Leukotriene B4 Receptor Antagonist LY293111 Inhibits Proliferation and Induces Apoptosis in Human Pancreatic Cancer Cells1

3232 Vol. 8, 3232–3242, October 2002 Clinical Cancer Research

Wei-Gang Tong, Xian-Zhong Ding, Rene Hennig lines, as indicated by morphology, TUNEL assay, and poly- Richard C. Witt, Jens Standop, Parviz M. Pour, (ADP-ribose) polymerase cleavage. In studies using AsPC-1 and Thomas E. Adrian2 and HPAC cell xenografts in athymic mice, LY293111 treatment markedly inhibited tumor growth over a 24-day treat- Department of Surgery, Northwestern University Medical School, ment period, as measured by both tumor volume and tumor Chicago, Illinois 60611 [W-G. T., X-Z. D., R. H., R. C. W., T. E. A.], and The Eppley Institute for Research in Cancer and Allied Diseases, weight. In situ tissue TUNEL assay showed massive apo- University of Nebraska Medical Center, Omaha, Nebraska 68198 ptosis in LY293111-treated tumor tissues. [J. S., P. M. P.] Conclusions: LTB4 can directly regulate the growth of human pancreatic cancer cells and control their survival. Additional studies will clarify the underlying mechanisms of ABSTRACT LTB4-regulated pancreatic cancer cell growth and apopto- Purpose: The effects of leukotriene (LT) B4 and its sis. LTB4 receptor blockade and inhibition of the down- receptor antagonist LY293111 on proliferation and apopto- stream signal pathway are likely to be valuable for the sis of human pancreatic cancer cells were investigated, both treatment of human pancreatic cancer. in vitro and in vivo. Experimental Design: Six human pancreatic cancer cell INTRODUCTION lines (MiaPaCa-2, HPAC, Capan-1, Capan-2, PANC-1, and AsPC-1) were used. Expression of LTB4 receptors, BLT1 Pancreatic adenocarcinoma is characterized by poor prog- and BLT2, was measured by reverse transcription-PCR. nosis and lack of response to conventional therapy. Although its 3 incidence is lower than that of many tumor types, it ranks as the Cell proliferation was measured by [methyl- H]thymidine fourth leading cause of cancer death for both men (after lung, incorporation and cell number counting. Extracellular prostate, and colon cancer) and women (after lung, breast, and signal-regulated kinase (ERK) 1/2 activation was measured colon cancer) in the United States (1, 2). The 5-year survival by Western blotting. Apoptosis was assessed by morphology, rate for it is Ͻ2%, and the median survival after diagnosis is Ͻ6 terminal deoxynucleotidyl transferase-mediated nick end la- months, hence pancreatic cancer poses one of the greatest chal- beling (TUNEL) assay, and poly(ADP-ribose) polymerase lenges in oncology (3, 4). cleavage. The effect of LY293111 on growth of AsPC-1 and Accumulating evidence has linked pancreatic cancer HPAC cell xenografts was assessed in BALB/c nu/nu athy- growth with dietary fat, especially intake of ␻-6 PUFAs,3 mic mice. such Results: Both LTB4 receptor types were found to be as linoleic acid and arachidonic acid, which enhance tumorigen- expressed in human pancreatic cancer cells. The LTB4 re- esis through the production of eicosanoids (5, 6). On the other ␻ ceptor antagonist LY293111 caused both time- and concen- hand, -3 PUFAs such as eicosapentaenoic acid and docosa- tration-dependent inhibition of proliferation of all six hu- hexaenoic acid, which are found in high concentrations in fish ␻ man pancreatic cancer cell lines studied. In contrast, LTB4 oils, have anticancer effects (5, 6). It is widely held that -3 stimulated proliferation of these cell lines and induced PUFAs inhibit cancer cell growth and induce apoptosis by ERK1/2 phosphorylation. The growth-stimulatory effect prevention of lipid peroxidation (7). There are two major met- and ERK1/2 phosphorylation induced by LTB4 were inhib- abolic pathways of arachidonic acid, the COX pathway and the ited by LY293111. Coincident with growth inhibition, LOX pathway. Early investigations into the role of arachidonic LY293111 induced apoptosis in these pancreatic cancer cell acid metabolism in cancer have focused mainly on the COX pathway, particularly that involving the inducible COX-2 enzyme, because of the epidemiological observation that the incidence of colonic cancer was significantly reduced in regular users of aspirin and other nonsteroidal anti-inflammatory drugs Received 3/13/02; revised 5/16/02; accepted 6/14/02. (8). However, several groups have shown that both the 5-LOX 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. 1 Supported by grants from the National Cancer Institute Specialized 3 The abbreviations used are: PUFA, polyunsaturated fatty acid; ERK, Programs of Research Excellence program (P50 CA72712) and the extracellular signal-regulated kinase; TUNEL, terminal deoxynucleoti- Lustgarten Foundation for Pancreatic Cancer Research. dyl transferase-mediated nick end labeling; PARP, poly(ADP-ribose) 2 To whom requests for reprints should be addressed, at Department of polymerase; COX, cyclooxygenase; LOX, lipoxygenase; HETE, Surgery, Northwestern University Medical School, 303 East Chicago hydroxyeicosatetraenoic acid; LT, leukotriene; TdT, terminal de- Avenue, Tarry 4-711, Chicago, IL 60611. Phone: (312) 503-3489; Fax: oxynucleotidyltransferase; NDGA, nordihydroguaiaretic acid; BLT1, (312) 503-3491; E-mail: [email protected]. LTB4 receptor 1; BLT2, LTB4 receptor 2.

and 12-LOX pathways also play important roles in promoting tumor growth (9–11). Previous studies in our laboratory revealed that both 5-LOX and 12-LOX mRNA and protein are expressed in human pancreatic cancer cells but not in normal human pancreatic ductal cells (12). The products of 5-LOX and 12-LOX, 5-HETE and 12-HETE, stimulate growth of human pancreatic cancer cells through activation of the ERK1/2 and Akt/protein kinase B pathways (13). Ding et al. (12, 14) have also shown that the general LOX inhibitor NDGA, the 5-LOX Fig. 1 Expression of LTB4 receptor (BLT1 and BLT2) mRNA in inhibitor Rev-5901, and the 12-LOX inhibitor baicalein inhibit human pancreatic cancer cells. Total RNA was isolated from human pancreatic cancer cell proliferation and induce apoptosis. MiaPaCa-2 and AsPC-1 pancreatic cancer cells as well as leukocytes. LTs constitute a class of potent biological mediators of The RNA was reverse transcribed and then amplified by PCR using inflammation and anaphylaxis. Their biosynthesis from 5-LOX specific primers. The PCR products were separated on a 1% agarose gel with ethidium bromide and visualized under UV light. The PCR product is catalyzed by oxygenation of arachidonic acid in granulocytes, for BLT1 is 830 bp, and the PCR product for BLT2 is 312 bp. A monocytes, and mast cells. LTB4 is produced from LTA4 representative gel from three separate experiments is shown. through the LTA4 hydrolase, whereas LTC4, LTD4, and LTE4, which are formerly known as slow-reacting substance of anaphylaxis, are produced through a different pathway. LTC4 is first produced from LTA4 by LTC4 synthetase, and LTC4 can were from PharMingen (San Diego, CA). The DeadEnd Color- be further converted in turn to LTD4 and LTE4 by the succes- imetric Apoptosis Detection System for tissue sections was from sive elimination of glutamic acid and glycine residues (15, 16). Promega (Madison, WI). LTB4 and a selective LTB4 receptor LTB4 has been widely implicated in the pathogenesis of several antagonist, U75302, were from Cayman Chemicals (Ann Arbor, inflammatory diseases, such as asthma, psoriasis, rheumatoid MI). The selective LTB4 receptor antagonist LY293111 was arthritis, and inflammatory bowel disease (17). Although LTB4 provided by Eli Lilly and Co. (Indianapolis, IN). The selective is a final product of the 5-LOX pathway of arachidonic acid LTD4 antagonist LY171883 was purchased from Biomol Re- 3 metabolism, its function in tumorigenesis has not been fully search Laboratories (Plymouth Meeting, PA). [methyl- H]Thy- investigated. However, several pieces of evidence suggest that midine was from Amersham (Arlington Heights, IL). The 202 204 LTB4 may be as important as 5-HETE and 12-HETE in prolif- monoclonal mouse phospho-ERK1/2 (Thr /Tyr ) antibody eration of cancer cells. Earashi et al. (18) found that LTB4 could and the monoclonal mouse ERK1/2 antibody were purchased reverse the inhibitory effect of NDGA on proliferation of the from New England BioLabs (Beverly, MA). The monoclonal breast cancer cell line MDA-MB-231, whereas eicosapentaenoic mouse antihuman PARP antibody was from Biomol Research acid and docosahexaenoic acid suppressed MDA-MB-231 cell Laboratories. All other chemicals were purchased from Sigma. growth and reduced the secretion of prostaglandin and LTB4. Human Pancreatic Cancer Cell Lines and Cell Culture. Okano et al. (19) showed that human melanoma cells express Six human pancreatic cancer cell lines were used: MiaPaCa-2 LTA4 hydrolase and generate LTB4 from LTA4. Bittner et al. and HPAC (poorly differentiated), Capan-1 and Capan-2 (well (20)showedthatLTB4playedanimportantroleinglucocorticoid- differentiated), and PANC-1 and AsPC-1 (pleomorphic). These induced inhibition of lymphoma growth, whereas dexametha- cell lines were purchased from American Type Culture Collec- sone treatment almost completely inhibited LT production. A tion (Manassas, VA). HPAC and MiaPaCa-2 were grown in study by El-Hakim et al. (21) revealed a 10–30-fold increase in DMEM, PANC-1 and AsPC-1 were grown in MEM, and Ca- the level of LTB4 in oral squamous cell cancers compared with pan-1 and Capan-2 were grown in McCoy’s 5A medium. Cells control normal tissues, suggesting a possible role of LTB4 in the were plated as monolayers in the medium supplemented with pathogenesis of head and neck carcinoma. Cristiana et al. (22) 10% fetal bovine serum in a humidified atmosphere of 95% O2 found that LTB4 stimulated the proliferation of colon cancer and 5% CO2 at 37°C. The cells were regularly seeded into 2 cell lines HT-29 and HCT-15 in a time- and concentration- 75-cm flasks with media changes every other day. For exper- dependent manner and that LTB4 receptors are expressed in iments, cells were grown to 70% confluence, digested with colonic epithelial cells. trypsin-EDTA, and plated in either 12- or 24-well plates at a Based on the above evidence, the current study was aimed concentration of 50,000 cells/ml as appropriate. at investigating the importance of the LTB4 metabolic pathway Reverse Transcription-PCR Analysis of BLT1 and in pancreatic cancer cell growth and the effect of a selective BLT2 mRNA Expression in Pancreatic Cancer Cells. RNA LTB4 receptor antagonist, LY293111, on pancreatic cancer cell was isolated from MiaPaCa-2 and AsPC-1 cells and leukocytes proliferation and apoptosis both in vitro and in vivo. separated from human whole blood as described previously using RNazol B (TEL-TEST Inc., Friendswood, TX). RNA was reversed transcribed into cDNA using random hexamer and MATERIALS AND METHODS reverse transcriptase according to the manufacturer’s protocol Materials. DMEM, MEM, McCoy’s 5A medium, peni- (Perkin-Elmer GeneAmp kit; Perkin-Elmer, Foster City, CA). cillin-streptomycin solution, trypsin-EDTA solution, proteinase After the reverse transcription reaction, cDNA was amplified K, propidium iodide, and RNase A were purchased from Sigma to determine both BLT1 and BLT2 mRNA expression using (St. Louis, MO). Fetal bovine serum was from Atlanta Biologi- their specific primers. Primers for BLT1 are 5Ј-CACTGCT- cals (Norcross, GA). The APO-BRDU kits for TUNEL assay CCCTTTTTCCTTCA-3Ј (forward) and 5Ј-CTAGTTCAG-

Fig. 2 Effects of different concentrations of LY293111 on proliferation of (A) MiaPaCa-2 and (B) AsPC-1 human pancreatic cancer cells after 24 h, as measured by [methyl-3H]thymidine incorporation. Time course effects of 500 nM LY293111 on proliferation of (C) MiaPaCa-2 and (D) AsPC-1 human pancreatic cancer cells after 6, 12, and 24 h. Results are expressed as a percent- ;P Ͻ 0.05 compared with control ,ء .age of control P Ͻ ,ءءء ;P Ͻ 0.01 compared with control ,ءء 0.001 compared with control. Data represent results from four separate experiments.

Table 1 Concentration-dependent growth inhibition induced by LY293111 in human pancreatic cancer cells at 24 h LY293111 (% control, mean Ϯ SE)

Cells 0 M 62.5 nM 125 nM 250 nM 500 nM 1000 nM HPAC 100 103.6 Ϯ 8.9 97.6 Ϯ 4.5 49.3 Ϯ 12.5a 10.3 Ϯ 4.8b 2.3 Ϯ 0.8b Capan-1 100 99.1 Ϯ 6.7 86.9 Ϯ 8.5 56.4 Ϯ 8.8a 12.7 Ϯ 4.2c 0.2 Ϯ 0.1b Capan-2 100 95.4 Ϯ 6.9 87.3 Ϯ 9.8 51.2 Ϯ 7.9a 15.8 Ϯ 2.5c 8.9 Ϯ 3.3b PANC-1 100 89.4 Ϯ 10.3 75.2 Ϯ 11.2a 43.5 Ϯ 11.2a 12.4 Ϯ 3.7b 5.6 Ϯ 2.4b a P Ͻ 0.05. b P Ͻ 0.001. c P Ͻ 0.01.

TTCGTTTAACTT-3Ј (reverse). Primers for BLT2 are 5Ј- Cell Proliferation Assay. Cells were regularly seeded AGCCTGGAGACTCTGACCGCTTTCG-3Ј (forward) and into 12-well microplates and incubated at 37°C for 24 h. Cells 5Ј-GACGTAGAGCACCGGGTTGACGCTA-3Ј (reverse). The were then cultured in serum-free medium for another 24 h and PCR products for BLT1 and BLT2 are 830 and 321 bp, respec- treated in fresh serum-free medium with or without 250 nM tively. The PCR profile was 94°C for 30 s, 58°C (for BLT1) or LY293111 for 24, 48, and 72 h. At the end of each time period, 68°C (for BLT2) for 30 s, and 72°C for 30 s for 30 cycles. The the cells were trypsinized to produce a single cell suspension, final PCR products were separated on a 1% agarose gel with and the cell number in each well was determined using a ethidium bromide and visualized under UV light. Z1-Coulter Counter (Luton, Bedfordsire, United Kingdom). DNA Synthesis by [methyl-3H]Thymidine Incorpora- Morphological Changes Using Light Microscopy. tion. Cells were plated in either 12- or 24-well plates at a Pancreatic cancer cells grown in 75-cm2 flasks were treated with concentration of 50,000 cells/well. After reaching 50% conflu- different concentrations of LY293111 for different periods of ence, they were incubated in serum-free medium for 24 h, which time. Cells were then viewed using light microscopy, and digital was then replaced with fresh serum-free medium with or with- images were taken with a Kodak DC120 zoom digital camera out the appropriate treatments. After the required period of (Eastman Kodak Co., Rochester, NY). culture, cellular DNA synthesis was assayed by adding 0.5␮Ci TUNEL Assay. Cells were treated with LY293111 in [methyl-3H]thymidine/well and incubating cells for another 2 h. serum-free medium for the appropriate period of time and then Then the cells were washed twice with PBS, fixed with 10% digested with trypsin-EDTA, washed twice with ice-cold PBS, trichloroacetic acid for 30 min, and solubilized by adding 250 ␮l and fixed in 10% paraformaldehyde on ice for 20 min. Cells of 0.4 M NaOH to each well. Radioactivity, indicating incorpo- were then washed with PBS, permeabilized with 70% ethanol ration of [methyl-3H]thymidine into DNA, was measured by for at least 4 h, washed again with PBS, and incubated with 2.5 adding scintillation mixture and counting on a scintillation units of TdT enzyme and 100 pmol of bromo-dUTP in DNA counter (LKB RackBeta; Wallac, Turku, Finland). labeling solution for1hat37°C. Cells were then rinsed twice in

Table 2 Time-dependent growth inhibition induced by 250 nM LY293111 in human pancreatic cancer cells LY293111 (% control, mean Ϯ SE) Cells 0 6 h 12 h 24 h HPAC 100 67.5 Ϯ 12.4a 35.7 Ϯ 9.8b 12.4 Ϯ 4.6c Capan-1 100 70.5 Ϯ 11.3a 44.5 Ϯ 7.8b 15.7 Ϯ 7.7b Capan-2 100 57.8 Ϯ 9.8b 32.1 Ϯ 11.9b 10.9 Ϯ 5.4c PANC-1 100 60.2 Ϯ 15.4a 48.9 Ϯ 10.6b 13.6 Ϯ 3.8c a P Ͻ 0.05. b P Ͻ 0.01. c P Ͻ 0.001.

PBS and resuspended in 0.1 ml of fluorescein labeled anti- bromodeoxyuridine antibody solution in the dark for 30 min. Then, 0.5 ml of propidium iodide/RNase A solution was added, and the cells were analyzed by flow cytometry at 488 nm excitation. Western Blotting. For analysis of ERK1/2 phosphorylation, cells were either treated with LTB4 and LY293111 alone or pretreated with LY293111 for 2 h and then treated with LTB4 Fig. 3 Time course effects of LY293111 on proliferation of (A) for 10 min. For measurement of PARP cleavage, cells were MiaPaCa-2 and (B) AsPC-1 human pancreatic cancer cells, as measured treated with 250 nM LY293111 for 6, 12, 24, and 48 h. After by cell number. Cells cultured in triplicate in 12-well plates in serum- treatment, cells were scraped into lysis buffer [20 mM Tris-HCl free medium were treated with 500 nM LY293111 for 24, 48, and 72 h. The cells were then trypsinized at each time point, and the cell number (pH 7.4), 2 mM sodium vanadate, 1.0 mM sodium fluoride, 100 ,ء .was counted in each well. Results are expressed as cell numbers/well ;P Ͻ 0.01 compared with control ,ءء ;mM NaCl, 2.0 mM phosphate substrate, 1% NP40, 0.5% sodium P Ͻ 0.05 compared with control P Ͻ 0.001 compared with control. Data represent results from four ,ءءء deoxycholate, 25 ␮g/ml aprotinin, 25 ␮g/ml leupeptin, 25.0 ␮g/ml pepstatin, 2.0 mM EDTA, and 2.0 mM EGTA] at 4°C. separate experiments. Cell lysates were clarified by microcentrifugation at 12,000 ϫ g after incubation on ice for 25 min. The supernatants were recovered, and their protein concentrations were measured using Bio-Rad protein assay reagent. Equivalent amounts of cell lysate euthanized, the tumors were dissected carefully, and tumor protein (30 ␮g) were resolved by 15% SDS-PAGE. Proteins weights were measured. were transferred to nitrocellulose membranes by electroblotting In Situ Tissue TUNEL Assay. Paraffin-embedded tissue using a Bio-Rad semidry transfer blotting apparatus. Mem- sections were deparaffinized in xylene for 5 min, rehydrated in branes were subsequently blocked in Tris-buffered saline and gradient ethanol, and fixed with 4% paraformaldehyde. After 0.1% Tween 20 and then incubated with appropriate antibodies permeabilizing tissues with proteinase K, they were refixed with (phospho-ERK1/2 and ERK1/2, 1:2000 dilution; PARP, 1:1000 4% paraformaldehyde. The sections were then incubated with dilution) in 5% nonfat milk overnight at 4°C. The membrane- TdT enzyme and biotinylated nucleotide mix after endogenous bound protein-antibody complexes were then incubated with peroxidase activity was blocked with 0.3% hydrogen peroxide. Ј horseradish peroxidase-conjugated goat antimouse secondary Finally, the sections were incubated with 3,3 -diaminobenzidine antibodies at a dilution of 1:2000 for2hatroom temperature. and chromogen for 10 min and viewed microscopically with The membranes were then detected by chemiluminescence, and digital images recorded as described above. light emission was captured on Kodak X-ray films (Eastman Statistical Analysis. Data were analyzed by ANOVA Kodak Co.). with Dunnett’s or Bonferroni’s corrections for multiple compar- Animal Studies. Athymic nude mice (BALB/c nu/nu, isons, as appropriate. This analysis was performed with the 5-week-old females) were purchased from the Frederick Cancer Prism software package (GraphPad, San Diego, CA). Data were Ϯ Research and Development Center (Frederick, MD). Mice were expressed as mean SE. acclimatized to the animal facility for 1 week before receiving xenografts. Three million AsPC-1 or HPAC human pancreatic RESULTS cancer cells were injected into the flanks of nude mice, and once Expression of BLT1 and BLT2 mRNA in Pancreatic visible tumors were evidenced 4–5 days after injection, the Cancer Cells. Two G protein-coupled receptors for LTB4 animals were divided equally into two groups (6 animals/group) have been identified and cloned thus far, namely, BLT1 and and treated with LY293111 (250 mg/kg/day) or control vehicle BLT2. They have been shown to be different in their binding (sterile PBS, PBS) by daily gavage. Animal weight and tumor profile to LTB4 and also in their tissue distributions. BLT1, a size were recorded every 3 days. The formula used for tumor high-affinity LTB4 receptor, is exclusively expressed in leuko- volume is as follows: volume ϭ (length) ϫ (width) ϫ (length ϩ cytes, with a little expression in thymus and spleen (23, 24). width/2) ϫ 0.526. After 24 days of treatment, the animals were BLT2, a low-affinity receptor for LTB4, is expressed relatively

Fig. 4 Effects of a selective LTB4 receptor antagonist, U75302, on thymidine incorporation in (A) MiaPaCa-2 and (B) AsPC-1 pancreatic cancer cells. Effects of a selective LTD4 antagonist, LY171883, on thymidine incorporation in (C) MiaPaCa-2 and (D) AsPC-1 cells. Results are P Ͻ 0.05 ,ء .expressed as a percentage of control P Ͻ 0.01 compared ,ءء ;compared with control -P Ͻ 0.001 compared with con ,ءءء ;with control trol. Data represent results from four separate experiments.

Fig. 5 Concentration-dependent effects of LTB4 on thymidine incorporation in (A) MiaPaCa-2 and (B) AsPC-1 human pancreatic cancer cells. Time- course effects of 100 nM LTB4 on thymidine incorporation in (C) MiaPaCa-2 and (D) AsPC-1 human pancreatic cancer cells. Results are ex- P Ͻ 0.05 ,ء .pressed as a percentage of control P Ͻ 0.01 compared ,ءء ;compared with control -P Ͻ 0.001 compared with con ,ءءء ;with control trol. Data represent results from four separate experiments.

ubiquitously, with the highest expression in spleen, followed by kocytes separated from human blood, which serve as a positive ovary, liver, and leukocytes (25, 26). BLT2 open reading frame control (Fig. 1). is present in the promoter region of BLT1, thus weaving these The Selective LTB4 Receptor Antagonist, LY293111, two receptors tightly at the both genomic and functional levels Inhibits Proliferation of Human Pancreatic Cancer Cells. (25). Our results with reverse transcription-PCR have shown The selective LTB4 receptor antagonist LY293111 caused both that the mRNA for both types of LTB4 receptors is expressed in a concentration-dependent [Fig. 2, A and B; F(5,30) ϭ 77.30, MiaPaCa-2 and AsPC-1 pancreatic cancer cells as well as leu- P Ͻ 0.0001 for MiaPaCa-2; F(5,30) ϭ 53.73, P Ͻ 0.0001 for

Table 3 Concentration-dependent growth stimulation induced by LTB4 in human pancreatic cancer cells at 24 h LTB4 (% control, mean Ϯ SE) 0 Cells nM 10 nM 100 nM 500 nM HPAC 100 123.8 Ϯ 5.7a 209.4 Ϯ 11.3b 312.3 Ϯ 11.6c Capan-1 100 114.1 Ϯ 8.9 198.6 Ϯ 14.6b 299.6 Ϯ 8.9c Capan-2 100 136.4 Ϯ 10.5a 226.7 Ϯ 12.3b 313.7 Ϯ 14.6c PANC-1 100 106.7 Ϯ 3.7 189.6 Ϯ 13.7a 289.7 Ϯ 11.7c a P Ͻ 0.05. b P Ͻ 0.01. c P Ͻ 0.001.

AsPC-1] and time-dependent [Fig. 2, C and D; F(3,20) ϭ 331.1, P Ͻ 0.0001 for MiaPaCa-2; F(3,20) ϭ 328.8, P Ͻ 0.0001 for AsPC-1] inhibition of thymidine incorporation in both MiaPaCa-2 and AsPC-1 human pancreatic cancer cells. Similar effects were seen in the other cell lines studied (Tables 1 and 2). LY293111 inhibited proliferation by at least 50% at a concentration of 250 nM and by Ͼ95% at 1000 nM at 24 h. The inhibition of DNA synthesis and proliferation was followed by a decrease in cell number over a 3-day period with 250 nM LY293111. The cell number of untreated cells doubled about Fig. 6 Effect of LY293111 on LTB4-stimulated proliferation of (A) every 24 h, whereas the cell number of treated cells remained MiaPaCa-2 and (B) AsPC-1 human pancreatic cancer cells. Cells cul- unchanged or decreased slightly (Fig. 3, A and B). To confirm tured in triplicate in 12-well plates in serum-free medium were treated the involvement of LTB4 receptors in mediating the effect of with either 100 nM LTB4 or 250 nM LY293111 or a combination of both ء LY293111 on human pancreatic cancer cell proliferation, we for 24 h. Results are expressed as a percentage of control. , compared with control; #, compared with LTB4 alone. Data represent results from also treated the cells with another selective LTB4 receptor three separate experiments. antagonist, U75302, and with a selective LTD4 antagonist, LY171883. U75302 inhibited the proliferation of both MiaPaCa-2 and AsPC-1 pancreatic cancer cells but did so less potently than LY293111 as expected from the lower receptor also measured to confirm that ERK1/2 protein content did not affinity of this drug (Fig. 4, A and B). Inhibition of proliferation change after treatment. These results suggested that ERK1/2 of approximately 50% was seen with 5 ␮M U75302. In contrast, phosphorylation is involved in LTB4-stimulated pancreatic can- the selective LTD4 antagonist LY171883 had no effect on the cer cell proliferation. proliferation of either of the human pancreatic cancer cell lines LY293111 Induces Apoptosis in Human Pancreatic tested (Fig. 4, C and D). Cancer Cells. LY293111 treatment induced dramatic mor- LTB4 Stimulates Human Pancreatic Cancer Cell Pro- phological changes in human pancreatic cancer cells within 6 h liferation. Because blocking LTB4 receptor inhibits prolifer- after commencing treatment. Over time, the treated cells became ation of pancreatic cancer cells, we hypothesized that LTB4 may rounded and exhibited membrane blebbing, chromatin conden- be a growth factor for human pancreatic cancer growth. The sation, and nuclear fragmentation and finally detached from the effects of LTB4 on proliferation of human pancreatic cancer microplate (Fig. 8). These morphological changes have been cells were investigated. LTB4 stimulated proliferation in both a previously interpreted as reflecting apoptosis. concentration-dependent [Fig. 5, A and B; F(3,11) ϭ 45.57, P Ͻ LY293111-induced apoptosis was also evaluated by the 0.0001 for MiaPaCa-2; F(3,11) ϭ 63.99, P Ͻ 0.0001 for TUNEL assay, in which DNA strand breaks can be detected by AsPC-1] and time-dependent [Fig. 5, C and D; F(3,11) ϭ 88.34, the incorporation of fluorescence-labeled dUTP in the presence P Ͻ 0.0001 for MiaPaCa-2; F(3,11) ϭ 129.4, P Ͻ 0.0001 for of TdT enzyme. Treatment of both MiaPaCa-2 and AsPC-1 cells AsPC-1] manner in MiaPaCa-2, AsPC-1, and all of the other with 250 and 500 nM LY293111 for 24 h greatly increased pancreatic cancer cell lines studied (Table 3). LY293111 was apoptosis from 1.5% to 17.4% and 59.3% in MiaPaCa-2 cells able to inhibit the stimulatory effect of LTB4 on human pan- and from 0.4% to 8.9% and 48.3% in AsPC-1 cells, respectively creatic cancer cell proliferation when cells were treated with a (Fig. 9). Again, similar results were obtained in the other pan- combination of 100 nM LTB4 and 250 nM LY293111 (Fig. 6, A creatic cancer cell lines. and B). To further investigate LY293111-induced apoptosis, cleav- LTB4 induced marked phosphorylation of ERK1/2 after 10 age of the caspase-3 substrate PARP was measured by Western min of treatment. LTB4-induced ERK1/2 phosphorylation was blotting. Cleavage of the Mr 116,000 PARP protein into a Mr inhibited by pretreatment with LY293111 in both MiaPaCa-2 85,000 fragment by activated caspase-3 is a hallmark of apo- and AsPC-1 pancreatic cancer cells (Fig. 7). Total ERK1/2 was ptosis. LY293111 induced time-dependent cleavage of PARP

Fig. 7 Effect of LTB4 and the LTB4 receptor antagonist on phosphorylation of ERK1/2 in human pancreatic cancer cells. MiaPaCa-2 and AsPC-1 cells were serum-starved for 24 h and then treated with 250 nM LY293111 alone for2hor100nM LTB4 alone for 10 min or pretreated with LY293111 for 2 h and then treated with LTB4 for 10 min. Phosphorylation of ERK1/2 was detected by Western blotting using phospho-ERK1/2 antibody (top panels). The same membranes were reprobed with ERK1/2 antibody to show equal expression of ERK1/2 protein among all samples (bottom panels).

Fig. 8 Morphological changes induced by 250 nM LY293111 in MiaPaCa-2 cells (A and B) and AsPC-1 (C and D) human pancreatic cancer cells. Cells were treated with 250 nM LY293111 in serum-free medium for 12 h.

after 6, 12, 24, and 48 h in both MiaPaCa-2 and AsPC-1 cells LY293111-treated animals (Fig. 12). This indicates that (Fig. 10). LY293111 inhibits tumor growth in vivo by inducing tumor cell LY293111 Inhibits Growth of AsPC-1 and HPAC apoptosis. Cancer Cell Xenografts and Induces Apoptosis in Vivo. LY293111 not only inhibited proliferation of human pancreatic cancer cells in vitro but also inhibited growth of human pancre- DISCUSSION atic cancer xenografts in athymic mice. In experiments with Our previous studies and those of others have confirmed AsPC-1 and HPAC human pancreatic cancer cells, LY293111 the importance of the LOX metabolic pathways of arachidonic markedly inhibited tumor growth as measured by both tumor acid in the proliferation of cancer cells (9–14). Both 5-LOX volume (Fig. 11, A and B) and tumor weight (Fig. 11, C and D). mRNA and 12-LOX mRNA are expressed in pancreatic cancer Furthermore, 50% of control animals showed s.c. metastases, cells, but not in normal pancreatic ductal cells. 5-HETE and but none were seen in the treated animals. There was no signif- 12-HETE stimulate human pancreatic cancer cell proliferation, icant difference in body weight between the control and and blockade of either pathway using specific enzyme inhibitors LY293111-treated animals throughout the entire treatment pe- blocks proliferation and induces apoptosis. The effects of LTs riod (Fig. 11, E and F), although the untreated control animals such as LTB4, LTC4, or LTD4 have not previously been inves- were less active because of large tumor burden. tigated in pancreatic cancer cells. We hypothesized that one of Tissue sections from tumor xenografts were subjected to in these active metabolites derived from the 5-LOX pathway was situ tissue TUNEL assay for the detection of apoptosis. In the likely to be important in the proliferation of pancreatic cancer control tumors, few apoptotic cells were seen. In contrast, lo- cells. The present studies confirmed the expression of both types calized apoptosis was visualized by the dark brown staining of of LTB4 receptor mRNA in human pancreatic cancer cells. We the fragmented nuclei, especially in the middle of the tumors in also showed that LTB4 stimulated proliferation of human pan-

Fig. 9 TUNEL assay showing the percentage of apoptotic cells after treatment of MiaPaCa-2 (top panels) and AsPC-1 (bottom panels) cells with 250 and 500 nM LY293111 for 24 h. The increase of fluorescence events in the top right quadrants is due to UTP labeling of fragmented DNA. Data represent results from three separate experiments.

Fig. 10 Effect of LY293111 on cleavage of the caspase-3 substrate PARP in human pancreatic cancer cells. MiaPaCa-2 and AsPC-1 cells were treated with 250 nM LY293111 for 6, 12, 24, and 48 h. Thirty ␮g of protein extracts were resolved by SDS-PAGE and detected using monoclonal PARP antibody.

creatic cancer cells, whereas the selective LTB4 receptor antag- tested. LTB4 also induced marked activation of ERK1/2, and onist, LY293111, inhibited proliferation and induced apoptosis this effect was inhibited by LY293111, providing further evi- in these cells both in vitro and in vivo. Furthermore, this growth- dence that LTB4 may be an important growth factor for pan- stimulatory effect of LTB4 is inhibited by LY293111, confirm- creatic cancer. The actions of LTB4 appear to be mediated by a ing the specificity of this action on LTB4 receptors. The results specific G protein-coupled receptor, BLT1, originally termed also indicate that the LTD4 pathway is not involved in pancre- BLT. BLT1 is highly expressed in leukocytes and expressed to atic cancer growth because blockade of this pathway using a a much lesser extent in other tissues (23, 24). Recently, another selective LTD4 antagonist had no effect on cell proliferation. LTB4 receptor, termed BLT2, was discovered. BLT2 is related LTs, together with prostaglandins, thromboxanes, and li- to BLT1, with an amino acid identity of 45.2%. BLT2 is poxins, are the major constituents of a group of biologically expressed almost ubiquitously and is a low-affinity receptor for active oxygenated fatty acids known as eicosanoids. LTs have LTB4 (25, 26). In the current study, we have shown the mRNA been implicated in the pathogenesis of several inflammatory expression of both BLT1 and BLT2 receptors for LTB4 in diseases, such as asthma, psoriasis, rheumatoid arthritis, and human pancreatic cancer cells. Immunohistochemisty in our inflammatory bowel disease. Whereas there is increasing inter- previous study has also revealed that LTB4 receptors are ex- est in the role of COX and LOX pathways in tumorigenesis, few pressed in human pancreatic cancer tissues, but not in normal studies have focused on the relationship between LT metabo- human pancreas from multiple-organ donors (28). Whereas its lism and cancer development. A study by Cristiana et al. (22) effects on BLT1 are well characterized, at this time, it is not found that both LTB4 and 12(R)-HETE stimulated proliferation known whether LY293111 has any effect on the BLT2 receptor. of the colonic cancer cell lines HCT and HT29 in a time- and Which receptor mediates the growth-stimulatory effect of LTB4 concentration-dependent manner. Qiao et al. (27) studied the on human pancreatic cancer cells needs to be further investi- effect of several prostaglandins and LTB4 on the proliferation of gated. the human colon adenocarcinoma cell lines SW1116 and HT29 Apoptosis, or programmed cell death, is an intrinsic cell and found that selective prostaglandins and LTB4 stimulated suicide mechanism that plays an important role in the develop- growth. Our results confirm that LTB4 plays an important role ment and maintenance of healthy tissues. It is morphologically in the proliferation of human pancreatic cancer cells. Treatment distinct from necrotic cell death. Deregulation of this cell death with 100 nM LTB4 for 24 h almost doubled the thymidine pathway occurs in cancer, autoimmune disease, and neurode- incorporation in all of the human pancreatic cancer cell lines generative disorders. In recent years, emphasis has focused on

Fig. 11 Effect of LY293111 on growth of human pancreatic cancer xenografts in athymic mice. Three million HPAC and ASPC-1 cells were injected s.c. into flanks of athymic mice. After visible tumors had formed, animals were treated with LY293111 (250 mg/kg/day) or control vehicle daily for a total of 24 days. LY293111 treatment inhibited growth of tumor xenografts as measured by both tumor volume (A and B) and tumor weight (C and D). In contrast, LY293111 had no significant effect on body weight in treated animals, compared with controls (E and F). Results are expressed as means Ϯ -P Ͻ 0.05 compared with con ,ء .SE P Ͻ 0.01 compared with ,ءء ;trol P Ͻ 0.001 compared ,ءءء ;control with control.

the importance of apoptosis as the mechanism by which chem- trations of LY293111 (250 and 500 nM) induced substantial otherapy and irradiation kill cancer cells. A reduction in apo- apoptosis within 24 h in all of the human pancreatic cancer cell ptosis favors malignant transformation and proliferation of cancer lines investigated. We also found a time-dependent cleavage of cells, whereas most chemotherapeutic agents induce apoptosis PARP after LY293111 treatment, which indicated the activation (29, 30). Our studies and those of others have shown that LOX of the caspase cascade induced by LY293111. inhibitors such as NDGA, baicalein, and Rev-5901 can all Effects of LY293111 were also examined in vivo. Athymic inhibit proliferation and induce apoptosis in cancer cells. The mice bearing s.c. transplanted human pancreatic cancer cells present study shows that LY293111 also induces pancreatic have proven to be an effective model for studying in vivo effects cancer cell death through induction of apoptosis. Distinctive of anticancer treatments (32, 33). Oral administration of morphological changes associated with apoptosis were evident LY293111 at a dose of 250 mg/kg/day dramatically inhibited after LY293111 treatment, including membrane blebbing and tumor growth compared with that seen in vehicle-treated control nuclear fragmentation. Cleavage of nuclear DNA into 180– animals during the 24-day treatment period. There did not 200-bp internucleosomal fragments is considered a hallmark of appear to be any toxic effects of LY293111 at the dose used in the late stage of apoptosis. The 3Ј-end break of double-stranded these animals. All animals tolerated the treatment very well, and DNA during apoptosis can also be detected by incorporation of there was no significant change in body weight between control fluorescence-labeled dUTP in the presence of TdT enzyme and and treated animals. LY293111 treatment also induced massive analyzed by flow cytometry (31). Treatment with low concen- apoptosis in the xenografted tumors, as shown by in situ tissue

Fig. 12 In situ tissue TUNEL assay in tissues from xenografts of AsPC-1 (A and B) and HPAC (C and D) cells. Controls show no staining, whereas LY293111-treated tumors show marked brown staining of fragmented nuclei that is indic- ative of apoptosis.

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Wei-Gang Tong, Xian-Zhong Ding, Rene Hennig, et al.

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