less than 5% of patients with adenocarcinoma of the pancreas will be alive 5 years after their diagnosis. Recently, Yeo et al. have demonstrated that postoperative chemoradiation improves survival in patients who undergo pancreaticoduodenectomy for adenocarcinoma [2]. The beneficial effect is limited, however, as median survival improves from 13.5 to 19.5 months. A therapeutic modality that increases the effectiveness of current chemoradiation regimens is needed. In 1893, William Coley noted that patients with sarcomas of the head and neck demonstrated tumor regression following repeated bouts of erysipelas. Based on this observation, he injected streptococcal toxins (Coley’s toxin) into 10 cancer patients [3]. Toxicity to patients, however, led to discontinuation of these treatments. Years later, two groups of investigators separately demonstrated that serum from lipopolysaccharide-injected mice could induce hemorrhagic necrosis in tumors, and called the circulating protein tumor necrosis factor [4, 5]. Nearly a decade later, TNF-a was purified and sequenced, and soon made available in recombinant form for research [6 – 8]. Nevertheless, TNF-a has generally been clinically disappointing as an antitumor therapy [9]. Three separate groups have reported studies that explain the limited antitumor effects of TNF-a [10 –12].

1 To whom correspondence should be addressed at University of Massachusetts Medical School, 55 North Lake Avenue, Worcester, MA 01655. Fax: (508) 856-1102. E-mail: Mark.Callery@ banyan.ummed.edu.

0022-4804/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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These studies demonstrated that in addition to activating programmed cell death (apoptosis), TNF-a coactivates the NF-kB transcription factor pathway, which limits apoptosis through anti-apoptotic gene transcription. This negative feedback mechanism thereby provides a salvage pathway for cell survival. Using a human fibrosarcoma cell line, Wang et al. demonstrated that the dual activation of apoptosis and apoptoticresistance pathways seen with TNF-a stimulation also occurs with either ionizing radiation or daunorubicin [11]. Thus, blockade of NF-kB activation provides a potential biological strategy for enhancing tumor cell killing by radiotherapy, chemotherapy, or biotherapy. Sodium salicylate prevents the TNF-a-induced degradation of the NF-kB repressor, IkB-a, in some epithelial cell lines [13]. Because IkB-a degradation is necessary for NF-kB translocation to the nucleus, this correlates with decreased activation of the transcription factor. Inhibition of NF-kB by salicylates has also been shown in lymphocytes [14]. We have demonstrated that TNF-a similarly induces IkB-a phosphorylation and degradation in PANC-1 human pancreatic cancer cells [15]. Using a specific p38MAPK inhibitor (SB203580), we confirmed (unpublished data) that salicylates inhibit NF-kB in PANC-1 cells via a p38MAPK-mediated pathway [13, 16]. These studies now examine whether salicylates potentiate TNF-ainduced apoptosis in human pancreatic cancer cells (Fig. 1). MATERIALS AND METHODS

Cell Culture and Treatments The human pancreatic adenocarcinoma cell lines, BxPC-3 and PANC-1, were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in RPMI (BxPC-3) or DMEM (PANC-1) tissue culture media and supplemented with 10% fetal calf serum (Gibco BRL, Gaithersburg, MD), as well as penicillin and streptomycin. They were incubated and propagated at 37°C and 5% CO 2. After serum starvation for 12 h, some cells were pretreated for 1 h with sodium salicylate (NaSal) (Fisher, Springfield, NJ). The concentration of NaSal ranged from 5 to 20 mM, depending upon the experiment. All cells, with the exception of negative controls, were then exposed to human recombinant TNF-a (Genzyme, Cambridge, MA) at 400 units/ml (specific activity 5.88 3 10 4 units/mg). NaSal exposure was maintained at the desired concentration throughout TNF-a stimulation.

Western Blots Cytoplasmic lysates. Cells were plated in six-well culture plates at a concentration of 4 – 6 3 10 5 cells per well. Cells were harvested by the addition of ice cold RIPA buffer {1% NP-40, 50 mM Tris, 150 mM NaCl, 0.25% deoxycholate, 1 mM EGTA, 1 mM NaF, and added protease inhibitor cocktail (Sigma, St. Louis, MO)} at various time points following the addition of TNF-a. Samples were incubated on ice for 20 min, and after scraping of the wells lysates were centrifuged at 15,000g for 11 min. A Bradford protein quantification assay (Bio-Rad, Hercules, CA) was performed on the resulting supernatant. Samples were loaded onto a 10% SDS-PAGE gel at 9 mg per well for IkB-a Western blots and 12 mg per well for phosphorylated IkB-a

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FIG. 1. Schematic demonstrating NF-kB salvage pathway and hypothesized site of action of salicylate. Note IkB-a repressor must be degraded prior to NF-kB nuclear translocation for pathway activation. In PANC-1 cells, inhibition of NF-kB activation by salicylate is mediated by p38 MAPK (unpublished data). Western blots, and then electrophoresed at 200 V. The gel was transferred to Imobilon-P membrane (Millipore, Bedford, MA) at 100 V for 65 min. Membranes were dried by placing into methanol for 1 min and then at room temperature for 15 min. Membranes were developed with the PhosphoPlus IkB-a antibody kits (New England BioLabs, Beverly, MA). Briefly, membranes were blocked by incubating in 5% milk in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 to 3 h. After washing three times for 5 min in 0.05% TBST, membranes were incubated overnight at 4°C in primary antibody dilution buffer (5% bovine serum albumin in 0.05% TBST, with a 1:1000 dilution of primary antibody). After three washes in 0.05% TBST, membranes were incubated in horseradish peroxidaselinked secondary antibody for 1 h at room temperature. Membranes were again washed three times with 0.05% TBST and developed using enhanced chemiluminescence. Membranes were then exposed to film (Hyperfilm ECL, Amersham, Buckinghamshire, England). Nuclear protein extracts. Cells were plated in six-well culture plates at a concentration of 1.5 3 10 7 cells per well. Following treatment with NaSal and/or TNF-a, plated cells were washed three times with ice cold phosphate-buffered saline (PBS), trypsinized, and centrifuged at 600g for 10 min. Nuclear protein extracts were obtained via a modification of the methods developed by Dignam et al. [17]. Briefly, cell pellets were resuspended and incubated in the first of a series of three buffers {10 mM Hepes, 1.5 mM MgCl 2, 10 mM KCl, and added protease inhibitor cocktail (Sigma)} for 30 min on ice. Cells were mechanically lysed using a glass Dounce homogenizer and centrifuged to obtain pelleting of the nuclei. Nuclear pellets were resuspended in a second buffer {10 mM Hepes, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and added protease inhibitor cocktail (Sigma)}. Following serial incubations and centrifugations, in this and a third buffer {20 mM Hepes, 10% (v/v) glycerol, 400 mM KCl, 1.0 mM EDTA, 1.0 mM EGTA, and added protease inhibitor cocktail (Sigma)}, a Bradford protein quantification assay (Bio-Rad) was performed on the resulting supernatant. Samples were loaded onto a

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10% SDS-PAGE gel at 9 mg per well. Electrophoresis and membrane transfer were performed as above. Membranes were incubated with antibodies to the p65- and p50-binding subunits of NF-kB (Santa Cruz Biotechnology, Santa Cruz, CA). Incubation in primary antibody dilution buffer (5% bovine serum albumin in 0.05% TTBS, with 0.5 mg/ml dilution of primary antibody) was performed for 1.5 h at room temperature. Membranes were washed and incubated in horseradish peroxidase-linked secondary antibody and then developed and exposed to film as described above. Densitometry was performed using a Stratagene Eagle Eye System (Stratagene, La Jolla, CA).

Analysis of DNA Fragmentation by ELISA Cells were plated in 96-well plates at a concentration of 2 3 10 4 cells per well. Four and a half hours following treatment with TNF-a and/or pretreatment with NaSal, plated cells were harvested and analyzed using a Cell Death Detection ELISA (Boehringer Mannheim, Indianapolis, IN). Briefly, following exposure to lysis buffer and centrifugation, the cytoplasmic fraction is incubated with both anti-histone and anti-DNA antibodies. Multiples of 180-bp histoneassociated DNA fragments are detected with this sandwich-ELISA and correlate with the DNA ladder classically described for apoptosis. Quadruplicate samples were analyzed by Student’s t test, with P , 0.05 indicating statistical significance.

Apoptosis Analysis by FACS Cells were plated in a six-well plate at a concentration of 5 3 10 5 cells per well. Four and a half hours following treatment with TNF-a and/or pretreatment with NaSal, cells were harvested by trypsinization, with care to include any floating cells. They were washed in PBS. Cells were treated with a nuclear staining solution {NP-40, propidium iodide, sodium citrate} at room temperature in the dark for 30 min. Cells were then washed and resuspended in PBS. Samples were assessed using fluorescence activated cell sorting (FACS) and flow cytometric analysis. Apoptotic cells were defined as those with an FL3 greater than one, but less than the G0/G1 peak.

RESULTS

Salicylates Inhibit TNF-a-Induced IkB-a Phosphorylation and Degradation in Cultured Human Pancreatic Cancer Cell Lines In both BxPC-3 and PANC-1 cell lines, treatment with TNF-a at 400 units/ml induces brisk phosphory-

FIG. 2. Western blot analysis of cytoplasmic IkB-a and phosphorylated IkB-a in PANC-1 and BxPC-3 human pancreatic cancer cells. In both cell lines, there is brisk phosphorylation within 5 min, peaking at approximately 10 min, with subsequent degradation of both phosphorylated and nonphosphorylated IkB-a being apparent by 20 min following TNF-a exposure. Pretreatment with sodium salicylate (20 mM) for 1 h decreases and delays this effect.

FIG. 3. Western blot analysis of cytoplasmic IkB-a in BxPC-3 human pancreatic cancer cells. While pretreatment with NaSal at 5 mM has no appreciable effect, IkB-a degradation is significantly delayed by NaSal at 15 mM.

lation of the NF-kB repressor, IkB-a, within 5 min (Fig. 2). Phosphorylation peaks at approximately 10 min, and degradation of both phosphorylated and nonphosphorylated IkB-a is apparent by 20 min. Pretreatment of both cell lines with sodium salicylate, at a concentration of 20 mM, markedly inhibits both TNF-ainduced IkB-a phosphorylation and degradation. A lower concentration of salicylate (5 mM) did not appreciably alter TNF-a-induced IkB-a degradation in the BxPC-3 cell line (Fig. 3). Salicylates Inhibit TNF-a-Induced Nuclear Translocation of the p65 Subunit of NF-kB Although IkB-a phosphorylation and degradation in the cytoplasm strongly suggest NF-kB activation, it does not provide evidence of events further downstream in the pathway. For that reason, we assessed the presence of NF-kB, no longer bound to its IkB-a repressor, in the nucleus. Western blots for the p50and p65-binding subunits of NF-kB were performed on nuclear protein extracts. Thirty minutes following treatment with TNF-a, the presence of the p65-binding subunit of NF-kB is markedly increased when compared to that of untreated cells (Fig. 4). Pretreatment with sodium salicylate at 20 mM inhibits this translocation to the nucleus. In contrast, there was no change in the p50-binding subunit of NF-kB among treatment groups (data not shown).

FIG. 4. Western blot analysis of nuclear protein extracts obtained from PANC-1 cells using antibodies to the p65 DNA-binding subunit of NF-kB. Tumor necrosis factor stimulation leads to nuclear translocation of p65 by 30 min. This effect is diminished when cells are pretreated with salicylate.

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dium salicylate had a synergistic effect, increasing the apoptotic fraction to 23% (Table 1). DISCUSSION

FIG. 5. Histone-associated DNA fragmentation as a measure of apoptosis. Treatment groups are shown on the x-axis. The y-axis represents histone-associated DNA fragmentation, relative to that of untreated cells (normalized to 1.0), in BxPC-3 cells. Stimulation for 4.5 h with 400 units/ml TNF-a alone produces minimal apoptosis. Treatment with sodium salicylate alone at a dose of 5 mM produces a similar result. Higher doses of sodium salicylate alone (20 mM) produce statistically significant (P , 0.05 by Student’s t test) increases in levels of apoptosis. The highest levels of apoptosis (approximately 24-fold) are seen following sodium salicylate pretreatment (20 mM 3 1 h) and TNF-a treatment (400 units/ml), presumably through inhibition of NF-kB by the former. A synergistic response is present even at a lower dose of salicylate (5 mM) (P , 0.05).

Salicylates Enhance Apoptosis in BxPC-3 but Not in PANC-1 Cells In BxPC3 cells, neither treatment with TNF-a alone (400 units/ml) nor treatment with 5 mM NaSal alone produced statistically significant changes in histoneassociated DNA fragmentation at 4.5 h when compared to untreated cells. However, the synergistic effect of these same doses (5 mM NaSal as pretreatment to 400 units/ml TNF-a) produced a 2.5-fold increase in apoptosis (P , 0.05 by Student’s t test). This synergism increased dramatically with higher doses of sodium salicylate. When TNF-a-stimulated cells were pretreated with 20 mM NaSal, a 24-fold increase above baseline was observed in apoptotic DNA fragmentation. Moderate levels of apoptosis resulted when this concentration of NaSal was used without subsequent TNF-a treatment. These data are reported as an increase in the amount of fragmented DNA in the cytoplasm, relative to untreated cells (Fig. 5). In contrast to BxPC3 cells, PANC-1 cells did not undergo appreciable interhistone DNA fragmentation regardless of treatment group (data not shown). These results were confirmed by FACS analysis of treated BxPC3 cells. At baseline, the apoptotic fraction accounted for approximately 8% of cells. This level was unchanged by treatment with TNF-a alone. Sodium salicylate alone, at 15 mM, increased the apoptotic fraction to approximately 15%. Again, TNF-a and so-

In an in vitro model of human pancreatic cancer, we have demonstrated that TNF-a induces phosphorylation and degradation of IkB-a, the cytoplasmic NF-kB repressor molecule, with subsequent translocation of the p65 subunit of NF-kB to the nucleus. In both of the tested cell lines, PANC-1 and BxPC-3, sodium salicylate inhibits activation of the NF-kB pathway. In one of these two cell lines, salicylates also dramatically enhance TNF-a-induced apoptosis. When used separately, the effects of TNF-a or sodium salicylate are unimpressive. The effects of TNF-a alone are negligible, while those of salicylate alone are modest, and only at the higher doses. When used in combination, however, their effects are synergistic, being much greater than simply additive. These results parallel and add to those of other investigators using various cell lines. Salicylates prevent the degradation of IkB-a in a human colorectal cancer cell line [13] and in a human fibrosarcoma cell line [11]. Salicylates also prevent the activation of NF-kB in lymphocytes [14]. Induction of apoptosis by salicylate has also been demonstrated, in both normal human fibroblasts [16] and human colorectal tumor cells [18]. In addition, the use of salicylates has been shown in epidemiological studies to have a chemopreventive effect against colorectal cancer; patients who took aspirin had up to a 50% reduction in the incidence of, or mortality from, colorectal cancer [19, 20]. Two cultured human pancreatic cancer cell lines, PANC-1 and BxPC-3, were examined in this study. The most prevalent mutation in human adenocarcinoma of the exocrine pancreas involves the K-ras oncogene. In TABLE 1 Sodium Salicylate Pretreatment Leads to an Increase in TNF-a-Induced Apoptosis, as Determined by Flow Cytometric Analysis of Propidium IodideStained BxPC-3 Cells Treatment group

Sub-G0/G1 (at 4.5 h)

Untreated TNF-a alone (400 units/ml) 15 mM NaSal alone NaSal pre-TNF-a

8.5% 9.3% 14.5% 22.9%

Note. In ethanol-fixed cells, the intensity of propidium iodide staining correlates with DNA content. The apoptotic fraction, visible as a hypodiploid or sub-G0-G1 peak, was 8.5% in untreated BxPC-3 cells. Treatment with TNF-a altered this minimally (9.3%). Salicylate (20 mM) alone increased the apoptotic fraction to 14.5%. Salicylate (20 mM) had a synergistic effect with TNF-a, increasing the apoptotic fraction to 22.9%.

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TABLE 2 The PANC-1 and BxPC-3 Cultured Cell Lines Share a Number of Characteristics with Surgically Resected Specimens of Human Pancreatic Cancer [21] a

Degree of Differentiation K-ras mutation p53 mutation p16 deletion

Resected panc ca

PANC-1

BxPC-3

Variable Most common .70% Common

Poor 1 1 1

Moderate 2 1 1

a Known differences between the two include K-ras mutation status and degree of differentiation.

addition, mutation of the p53 tumor suppressor is present in greater than 70% of resected human pancreatic adenocarcinomas. Deletion of the p16 tumor suppressor is also prevalent [21]. Both the PANC-1 and BxPC-3 cell lines have characteristics similar to these (Table 2). BxPC-3, a moderately differentiated cell line, expresses wild-type K-ras while PANC-1, a poorly differentiated cell line, expresses mutant K-ras [22]. Both cell lines have deletions of the p16 tumor suppressor gene [23] and overexpress a mutated form of p53 [24]. Although salicylates inhibit TNF-a-induced NF-kB activation in both human pancreatic cell lines tested, enhancement of apoptosis was only seen in BxPC-3 cells. In PANC-1 cells, there were no such increases in apoptosis above constitutive levels, regardless of treatment group. Known differences between these two cell lines include both the degree of differentiation and the presence of the K-ras oncogene mutation. These, among others, may be important factors in allowing apoptosis to proceed once NF-kB has been inhibited. NF-kB/Rel is a family of transcription factors first described in B cells and shown to constitutively bind and transactivate the enhancer of the immunoglobulin kappa gene [25]. They are now known to regulate a variety of genes, controlling the cell cycle, immune function, and inflammatory processes [26]. The members of this family have in common a region of homology also found in the Drosophila melanogaster dorsal protein, therefore known as the NF-kB/Rel/dorsal region (NRD) [27]. It is the NRD region that provides the abilities for dimerization, binding with IkB repressors, and DNA binding [28]. There are five DNA-binding subunits in this family: NFKB1/p50 (50-kDa product of proteolytic cleavage of p105 precursor), NFKB2/p52 (52-kDa product of proteolytic cleavage of p100 precursor), RelA/p65, RelB, and c-Rel (85 kDa). Inducible complexes of various homo- and heterodimers of these subunits are referred to as NF-kB [26, 28]. While the roles of each of these subunits have not been entirely characterized, studies of mice with homozygous deficiencies have provided some informa-

tion. NFKB1/p50 is largely involved with immunologic function. c-Rel is primarily expressed in B and T lymphocytes, while RelB is primarily in the thymus. RelA/ p65 is extremely important in embryogenesis, most notably that involving apoptosis (RelA2/2 mouse embryos fail to fully develop secondary to hepatic apoptosis.) [28]. Of the five subunits, only RelA/p65, RelB, and c-Rel are transcriptionally active. NFKB1/p50 and NFKB2/p52 serve primarily helper functions as part of heterodimers [27]. In addition, evidence suggests that excess NFKB1/p50 present as inactive homodimers in the nucleus competitively downregulates gene expression [27, 29, 30]. In these studies of pancreatic cancer cell apoptosis, we present data investigating the best characterized form of NF-kB, consisting of the p50 and p65 subunits. Our results demonstrate that p65 is the transcriptionally active binding form that is important in TNF-ainduced BxPC-3 pancreatic cancer cell apoptosis. This is in agreement with data of other investigators showing RelA/p65 to be necessary for TNF-a-dependent gene induction in murine fibroblasts and macrophages [10]. Build-up of p50 homodimers during NF-kB inhibition was not noted with the techniques used in these studies. Follow-up studies of the other NF-kB-binding subunits are a logical next step. We utilize TNF-a for the dual activation of apoptosis and the NF-kB apoptotic-resistance pathway and observe a powerful synergistic effect producing apoptosis when sodium salicylate is used to block NF-kB activation. As previously noted, Wang et al. demonstrated that this coactivation, seen with TNF-a stimulation, also occurs with either ionizing radiation or daunorubicin [11]. Together, these data suggest that suppression of the NF-kB salvage pathway may increase the efficacy of available regimens, such as ionizing radiation or chemotherapy, while minimizing dose-limiting toxicities and tumor resistance, attractive features of a synergistic approach [31]. In addition, with appropriate selection of an NF-kB inhibitor, perhaps TNF-a biotherapy may finally earn the enthusiasm it historically produced. REFERENCES 1.

Parker, S. L., et al. Cancer statistics, 1997. CA Cancer J. Clinicians 47: 5, 1997.

2.

Yeo, C. J., et al. Pancreaticoduodenectomy for pancreatic adenocarcinoma: Postoperative adjuvant chemoradiation improves survival. Anna. Surg. 225: 621, 1997.

3.

Coley, W. B. The treatment of malignant tumors by repeated inoculations of erysipelas: With a report of ten original cases. Am. J. Med. Sci. 5: 487, 1893.

4.

O’Malley, W. E., Achinstein, B., and Shear, M. J. Action by bacterial polysaccharide on tumors. J. Natl. Cancer 29: 1169, 1962.

5.

Carswell, E. A., et al. An endotoxin-induced serum factor that

MCDADE ET AL.: SALICYLATES INHIBIT NF-kB ACTIVATION

6. 7.

8.

9.

10. 11.

12. 13.

14. 15.

16.

17.

18.

causes necrosis of tumors. Proc. Natl. Acad. Sci. USA 72: 3666, 1975. Beutler, B., et al. Identity of tumor necrosis factor and the macrophage-secreted factor cachectin. Nature 316: 552, 1985. Beutler, B., et al. Purification of cachectin, a lipoprotein lipasesuppressing hormone secreted by endotoxin-induced RAW 264.7 cells. J. Exp. Med. 161: 984, 1985. Pennica, D., et al. Human tumour necrosis factor: Precursor structure, expression, and homology to lymphotoxin. Nature 312: 724, 1984. Brophy, L. Tumor necrosis factor. In P. T. Rieger, (Ed.), Biotherapy: A Comprehensive Overview. Boston: Jones and Bartlett, 1995. Pp. 161–176. Beg, A. A., and Baltimore, D. An essential role for NF-kB in preventing TNF-a induced cell death. Science 274: 782, 1996. Wang, C. Y., Mayo, M. W., and Baldwin, A. S., Jr. TNF and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-kB. Science 274: 784, 1996. Van Antwerp, D. J., et al. Suppression of TNF-a induced apoptosis by NF-kB. Science 274: 787, 1996. Schwenger, P., et al. Activation of p38 mitogen-activated protein kinase by sodium salicylate leads to inhibition of tumor necrosis factor-induced IkBa phosphorylation and degradation. Mol. Cell. Biol. 18(1): 78, 1998. Kopp, E., and Ghosh, S. Inhibition of NF-kB by sodium salicylate and aspirin. Science 265: 956, 1994. McDade, T. P., et al. Sodium salicylate inhibits tumor necrosis factor-induced IkB-a phosphorylation and degradation in human pancreatic cancer. Owen H. Wangensteen Surg. Forum 49: 438, 1998. Schwenger, P., et al. Sodium salicylate induces apoptosis via p38 mitogen-activated protein kinase but inhibits tumor necrosis factor-induced c-jun N-terminal kinase/stress-activated protein kinase activation. Proc. Natl. Acad. Sci. USA 94: 2869, 1996. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. Accurate transcription initiation by polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11(5): 1475, 1983. Elder, D. E., et al. Differential growth inhibition by the aspirin metabolite salicylate in human colorectal tumor cell lines: En-

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30. 31.

61

hanced apoptosis in carcinoma and in vitro-transformed adenoma relative to adenoma cell lines. Cancer Res. 56: 2273, 1996. Trujillo, M. A., Garewal, H. S., and Sampliner, R. E. Nonsteroidal antiinflammatory agents in chemoprevention of colorectal cancer. At what cost? Dig. Dis. Sci. 39: 2260, 1994. Giardiello, F. M., Offerhaus, G. J. A., and DuBois, R. N. The role of non-steroidal anti-inflammatory drugs in colorectal cancer prevention. Eur. J. Cancer 31A: 1071, 1995. Perugini, R. A., et al. The molecular and cellular biology of pancreatic cancer. Crit. Rev. Eukaryotic Gene Expression 8(3, 4): 377, 1998. Berrozpe, G., et al. Comparative analysis of mutations in the p53 and K-ras genes in pancreatic cancer. Int. J. Cancer 58: 185, 1994. Naumann, M., et al. Frequent codeletion of p16/MTS1 and p15/MTS2 and genetic alterations in p16/MTS1 in human pancreatic cancer. Gastroenterology 110: 1215, 1996. Barton, C. M., et al. Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer. Br. J. Cancer 64: 1076, 1991. Sen, R., and Baltimore, D. Inducibility of k immunoglobulin enhancer-binding protein NF-kB by a posttranslational mechanism. Cell 47: 921, 1986. Finco, T. S., Beg, A. A., and Baldwin, A. S. Inducible phosphorylation of IkBa is not sufficient for its dissociation from NF-kB and is inhibited by protease inhibitors. Proc. Natl. Acad. Sci. 91: 11884, 1994. Schmitz, M. L., and Baeuerle, P. A. Multi-step activation of NF-kB/Rel transcription factors. Immunobiology 193: 116, 1995. Beauparlant, P., and Hiscott, J. Biological and biochemical inhibitors of the NF-kB/Rel proteins and cytokine synthesis. Cytokine Growth Factor Rev. 7(2): 175, 1996. Brown, A., et al. Function of NF-kB/Rel binding sites in the major histocompatability complex class II invariant chain promoter is dependent on cell-specific binding of different NK-kB/ Rel subunits. Mol. Cell. Biol. 14: 2926, 1994. Kang, S., et al. NF-kB subunit regulation in nontransformed CD41 T lymphocytes. Science 256: 1452, 1992. Mauceri, H., et al. Combined effects of angiostatin and ionizing radiation in antitumor therapy. Nature 394: ix, 287, 1998.