Abstract
Cyclooxygenase (COX)-2 mRNA and protein expression were found to be frequently elevated in human pancreatic adenocarcinomas and cell lines derived from such tumors. Immunohistochemistry demonstrated cytoplasmic COX-2 expression in 14 of 21 (67%) pancreatic carcinomas. The level of COX-2 mRNA was found to be elevated in carcinomas, relative to histologically normal pancreas from a healthy individual, as assessed by reverse transcription-PCR. COX-2 protein expression was detected by the Western blot assay in three of five pancreatic carcinoma cell lines (BxPC-3, Capan-1, and MDAPanc-3), whereas COX-1 protein was detected in two of the five cell lines (BxPC-3 and Capan-1). Increased levels of COX-2 mRNA were found in four of five cell lines, and only in PANC-1 cells was the low level of transcript comparable to that in the normal pancreas. The level of COX-2 mRNA was positively correlated with the differentiation status of the tumor of origin for each cell line, COX-2 protein expression was up-regulated by epidermal growth factor when the cells were grown in absence of serum. Finally, two nonsteroidal anti-inflammatory drugs, sulindac sulfide and NS398, produced a dose-dependent inhibition of cell proliferation in all pancreatic cell lines tested. No correlation was found between the level of COX-2 or COX-1 expression and the extent of growth inhibition. Treatment of BxPC-3 cells with sulindac sulfide and NS398 resulted in an induction of COX-2 expression. Our findings indicate that COX-2 up-regulation is a frequent event in pancreatic cancers and suggest that nonsteroidal anti-inflammatory drugs may be useful in the chemoprevention and therapy of pancreatic carcinoma.
INTRODUCTION
Pancreatic cancer is the fourth leading cause of cancer deaths in the United States. Five % of all cancer deaths in men and 6% in women are due to this malignancy (1) . Despite some advances in diagnosis, staging, and treatment, the prognosis remains extremely poor with a 5-year survival rate of <1% (2) . Early detection of pancreatic cancer is difficult, and most patients are found to have unresectable disease at presentation (2) . An increased understanding of the molecular biology of pancreatic carcinoma is needed to develop new diagnostic and treatment approaches.
Recent studies have led to the recognition of the importance of COX-2 4 in colorectal tumorigenesis (3, 4, 5, 6, 7, 8) . COX-2 has been shown to play a role in the development of intestinal neoplasms in genetically manipulated animal models (5) . Specifically, the formation of intestinal polyps in ApcΔ716 knockout mice was dramatically suppressed by crossing these animals with COX-2 knockout mice (7) . Up-regulation of COX-2 has been detected in human colorectal carcinomas relative to normal epithelium (3 , 4 , 9) , and COX-2 expression has also been detected in gastric (10) , esophageal (11 , 12) , and lung (13 , 14) carcinomas. COX enzymes catalyze the rate-limiting step in arachidonate metabolism, resulting in prostaglandin H2 production. This molecule is the precursor of other prostaglandins, prostacyclin, and thromboxanes. Two cyclooxygenase isoforms have been identified: COX-1 and COX-2 (15) . COX-1 is expressed constitutively in several cell types in normal mammalian tissues, where it is involved in the maintenance of tissue homeostasis. In contrast, COX-2 is an inducible enzyme responsible for prostaglandin production at sites of inflammation (15 , 16) . Growth factors, tumor promoters, cytokines, and other inflammatory mediators have been found to induce COX-2 expression (15 , 16) . EGF is one of the growth factors known to up-regulate the expression of the COX-2 enzyme in several cell types such as human squamous carcinoma cells (17 , 18) .
NSAIDs, including aspirin and sulindac, inhibit both COX-1 and COX-2 isoforms and have been shown to prevent colon cancer in animal models (8) . In a clinical trial, sulindac prevented new polyp formation and produced a significant regression of existing colorectal polyps in patients with familial adenomatous polyposis (19) . Furthermore, numerous epidemiological studies have shown that chronic NSAID usage is associated with a reduction in the incidence of colorectal cancer (8 , 20) . The NSAIDs indomethacin and phenylbutazone have also been shown to reduce the development of pancreatic cancer in a hamster model of this disease, in which tumors were initiated by treatment with N-nitrosobis(2-oxopropyl)amine (21) . Nonselective and selective COX-2 inhibitors have been shown to inhibit proliferation and to induce apoptosis of several cultured tumor cell lines, suggesting a mechanism for their antitumor effects (22, 23, 24, 25, 26) . The antiproliferative and chemopreventive properties of NSAIDs have been traditionally attributed to COX inhibition and a consequent reduction in prostaglandin levels. However, the sulfone metabolite of sulindac, which lacks COX-inhibitory activity, has been shown to have antiproliferative and proapoptotic properties and is an effective chemopreventive agent in an animal model of colon cancer (22 , 27) . Sulindac is a prodrug that is metabolized to its sulfide and sulfone derivatives (22) . The sulfide is a potent inhibitor of COX and is responsible for the anti-inflammatory effects of sulindac. NSAIDs can produce toxic effects, including gastrointestinal mucosal injury and renal dysfunction due to inhibition of COX-1. For this reason, a new class of NSAIDs that includes NS398 and SC58635 (Celecoxib) has recently been developed; these NSAIDs selectively inhibit the COX-2 isoform (24, 25, 26) . Available evidence suggests that these drugs retain the capacity to prevent experimental colorectal cancer (6) and are devoid of gastrointestinal mucosal toxicity, at least in short-term studies (28) .
To date, the expression of COX-2 in pancreatic cancer and the effects of NSAIDs on the growth of pancreatic carcinoma cell lines have not been analyzed. In this study, we report that human pancreatic tumors and cell lines express increased levels of COX-2 mRNA and protein and that both nonselective and selective COX-2 inhibitors reduce the growth of cultured pancreatic carcinoma cell lines.
MATERIALS AND METHODS
Tissue Specimens.
All tissues in this study were surgical resection specimens obtained from the Surgical Pathology Laboratory at The University of Texas M. D. Anderson Cancer Center. All pancreatic adenocarcinomas were of ductal origin (n = 21). Paired tumor and histologically normal pancreas were analyzed from each patient. Normal pancreatic tissue from a healthy individual was also studied. For immunohistochemical analysis, formalin-fixed, paraffin-embedded blocks were obtained from tumors. Consecutive 4–6-μm tissue sections were cut from each tumor block. For RNA purification, fresh tissues were used.
Cultured Tumor Cell Lines.
Pancreatic adenocarcinoma cell lines used in this study were Capan-1 (29) and MDAPanc-3 (30) , derived from liver metastases of well-differentiated and moderately differentiated tumors, respectively; BxPC-3, established from a moderately well-differentiated adenocarcinoma (31) ; MDAPanc-28, established from a poorly differentiated adenocarcinoma (32) ; and PANC-1, derived from a mostly undifferentiated carcinoma (33) . HCA-7 colon cancer cells were obtained from Dr. S. Kirkland (Imperial Cancer Research Fund, London, United Kingdom). HCT-116 colon cancer cells and Capan-1, PANC-1, and BxPC-3 pancreatic cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). All cells were grown in DMEM/F-12 supplemented with 10% FBS, 2 mm glutamine, and 20 μg/ml gentamycin, unless otherwise indicated.
Immunohistochemistry.
Slides were deparaffinized, and endogenous peroxidase activity was blocked by incubation in 3% H2O2 in methanol for 10 min at room temperature. Sections were then microwaved in PBS for 4 min for antigen retrieval and incubated with avidin and then biotin (Vector Laboratories, Burlingame, CA) for 15 min each to block nonspecific binding. An immunoperoxidase technique was performed using the Vectastain ABC Elite kit (Vector Laboratories). A mouse monoclonal antibody against human COX-2 (Cayman Chemical Co., Ann Arbor, MI) was then applied at a dilution of 1:500 overnight at 4°C (9) . This antibody recognizes a 19-amino acid sequence at the COOH terminus of COX-2 that is absent in COX-1. As a control for nonspecific staining, a COX-2 sequence-specific blocking peptide (Cayman Chemical) was used, which, when combined with the primary COX-2 antibody, completely suppressed COX-2 staining. Following rinsing with PBS, the biotinylated secondary IgG antibody was applied for 30 min at room temperature. Hematoxylin was used as a counterstain. As an additional negative control, the primary antibody was omitted. As a positive control, we used slides from a human colon carcinoma known to overexpress COX-2 proteins.
Neoplasms displaying COX-2 immunoreactivity in >5% of tumor cells were regarded as positive. Study specimens were evaluated independently by two examiners (M. A. M. and F. A. S.) and were reviewed by a pathologist.
Western Blot Assay.
Cells grown in 100-mm dishes in presence of DMEM/F-12 plus 10% FBS were washed twice with cold PBS, harvested in 0.5 ml of RIPA B lysis buffer [20 mm sodium phosphate (pH 7.4), 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 5 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 250 μg/ml sodium vanadate] and lysed using a glass homogenizer. After removal of cell debris by centrifugation, the protein concentration in cell lysates was determined by the Bradford assay. Frozen tissue samples were homogenized in Tris-HCl buffer (pH 7.4) containing 0.5% NP40 and protease inhibitors (Boehringer Mannheim, Indianapolis, IN). Samples containing 150 μg of protein (for BxPC-3 and Capan-1 cells) or 300 μg (for MDAPanc-3, MDAPanc-28, and PANC-1 cells) were then added to SDS-PAGE loading buffer with 5% β-mercaptoethanol, heated for 5 min at 100°C, and loaded in a 12% gel. For HCA-7 and HCT-116 colon cancer cell lines, blots were loaded with 100 μg of protein. Electrophoretic transfer to polyvinylidene difluoride membranes was followed by immunoblotting with the anti-COX-2 or anti-COX-1 antibodies (Cayman Chemical), a monoclonal antiactin antibody (Amersham, Arlington Heights, IL), and hybridization with a secondary antibody conjugated with peroxidase (Amersham). Signal was detected by chemiluminiscence using the ECL detection system (Amersham).
RNA Purification and RT-PCR.
RNA purification from human pancreatic tumors, paired normal tissues, and cell lines was performed by the acid guanidinium thiocyanate-phenol-chloroform method (34) . The RNA was quantified by determining absorbance at 260 nm, and its quality assessed by subjecting it to electrophoresis in citric acid urea gels. Total RNA (1 μg) was converted to cDNA by incubation at 42°C for 1 h with 40 units of avian myeloblastosis virus reverse transcriptase in buffer I, 25 units of RNAse inhibitor, 0.4 mm dNTPs, and 100 ng of random primer p(dN)6. Final reaction volume was 20 μl. All reagents for cDNA synthesis were obtained from Boehringer Mannheim. The cDNA was then subjected to PCR. To obtain semiquantitative results, we optimized three parameters: cDNA concentration, number of cycles, and concentration of primers. The cDNA was diluted to 250 μl, and RT-PCR was performed using 15 μl of diluted cDNA (for COX-2) or 1 μl of a further 1:20 dilution of the already diluted cDNA (for β-actin). In both cases, the reaction mixture contained 2 units of Taq polymerase Gold (Perkin Elmer/Applied Biosystems, Foster City, CA), 0.4 mm dNTPs, and 0.5 μg of the forward (F) and reverse (R) primers in a final volume of 50 μl. Amplification of COX-2 and β-actin was performed at the same time so that the latter served as a control for efficiency of RT-PCR and amount of RNA. After a polymerase activation step (94°C for 12 min), samples were amplified for 35 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 2 min, and extension at 72°C for 3 min, followed by an additional extension step (72°C for 5 min). The primer sequences and PCR product sizes were as follows: for COX-2, 5′-TTC AAA TGA GAT TGT GGG AAA AT-3′ (F) and 5′-AGA TCA TCT CTG CCT GAG TAT CTT-3′ (R), 304 bp; and for β-actin, 5′-CGA GCG GGA AAT CGT GCG TGA CAT TAA GGA GA-3′ (F) and 5′-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3′ (R), 479 bp. The COX-2-specific primers possess <50% homology to the respective sites in the DNA sequence of COX-1 (35) . Amplified cDNAs were run on 2% agarose gels with 0.5 μg/ml ethidium bromide, and visualized under UV light. RNAs from the colonic adenocarcinoma cell lines HCA-7 and HCT-116 were used as positive and negative controls, respectively.
Cell Proliferation Assay.
The effects of sulindac sulfide (Cell Pathways, Inc., Horsham, PA) and NS398 (Cayman Chemical) on cell growth were determined using a cell proliferation assay. Both drugs were dissolved in 100% DMSO as 1000× stock solutions and then diluted with DMEM/F-12 for cell culture experiments. The final concentration of DMSO for all treatments (including controls, where no drug was added) was maintained at 0.1%. All drug solutions were prepared fresh on the day of testing. Cells were seeded at a density of 5 × 103 per well for BxPC-3 and PANC-1, 2.5 × 103 per well for MDAPanc-3, or 2 × 103 per well for Capan-1 and MDAPanc-28, in 96-well plates in DMEM/F-12 containing 10% FBS. After 24 h, fresh medium was added containing sulindac sulfide or NS398 at concentrations of 0 to 200 μm. After a 4-day incubation, the MTS assay (Promega, Madison, WI) was performed to estimate the number of viable cells according to the manufacturer’s instructions. In the case of Capan-1 cells treated with NS398, the cell number was also estimated by direct cell counting after trypan blue staining due to a pronounced change in their cell morphology. The data were analyzed by the Student’s t test.
The effect of sulindac sulfide and NS398 upon the level of COX-2 expression was also studied. BxPC-3 cells, grown to 50% confluence in 100-mm dishes, were incubated in the presence of drug for 48 h in medium containing sulindac sulfide (75 μm) or NS398 (100 μm) or medium alone (control). COX-2 protein expression was then analyzed by the Western blot assay.
RESULTS AND DISCUSSION
Expression of COX-2 Protein and mRNA in Pancreatic Adenocarcinomas.
In histologically normal human pancreas, cytoplasmic COX-2 staining was detected in islets but not in acinar or duct cells (Fig. 1A) ⇓ . Pancreatic islets have been shown to constitutively express COX-2 (36) . Expression of COX-2 protein was detected in 14 of 21 (67%) pancreatic adenocarcinomas analyzed. In these tumors, granular staining was seen in the cytoplasm of malignant cells (Fig. 1B and D–F) ⇓ . Lymphocytes and other interstitial mononuclear cells were consistently negative for COX-2. A COX-2 sequence-specific blocking peptide was used as a control for antibody specificity. In all cases analyzed, the peptide completely suppressed COX-2 staining (Fig. 1C) ⇓ .
Representative results of COX-2 immunostaining in normal human pancreas (A) and pancreatic adenocarcinomas (B–F). A, COX-2 immunoreactivity was detected in the islets of the normal pancreas. B, cytoplasmic COX-2 staining is shown in an adenocarcinoma at a low magnification. C, use of a COX-2 sequence-specific blocking peptide completely suppressed COX-2 staining in this same tumor. D–F, three pancreatic carcinomas expressing COX-2. Intense immunoreactivity can be seen in tumor cells, whereas interstitial mononuclear cells and nerve cells (as in F) were negative for COX-2.
Semiquantitative RT-PCR revealed a low level of COX-2 mRNA in pancreatic tissue from a healthy individual, consistent with the findings of O’Neill et al. (37) . The source of transcripts is likely the islets, given their constitutive expression of COX-2 (36) . We determined the level of COX-2 mRNA in tumors relative to adjacent normal pancreas. Four pancreatic carcinoma specimens and paired histologically normal pancreas were analyzed, and three of the tumors expressed significantly increased levels of COX-2 mRNA relative to their normal counterparts (Fig. 2) ⇓ . When compared to the healthy pancreas, the level of COX-2 transcript was elevated in all four carcinomas analyzed.
RT-PCR analysis of COX-2 mRNA expression in pancreatic adenocarcinomas. Total RNA was extracted from four pancreatic tumors (Lanes T) and their paired normal tissues (Lanes C), pancreatic tissue from a healthy individual (Lane NP), and the pancreatic adenocarcinoma cell line BxPC-3 (Lane Bx). β-Actin was used as a control. PCR product sizes (in bp) were 304 for COX-2 and 479 for β-actin. The DNA markers (Lane M) were 1353, 1078, 872, 603, 310, 281, 271, 234, and 194.
COX-2 mRNA levels where increased in two of four specimens of normal pancreas adjacent to tumor, relative to pancreas from a healthy individual (Fig. 2) ⇓ . The explanation for this observation is unclear, but it may be a consequence of tumor cell contamination or the induction of COX-2 expression in normal tissue by the adjacent tumor. Although COX-2 has not been detected in normal epithelial cells adjacent to colorectal (4 , 9) or esophageal (11) adenocarcinomas, we reported COX-2 staining in interstitial mononuclear cells adjacent to malignant glands in some colorectal cancers (9) .
In separate experiments, we examined COX-2 and COX-1 protein expression in human colorectal carcinomas by Western blot analysis. Similar to results in pancreatic cancer, we found that COX-2 was up-regulated in colorectal cancers relative to normal epithelia from these same cases (Fig. 3 ⇓ ; Refs. 3 ,, 4 , and 9 ). COX-1 was detected at an equivalent level in tumors and normal epithelia, consistent with its constitutive expression (4) .
Immunoblot analysis of COX-2 and COX-1 protein expression in human colorectal carcinomas and cell lines. Paired tumor and normal mucosa were analyzed, as were HCA-7 and HCT-116 colon carcinoma cell lines. HCA-7 cells constitutively express COX-2 (positive control) and HCT-116 cells are known to lack COX-2 (negative control). Up-regulation of COX-2 is seen in representative tumors relative to normal mucosa. Constitutive COX-1 is equally expressed in tumor and normal tissue.
Expression of COX-2 and COX-1 in Pancreatic Adenocarcinoma Cell Lines.
The expression of COX-2 was studied in five human pancreatic adenocarcinoma cell lines and two colon carcinoma cell lines by Western blotting, immunohistochemistry, and RT-PCR. The results of the three techniques yielded concordant results. COX-2 protein was detected by Western blotting in three of the five cell lines and the highest level of expression was found in BxPC-3 cells, followed by Capan-1 and MDAPanc-3. No COX-2 expression was detected in MDAPanc-28 or PANC-1 cells by immunoblotting, even when 300 μg of protein was loaded (Fig. 4A) ⇓ . Expression of COX-1 protein was detected only in BxPC-3 and Capan-1 cells (Fig. 4B) ⇓ . RT-PCR demonstrated that four of five pancreatic cancer cell lines expressed increased COX-2 mRNA levels when compared with normal pancreas from a healthy individual (Fig. 4B) ⇓ . BxPC-3 showed the highest level of mRNA expression, followed by Capan-1, MDAPanc-3, and MDAPanc-28. In the case of PANC-1 cells, the low level of COX-2 transcript was not increased over normal pancreas. The human colon cancer cell lines HCA-7, known to constitutively express high levels of COX-2 protein, and HCT-116, which lack COX-2 protein, were used as controls (24) . COX-2 mRNA was detected in HCA-7 but not in HCT-116 cells (Fig. 4B) ⇓ . The high level of COX-2 mRNA detected in BxPC-3 cells was similar to that found in HCA-7 cells. Although not run on the same blot, the level of COX-2 protein in BxPC-3 cells approximated that found in HCA-7 colon cancer cells (Figs. 3 ⇓ and 4A ⇓ ). Taken together, our data indicate that COX-2 is up-regulated in pancreatic and colorectal cancer cell lines and human tumors. Findings in BxPC-3 and HCA-7 cell lines for both mRNA and protein suggest that similar levels of COX-2 expression may occur in these tumor types.
COX-2 expression in human pancreatic adenocarcinoma cell lines. A, representative immunoblot analysis of COX-2 and COX-1 protein expression in five cell lines. One hundred-fifty μg of protein was loaded for BxPC-3 and Capan-1, compared to 300 μg for the remainder of the cell lines. B, RT-PCR analysis of COX-2 mRNA expression in the same five cell lines, together with pancreatic tissue from a healthy individual (Lane NP) and two colon cancer cell lines (Lanes HCA-7 and HCT-116). The PCR product sizes are the same as those in Fig. 2 ⇓ .
The level of COX-2 expression in pancreatic cancer cell lines was associated with the degree of differentiation of the tumors from which these cell lines were derived. Cell lines with the highest COX-2 levels were BxPC-3 and Capan-1, which derive from moderately well- and well-differentiated carcinomas, respectively. MDAPanc-3 derives from a moderately differentiated tumor, MDAPanc-28 from a poorly differentiated carcinoma, and PANC-1 was established from an undifferentiated tumor.
Effect of EGF on COX-2 Expression.
COX-2 is an intermediate-early response gene that has been shown to be regulated at the level of transcription (8 , 15) . One growth factor known to induce COX-2 expression in certain cell types is EGF (17 , 18) . Given the importance of the EGF/transforming growth factor-α signal transduction pathway in pancreatic cancer (38) , we determined whether COX-2 can be induced by EGF in pancreatic cancer cell lines. BxPC-3 cells were grown in presence of 50 ng/ml EGF for 5 h, and COX-2 protein expression was then analyzed by Western blotting. EGF significantly induced COX-2 expression in serum-starved cells but not in the presence of serum (Fig. 5) ⇓ . The fact that a further induction of COX-2 by EGF did not occur in cells grown with 10% FBS suggests that growth factors present in serum may be sufficient to induce a high level of COX-2 expression. In this regard, the levels of COX-2 protein in cells grown in serum-free medium containing EGF and in FBS-supplemented medium were comparable. A similar induction of COX-2 expression by EGF was observed in serum-starved Capan-1 and MDAPanc-3 cells (data not shown). In these two cell lines and in BxPC-3 cells, COX-2 protein levels were very low but still detectable in cells growing in absence of serum and EGF. These results suggest a paracrine effect by exogenous growth factor on the induction of COX-2. Pancreatic tumor cell lines have also been shown to have an autocrine transforming growth factor-α/EGF receptor loop that may contribute to COX-2 induction in absence of growth factors (38) . In MDAPanc-28 and PANC-1 cells, EGF failed to induce COX-2 expression.
Effects of EGF on COX-2 protein expression in the BxPC-3 tumor cell line. Cells grown in serum-free or supplemented (10% FBS) medium were incubated without (Lanes C) or with 50 ng/ml EGF (Lanes EGF) for 5 h, and the level of COX-2 protein was determined by Western blot analysis.
Inhibition of Cell Growth by Sulindac Sulfide and NS398.
NSAIDs have been shown to inhibit cell growth and to induce apoptosis in several tumor cell types, including colon carcinoma cells (22, 23, 24, 25, 26, 27) . To date, their effects on pancreatic cancer cell lines have not been studied. We analyzed the effects of two of these drugs, sulindac sulfide and NS398, on cell proliferation in cultured pancreatic cancer cell lines after 4 days of treatment. Sulindac sulfide, a nonselective COX inhibitor, produced a dose-dependent inhibition of growth of all five pancreatic adenocarcinoma cell lines (Fig. 6A) ⇓ . IC50s for sulindac sulfide were: ∼70 μm for BxPC-3 and Capan-1, 90 μm for PANC-1, 130 μm for MDAPanc-3, and 140 μm for MDAPanc-28 cells. The number of remaining viable cells at the highest concentrations of sulindac sulfide tested (160 and 200 μm) was markedly reduced compared to the initial cell number, suggesting that this drug may be inducing apoptosis. Studies in these cell lines are ongoing to verify induction of apoptosis; however, experiments in several colon carcinoma cell lines treated with sulindac sulfide and NS398 demonstrate a parallel inhibition of growth and induction of apoptosis (22 , 23 , 25 , 39) . In these studies, evidence of apoptosis included characteristic morphology and DNA fragmentation and degradation. Finally, in the case of MDAPanc-28 cells, a change in morphology was observed at concentrations of the drugs exceeding 80 μm with cells becoming more elongated and enlarged.
Dose-dependent inhibition of pancreatic adenocarcinoma cell growth by sulindac sulfide (A) and NS398 (B). The cell lines used were BxPC-3 (□), Capan-1 (▪), MDAPanc-3 (•), MDAPanc-28 (▴), and PANC-1 (○). Cells growing in 96-well plates were treated with either drug for 4 days, and the MTS assay was performed to determine the number of viable cells as described in “Materials and Methods.” Data points, means from six replicate wells; bars, SD. The effect of each treatment was calculated relative to vehicle (0.1% DMSO).
We failed to detect a direct association between the level of COX-2 or COX-1 expression and the extent of sulfide-induced growth inhibition, suggesting that the mechanism of the inhibitory effect of this drug on pancreatic adenocarcinoma cell growth may be independent of COX-2. However, in cell lines overexpressing COX-2, such as BxPC-3 or Capan-1, COX-2 inhibition may contribute to their growth inhibition. Recent studies in fibroblasts indicate that sulindac binds to the Ras gene product p21ras, blocking its interaction with Raf and leading to inhibition of the Ras signaling pathway and p21ras-mediated cellular processes (40) . Potentially, inhibition of the Ras pathway may contribute to the growth inhibitory effects of sulindac sulfide in pancreatic adenocarcinoma cells, explaining why cell lines expressing very low levels of COX-2, such as PANC-1, were strongly growth inhibited by this drug. Interestingly, suppression of p21ras levels by a plasmid expressing an antisense Ki-ras gene fragment has been shown to inhibit the growth of pancreatic cancer cell lines carrying Ki-ras mutations such as PANC-1 (41) . In contrast, cell lines with a wild-type Ki-ras p21 gene such as BxPC-3 were unaffected.
NS398, a selective COX-2 inhibitor, also had growth-inhibitory effects on all pancreatic cancer cell lines tested (Fig. 6B) ⇓ . A similar effect has been reported in colon and prostate carcinoma cell lines, where NS398 was shown to inhibit proliferation and to induce apoptosis (25 , 26) . Except in Capan-1 cells, the growth-inhibitory effects of NS398 were less pronounced than those produced by sulindac sulfide at the same concentrations. NS398 induced a change in cell morphology in all cell lines tested except BxPC-3. Cells became more elongated and enlarged and developed cytoplasmic projections, suggesting an effect of differentiation. These changes were very pronounced in Capan-1 cells. As with sulindac sulfide, no clear relationship was found between the level of COX-2 expression and the extent of growth inhibition by NS398, again suggesting that the effects of NSAIDs on pancreatic cancer cell growth could be mediated, at least in part, by a COX-2-independent pathway.
We also examined the effect of sulindac sulfide and NS398 on COX-2 protein expression by Western blot assay. We found that both drugs induced COX-2 expression in BxPC-3 cells and that the selective COX-2 inhibitor, NS398, produced a greater induction than did the nonselective drug (Fig. 7) ⇓ . This result is consistent with the data of Meade et al. (42) , who showed that NSAIDs induced COX-2 expression in mammary epithelial cells and in colon carcinoma cell lines by increasing COX-2 transcription. Specifically, a region of the COX-2 promoter was identified that contains a peroxisome proliferator response element that appears to be responsible for COX-2 induction by these agents (42) . Meade et al. (40) found no effect of NSAIDs on the level of COX-1 expression. Greater induction of COX-2 by NS398 versus sulindac sulfide was also reported in the CaCo-2 colon cancer cell line (42) . Because inhibition of COX-2 enzymatic activity is likely related to the antineoplastic effects of these drugs, the clinical significance of COX-2 protein induction by these agents is unclear.
Effect of sulindac sulfide or NS398 on COX-2 protein expression in the human pancreatic adenocarcinoma cell line BxPC-3. Cells were incubated with sulindac sulfide (75 μm), NS398 (100 μm), or medium alone (control) for 48 h, and the level of COX-2 was analyzed by the Western blot assay. COX-2 expression was induced by both drugs and a greater degree of induction was seen with the selective COX-2 inhibitor, NS398.
Contradictory reports exist regarding the mechanism responsible for the growth inhibitory properties of specific COX-2 inhibitors. Sheng et al. (24) evaluated the effects of SC58635, a selective COX-2 inhibitor, on the colon cancer cell lines HCA-7, which constitutively expresses COX-2, and HCT-116, which lack detectable COX-2 expression. SC58635 was shown to inhibit HCA-7 but not HCT-116 colony formation and growth of tumor implants in nude mice, suggesting a direct link between COX-2 inhibition and antitumor effect. On the contrary, Elder et al. (25) found that NS398 inhibited cell growth and induced apoptosis in both HT-29 colon carcinoma cells, which express COX-2, and in S/KS cells, which lack detectable COX-2 expression. The authors concluded that NS398’s effects can be independent of COX-2 inhibition. Of note, HCT-116 and S/KS cell lines were reported to lack COX-2 based on Western blots loaded with 100 μg of protein or 1 × 106 cells, respectively. This amount of protein may not be sufficient to allow detection of COX-2 in some cell lines. For example, a COX-2 band was apparent in MDAPanc-3 cells only after loading 300 μg of protein (corresponding to ∼3 × 106 cells). Furthermore, more sensitive methods such as RT-PCR can detect COX-2 expression in cell lines lacking detectable COX-2 protein levels, as shown for MDAPanc-28 and PANC-1 in this study. It is important to note that NSAID-induced growth inhibition and apoptosis in colon carcinoma cell lines was not reversed by addition of prostaglandin E2, the major COX product produced by colonic tumors (39 , 43) . Chan et al. (43) observed that sulindac sulfide and indomethacin treatment of colon carcinoma cells produced an elevation of arachidonic acid, the precursor of prostaglandins, which stimulated the conversion of sphingomyelin to ceramide, a potent inducer of apoptosis. These results suggest a COX-related mechanism for the chemopreventive efficacy of NSAIDs and support the ability of these compounds to reduce tumor formation in animal models systems.
In conclusion, we have shown that COX-2 levels are frequently elevated in human pancreatic adenocarcinomas and cell lines derived from such tumors. In addition, EGF was found to induce COX-2 expression in several of these cell lines in the absence of serum. We demonstrate for the first time that nonselective (sulindac sulfide) and selective COX inhibitors (NS398) have antiproliferative effects on pancreatic cancer cells that were not predicted by the level of COX-2 expression. These drugs also induced the expression of their target enzyme, COX-2. The up-regulation of COX-2 in pancreatic cancers and the ability of NSAIDs to inhibit the growth of cell lines derived from these tumors is consistent with results in colon cancers and suggests that NSAIDs may also be effective chemopreventive agents in this malignancy.
Acknowledgments
We thank Dr. Karen Cleary of the Division of Pathology and Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, for her review of the immunostaining results. We also appreciate the very capable secretarial support provided by Rebecca Russell.
Footnotes
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 Supported by NIH Grant CA 70759; NIH Cancer Center Support Grant CA 16672; and a Human Cancer Genetics grant (The University of Texas M. D. Anderson Cancer Center) and a Career Development Award from the American Cancer Society (both to F. A. S.). M. A. M. was supported by a Rotary Foundation Research Fellowship, and M. S-A. was a fellowship recipient from the Autonomous Government of Catalonia (Spain).
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↵2 These two authors contributed equally to this work.
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↵3 To whom requests for reprints should be addressed, at Department of Gastrointestinal Medical Oncology and Digestive Diseases, Box 78, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-2828; Fax: (713) 792-5010; E-mail: fsinicro{at}notes.mdacc.tmc.edu
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↵4 The abbreviations used are: COX, cyclooxygenase; EGF, epidermal growth factor; NSAID, nonsteroidal anti-inflammatory drug; FBS, fetal bovine serum; RT-PCR, reverse transcription-PCR.
- Received April 6, 1999.
- Accepted July 8, 1999.
- ©1999 American Association for Cancer Research.
References
Volume 59, Issue 17
