And Actinomycin D Induces Apoptosis Even in TRAIL-Resistant Human Pancreatic Cancer Cells1

Vol. 7, 407–414, February 2001 Clinical Cancer Research 407

Combination of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) and Actinomycin D Induces Apoptosis Even in TRAIL-resistant Human Pancreatic Cancer Cells1

Hosei Matsuzaki, Bruno M. Schmied, resistance of Aspc1 cells to TRAIL was not related to the Alexis Ulrich, Jens Standop, lack of TRAIL receptors. The combination of actinomycin D and TRAIL induced an almost complete lysis of Aspc1 cells, Matthias B. Schneider, Surinder K. Batra, 2 whereas actinomycin D alone had no effect on cell survival Kathleen S. Picha, and Parviz M. Pour but inhibited the expression of the Flice inhibitory protein, UNMC Eppley Cancer Center, University of Nebraska Medical which is assumed to play a role in the apoptotic pathway of Center, Omaha, Nebraska 68198-6805 [H. M., B. M. S., A. U., J. S., TRAIL. Thus, the combination of actinomycin D and M. B. S., P. M. P.]; Department of Surgery II, Kumamoto University School of Medicine, Kumamoto, Japan 860 [H. M.]; Department of TRAIL appears to be a promising approach for the therapy Visceral and Transplantation Surgery, Insel Hospital, Bern, of pancreatic cancers resistant to TRAIL. Switzerland 3010 [B. M. S., M. B. S.]; Department of Surgery, Rheinische Friedrich-Wilhelms-University, Bonn, Germany [A. U., J. S.]; Departments of Biochemistry and Molecular Biology [S. K. B.] INTRODUCTION and Pathology and Microbiology [P. M. P.], University of Nebraska Cytokines are a family of proteins that regulate cellular Medical Center, Omaha, Nebraska 68198-6805; and Immunex Corp., proliferation and differentiation by binding to their specific Seattle, Washington 98101 [K. S. P.] receptors on target cells. They are grouped into at least three subfamilies: (a) cysteine knot factors; (b) TNFs;3 and (c) helical ␣ ABSTRACT cytokines. The TNF family includes TNF- , FasL, lympho- toxin, CD30 ligand, 4-1BB ligand, CD40 ligand, CD27 ligand, Tumor necrosis factor-related apoptosis-inducing li- and TRAIL (1). Most TNF family cytokines are expressed as a gand (TRAIL) is a novel member of the tumor necrosis type II transmembrane protein with its NH terminus in the factor superfamily of cytokines that induces cell death by 2 cytoplasm and its COOH-terminal region extending into the apoptosis. TRAIL has been shown to be effective in almost extracellular space. The COOH-terminal extracellular domain is two-thirds of solid tumors tested thus far, but its effect on processed proteolytically to form a soluble homotrimeric mol- pancreatic cancer cells is unknown. We tested the effect of ecule (2). TRAIL (also called APO2 ligand) is a newly discov- TRAIL on seven human pancreatic cancer cell lines (HPAF, ered TNF superfamily member initially cloned from human Panc1, Miapaca2, Bxpc3, Panc89, SW979, and Aspc1) in heart and lymphocyte cDNA libraries (3). With a predicted vitro. Of these cell lines, all but Aspc1 showed a significant molecular weight of 32,000, human TRAIL is 281 aa residues dose-dependent increase in apoptosis. The apoptotic rate, as long, with a 17-aa residue cytoplasmic tail, a 21-aa residue detected by a terminal deoxynucleotidyl transferase-medi- transmembrane segment, and a 243-aa residue extracellular re- ated nick end labeling assay, was highest in Bxpc3 (71.5%), gion (2, 3). Human TRAIL is 65% identical to mouse TRAIL at followed by HPAF (38.0%), Miapaca2 (24.9%), Panc1 the aa sequence level across the entire molecule, and there is a (16.1%), Panc89 (15.8%), SW979 (13.9%), and Aspc1 complete species cross-reactivity (3). Although TRAIL is (5.2%). Multiple treatments were more effective than a sin- known to be expressed by lymphocytes, many tissues seem to gle treatment and caused a sustained and profound cell express the ligand, and its broad expression pattern suggests an death in all but Aspc1 cells. There was no correlation be- intriguing function for the molecule (3). tween the effect of TRAIL and the differentiation grade of TNF3 family members induce cell death by apoptosis; the cell lines, p53 mutation, or bcl-2 or bax expression. The hence, their receptors are also called “DRs.” T-cell cytotoxicity is mediated primarily by FasL and TRAIL (4, 5). Their apoptotic properties have prompted investigation of the use of certain TNF family members in treating melanomas; mammary, Received 8/18/00; revised 11/2/00; accepted 11/2/00. colorectal, ovarian, cervical, bladder, and renal cancers; and The costs of publication of this article were defrayed in part by the hematological and some mesenchymal malignancies (4, 6–11). payment of page charges. This article must therefore be hereby marked In this aspect, TRAIL has gained a central role because it also advertisement in accordance with 18 U.S.C. Section 1734 solely to induces apoptosis in FasL-resistant tumor cells (4). Further- indicate this fact. 1 Supported by National Cancer Institute Grants CA60479 and CA367127, SPORE Grant P5O CA72712, and a Special Institution Grant from the American Cancer Society. A. U. is a recipient of a scholarship from the Deutsche Forschungsgemeinschaft, Germany. 3 The abbreviations used are: TNF, tumor necrosis factor; TRAIL, tumor 2 To whom requests for reprints should be addressed, at The Eppley necrosis factor-related apoptosis-inducing ligand; TUNEL, terminal de- Institute for Research in Cancer and Allied Diseases, University of oxynucleotidyl transferase-mediated nick end labeling; FLIP, Flice in- Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, hibitory protein; aa, amino acid(s); FasL, Fas ligand; RT-PCR, reverse NE 68198-6805. Phone: (402) 559-4495; Fax: (402) 559-4651; E-mail: transcription-PCR; PBS-T, PBS-Tween 20; Ab, antibody; DR, death [email protected]. receptor; DcR, decoy receptor.

Table 1 Antibodies used for immunohistochemistry Antibody Pretreatment Dilution Supplier P53 (Pab240) Microwave heat 1:100 Biogenex (San Ramon, CA) DU-PAN 2 None 1:10 Dr. Colcher (Omaha, NE) CA 19-9 None 1:10 Wistar Institute (Philadelphia, PA) B72.3 None 1:50 NIH (Bethesda, MD) TGF-␣a 0.05% saponin 1:20 Oncogene Research Products (Cambridge, MA) EGFR 0.05% saponin 1:40 Sigma (St Louis, MO) P16 Microwave heat 1:40 Oncogene Research Products (Cambridge, MA) Bcl-2 Microwave heat 1:100 Oncogene Research Products (Cambridge, MA) Bax Microwave heat 1:20 Oncogene Research Products (Cambridge, MA) Ki 67 Microwave heat Ready to use Biogenex (San Ramon, CA) a TGF-␣, transforming growth factor ␣; EGFR, epidermal growth factor receptor.

more, contrary to FasL, TRAIL is not toxic to normal cells (6), 800 ng/ml. Aspc1 cells were also treated with 1600 ng/ml although a new report indicates toxicity in normal human hepa- TRAIL. Control cells were cultured in the absence of TRAIL. tocytes (12), and it does not have a graft-versus-host disease After 24 h, the cells were washed with PBS, and the cells that effect but mediates a favorable graft-versus-tumor effect (6). were still attached were considered to be viable cells and Pancreatic cancer is the fifth leading cause of cancer deaths counted by a hemocytometer. The IC50 of TRAIL was deter- in the United States, killing about 28,000 people every year in mined by a 50% decrease of the viable cell number compared the United States alone (13). The etiology of the diseases is still with untreated cells by a given concentration. obscure, and early diagnosis and treatment remain disappoint- For a multiple treatment of TRAIL, 4 ϫ 104 cells from ing. Consequently, it was of interest to examine the effect of each cell line were cultured per well in a 6-well plate in regular TRAIL on pancreatic cancer cells. Although Fas and FasL have culture medium (3 ml/well) without fetal bovine serum and been shown to be expressed by human pancreatic cancer cells, treated with TRAIL at a dose of 200 ng/ml eight times, each they have been resistant to Fas-mediated apoptosis (14). Studies time for 3 days. The cell number of each group was determined by Raitano et al. (15) have shown that TNF in combination with at the end of each treatment. INF-␥ inhibited the growth of some pancreatic cancer lines. However, to our knowledge, the effect of TRAIL on pancreatic We also examined the effect of actinomycin D alone or in cancer has not been tested. We examined the effect of TRAIL on combination with TRAIL on Aspc1, HPAF, and Bxpc3 cells. ϫ 4 seven pancreatic cancer lines in vitro and found marked apo- Cells of each cell line (4 10 ) were cultured in a 6-well plate. ptosis in six of these cell lines. After 36 h of incubation, the cells were treated with TRAIL (200 ng/ml) and actinomycin D (0.1 or 1 ␮g/ml). After the treatment MATERIALS AND METHODS for 24 h, viable cells were counted. The effect of actinomycin D alone at doses of 0.1 or 1 ␮g/ml on Aspc1, HPAF, and Bxpc3 Reagents and Abs. Recombinant human TRAIL was also examined similarly. Aspc1, HPAF, and Bxpc3 were (leucine-zipper construct; Ref. 8) was provided by the Immunex chosen because of their differing sensitivities to TRAIL. Corp. (Seattle, WA). Actinomycin D was purchased from Sigma TUNEL Assay. TUNEL assay was performed to deter- (St. Louis, MO). The Abs for the four TRAIL receptors were mine the percentage of cells undergoing apoptosis after treat- provided by the Immunex Corp. Abs used for immunohistochemistry are summarized in Table 1. ment with TRAIL or treatment with the combination of TRAIL and the protein synthesis inhibitor actinomycin D. Briefly, 1 ϫ Cell Lines. Human pancreatic cancer cell lines Panc1, 6 Aspc1, Miapaca2, and Bxpc3 were obtained from American 10 cells from each cancer cell line were seeded in T75 flasks Type Culture Collection; Panc89 and SW979 were obtained and cultured for 2 days at 37°C before TRAIL was added at a from Dr. S. K. Batra (University of Omaha, Nebraska, NE); and concentration of 200 ng/ml. In the combined treatment, TRAIL HPAF was obtained from Dr. M. A. Hollingsworth (The Eppley was given at a dose of 200 ng/ml, and actinomycin D was given ␮ Institute, University of Nebraska, Omaha, NE). Panc1 and at a dose of 0.1 or 1 g/ml. After 24 h of incubation, the cells HPAF cells were cultured in RPMI 1640 (Life Technologies, were collected and fixed with 1% paraformaldehyde in PBS for Inc., Gaithersburg, MD), and the remaining cells were cultured 20 min on ice. After washing with PBS, the cells were perme- in DMEM (Life Technologies, Inc.), and each was supple- abilized with 0.2% Triton X-100 in PBS for 5 min on ice. After mented with 10% fetal bovine serum (Summit, CO), 100 washing, the cells were incubated with FITC-conjugated dUTP units/ml penicillin, and 100 ␮g/ml streptomycin (Life Technol- in the presence of terminal deoxynucleotidyl transferase enzyme

ogies, Inc.) in a humidified atmosphere of 5% CO2 in air. solution for1hat37°C using the Apoptosis Detection System Treatment. To assess the optimal concentration of (Promega, Madison, WI). After incubation, cells were washed TRAIL, in a pilot study 4 ϫ 104 cells from each cell line were twice with 0.1% Triton X-100 in PBS containing 5 mg/ml BSA seeded in 24-well plates. After 36–48 h of incubation at 37°C, and then incubated with 5 ␮g/ml propidium iodide solution in the medium was changed, and TRAIL was added to the regular PBS containing 250 ␮g of Dnase-free RNase A (Sigma) for 30 culture medium (1 ml/well) without fetal bovine serum at con- min at room temperature. Ten thousand cells were assessed by centrations of 0.1, 1, 3.13, 6.25, 12.5, 25, 50, 100, 200, 400, and two-color FACScan analysis (Becton Dickinson, San Jose, CA).

Fig. 2 The time course of cell death in the pancreatic cancer cells after TRAIL treatment. The cells were treated with TRAIL (200 ng/ml). The Fig. 1 Dose-response effect of TRAIL in human pancreatic cancer cell cell number rate was shown compared to untreated control. F, Aspc1; lines. The cells were treated with different concentrations of TRAIL Œ, Panc1; E, HPAF; Ⅺ, Miapaca2; ‚, Panc89; ϫ, SW979; f, Bxpc3. (0.1, 1, 3.12, 6.25, 12.5, 25, 50, 100, 200, 400, and 800 ng/ml) for 24 h. The cells were then counted, and the percentage of viable cells was calculated in comparison with the control cells. F, Aspc1; Œ, Panc1; E, HPAF; Ⅺ, Miapaca2; ‚, Panc89; ϫ, SW979; f, Bxpc3. Immunocytochemistry. Cells (5 ϫ 106) from each cell line were pelleted, fixed in 10% buffered formalin for 24 h, embedded in paraffin, cut in serial sections, and processed for FITC-propidium iodide double-positive cells were considered immunocytochemistry by the Avidin Biotin Complex method apoptotic. (20). The cells were also grown on a Lab-TEK chamber slide DNA Fragmentation Analysis. Induction of apoptosis (Nalge Nunc International, Naperville, IL), fixed with acetone, was also determined by the internucleosomal DNA fragmenta- and processed for immunohistochemistry. tion method as described previously (16). Briefly, purified DNA Western Blot. The cells were lysed in PBS containing was analyzed by electrophoresis on a 1% agarose gel at 25 V 1% NP40 and protease inhibitor mixture (Boehringer Mann- for 16 h. heim, Mannheim, Germany). The lysates were centrifuged at ϫ RT-PCR Analysis. The expression of the mRNA for 14,000 g to remove the debris, and the protein concentration TRAIL receptors DR4, DR5, DcR1, and DcR2 in all seven cell was determined by using the Bio-Rad protein assay kit (Bio- lines was examined. Total RNA was purified using the guanidine Rad, Hercules, CA). Equal amounts of protein were separated thiocyanate acid phenol method (17). Briefly, 1 ␮g of total RNA by SDS-PAGE, transferred to a nitrocellulose membrane (Bio- was mixed with 100 ng of random hexamer primers, and the Rad), and blocked with 5% NFDM in PBS-T overnight at 4°C. solution was heated at 72°C for 5 min and then placed on ice. After washing with PBS-T, the membrane was incubated with Deoxynucleotide triphosphates (10 mM each), reverse transcriptase, TRAIL receptor monoclonal Abs or FLIP antiserum (diluted ϫ 1:1,000) for1hatroom temperature. After washing with Rnasin, 10 PCR buffer, 25 mM MgCl2, and water were added according to the manufacturer’s instructions (Perkin-Elmer, Cy- PBS-T, the membrane was incubated with antimouse horserad- press, CA). The mixture was incubated at 42°C for 60 min. cDNA ish peroxidase-conjugated Ab (Amersham, Arlington Heights, synthesis reaction was terminated by heating at 95°C, and the IL). After washing with PBS-T, the blots were developed by mixture was stored at Ϫ70°C. Primer sequences for all four TRAIL chemiluminescence (ECL protein detection reagent; Amer- receptors and the PCR protocol used in this study were published sham). previously (18). Glyceraldehyde-3-phosphate dehydrogenase prim- Statistical Analysis. Statistical significance was deter- ers were used for the internal control (19). Primers for RT-PCR mined by the Student’s t test. were synthesized by the UNMC Eppley Molecular Biology Core Laboratory (Omaha, NE). The sequences were as follows: (a) DR4, RESULTS 5Ј-CGATGTGGTCAGAGCTGGTACAGC-3Ј (sense) and 5Ј- The correlation between the growth inhibition and the dose GGACACGGCAGAGCCTGTGCCATC-3Ј (antisense); (b) DR5, of TRAIL is illustrated in Fig. 1. After 24 h, Bxpc3 and SW979 5Ј-GGGAGCCGCTCATGAGGAAGTTGG-3Ј (sense) and 5Ј- cells were most affected, whereas the response of Miapaca2, GGCAAGTCTCTCTCCCAGCGTCTC-3Ј (antisense); (c) DcR1, Panc89, and HPAF was moderate. Panc1 showed a minimal Ј Ј Ј 5 -GTTTGTTTGAAAGACTTCACTGTG-3 (sense) and 5 - response, and Aspc1 showed no response at all. The IC50 values GCAGGCGTTTCTGTCTGTGGGAAC-3Ј (antisense); (d) DcR2, for Bxpc3, SW979, Panc89, Miapaca2, HPAF, Panc1, and 5Ј-CTTCAGGAAACCAGAGCTTCCCTC-3Ј (sense) and 5Ј-TT- Aspc1 were 12, 20, 33, 48, 66, 95, and Ͼ1600 ng/ml TRAIL, CTCCCGTTTGCTTATCACACGC-3Ј (antisense); and (e) glyc- respectively. eraldehyde-3-phosphate dehydrogenase, 5Ј-CGGATTTGGTCG- The time course of the cell death in the cell lines after TATTGG-3Ј (sense) and 5Ј-TCCTGGAAGATGGTGATG-3Ј treatment with TRAIL is shown in Fig. 2. TRAIL-induced cell (antisense). death was very rapid and was seen already after 2 h. In Bxpc3, PCR products were analyzed by electrophoresis on 1.8% Panc89, Miapaca2, and SW979 cells, most of the cells were agarose gels. dead, and only a few cells were still attached to the bottom of the

Fig. 3 The cell growth after treatment eight times with TRAIL. The treatment period was 3 days each time. The cell number was determined every 3 days. X axis represents the number of TRAIL treatments, and Y axis represents the cell number. E, control; f, TRAIL treatment; the broken lines .P Ͻ 0.01 ,ء .represent confluent state of the cells

Table 2 Cell growth after treatment with TRAIL The cell growth after eight times treatment with 0.2 ␮g/ml TRAIL. The treatment period was 3 days each time. The cell number (ϫ 102) was determined every 3 days. Values are the mean Ϯ SD from three independent experiments. A. Bxpc3, Panc89, Miapaca2 , and SW979 cells Bxpc3 Panc89 Miapaca2 SW979 Day TRAIL Control TRAIL Control TRAIL Control TRAIL Control 0 400 400 400 400 400 400 400 400 365Ϯ 7 555a Ϯ 35 132 Ϯ 9 925a Ϯ 30 53 Ϯ 3 900a Ϯ 40 50 Ϯ 3 700a Ϯ 30 641Ϯ 7 1,100a Ϯ 100 41 Ϯ 10 3,499a Ϯ 44 83 Ϯ 2 4,112a Ϯ 150 55 Ϯ 7 1,900a Ϯ 21 933Ϯ 4 3,960a Ϯ 1,500 50 Ϯ 11 10,513a Ϯ 1,650 50 Ϯ 2 6,471a Ϯ 1,000 50 Ϯ 6 5,120a Ϯ 1,000 12 38 Ϯ 4 11,000a Ϯ 2,300 45 Ϯ 9 12,000a Ϯ 1,000 49 Ϯ 3 10,000a Ϯ 2,100 49 Ϯ 7 11,100a Ϯ 2,000 15 45 Ϯ 7 Confluent 37 Ϯ 10 Confluent 48 Ϯ 5 Confluent 46 Ϯ 4 Confluent 18 48 Ϯ 447Ϯ 547Ϯ 448Ϯ 5 21 50 Ϯ 543Ϯ 449Ϯ 645Ϯ 4 24 45 Ϯ 443Ϯ 745Ϯ 446Ϯ 3 B. HPAF, Panc1, and Aspc1 cells HPAF Panc1 Aspc1 Day TRAIL Control TRAIL Control TRAIL Control 0 400 400 400 400 400 400 3 150 Ϯ 21 3,150a Ϯ 110 2,250 Ϯ 110 4,800a Ϯ 110 1,715 Ϯ 160 2,476 Ϯ 400 6 100 Ϯ 13 9,150a Ϯ 210 3,900 Ϯ 1,100 20,300a Ϯ 1,000 2,610 Ϯ 700 5,105 Ϯ 1,000 9 350 Ϯ 41 13,600a Ϯ 1,600 4,300 Ϯ 1,400 22,150a Ϯ 2,200 7,761 Ϯ 500 9,546 Ϯ 1,600 12 650 Ϯ 72 14,100a Ϯ 710 4,400 Ϯ 700 21,000a Ϯ 2,200 12,079 Ϯ 1,400 11,426 Ϯ 1,900 15 2,700 Ϯ 500 Confluent 4,900 Ϯ 1,000 Confluent 11,986 Ϯ 1,000 13,617 Ϯ 1,500 18 3,850 Ϯ 570 5,100 Ϯ 850 Confluent Confluent 21 3,950 Ϯ 1340 5,300 Ϯ 700 24 4,100 Ϯ 780 5,600 Ϯ 850 a P Ͻ 0.01.

dish. In HPAF and Panc1 cells, the survival rate was 15% and depleted the Bxpc3, Panc89, Miapaca2, and SW979 cells (Fig. 40%, respectively, compared with their respective untreated cell 3; Table 2). HPAF showed a significant cell number reduction controls, respectively. during the first four treatments, followed by a slight and sus- Treatment of cells with TRAIL for eight times almost tained recovery. In Panc1 cells, the cell number increased

Table 3 Effects of a combination of TRAIL with actinomycin D on TRAIL-resistant Aspc1 cells Values are mean Ϯ SD from three independent experiments. Treatment Viable cells ϫ 102 after 24 h Control 799 Ϯ 91 (100%) TRAIL (0.2 ␮g/ml) 788 Ϯ 84 (97.4%) Actinomycin D (0.1 ␮g/ml) 783 Ϯ 79 (100%) TRAIL & actinomycin D (0.1 ␮g/ml) 368 Ϯ 29 (47%) Actinomycin D (1 ␮g/ml) 751 Ϯ 86 (100%) TRAIL & actinomycin D (1 ␮g/ml) 54 Ϯ 14 (7%)a a P Ͻ 0.01.

Fig. 4 Induction of apoptosis in pancreatic cancer cell lines by TRAIL. .P Ͻ 0.05 ,ء .Apoptotic cells were detected by the TUNEL assay

Fig. 6 Expression of FLIP protein in: Lane 1, marker; Lane 2, Aspc1 cells treated with 0.1 ␮g/ml actinomycin D; Lane 3, untreated Aspc1 cells; Lane 4, marker; Lane 5, untreated Aspc1 cells; Lane 6, untreated HPAF cells; Lane 7, untreated Bxpc3 cells; Lane 8, marker; Lane 9, Bxpc3 cells treated with 0.1 ␮g/ml actinomycin D; and Lane 10, HPAF cells treated with 0.1 ␮g/ml actinomycin D.

cell lines. However, in Aspc1 cells, the fragmentation was very weak. The apoptotic potential of the combined treatment (TRAIL plus actinomycin D) was investigated on the TRAIL-resistant Aspc1 cells. After treatment for 24 h, almost all cells were detached and floated in the flask, whereas only a few cells were found detached after treatment with actinomycin D alone. The survival rate of Aspc1 cells treated with the combined treatment was 47% (actinomycin D, 0.1 ␮g/ml) or 7.2% (actinomycin D, 1 ␮g/ml) compared with the cells treated with actinomycin D (either concentration) alone (Table 3). Similar results were found for HPAF (survival rates of 17.9% and 6.7%, respectively), which was moderately sensitive to TRAIL, whereas in Bxpc3, due to the already strong effect of TRAIL alone, the survival rate (6%) could not be further decreased by combina- Fig. 5 DNA fragmentation of the cell lines after TRAIL-induced ap- tion with actinomycin D. The apoptosis rate was significantly optosis. DNA smear and the characteristic ladder can be seen in all of higher after combined treatment than it was in cells treated with the cell lines. C, control; T, TRAIL treatment. actinomycin D or TRAIL alone. Western blot analysis demon- strated the presence of FLIP in Aspc1 cells (resistant to TRAIL) treated with TRAIL but not in the cells treated with 0.1 ␮g/ml actinomycin D (Fig. 6). However, actinomycin D showed no slightly but gradually after one treatment and remained stable at such effect on FLIP in HPAF (moderately sensitive to TRAIL) around 500,000 cells until the last treatment, when the control and Bxpc3 cells (highly sensitive to TRAIL; Fig. 6). cells became confluent (2,100,000 cells). Treatment with Immunocytochemical examination showed no correlation TRAIL did not alter the cytological appearance of the cells in between TRAIL sensitivity and the expression of DU-PAN 2, any cell line. Aspc1 proved to be refractory also to the repeated CA19-9, B72.3, transforming growth factor ␣, epidermal TRAIL treatment. The effect of TRAIL on apoptosis, as meas- growth factor receptor, p16, p53, bcl-2, and bax expression, on ured by TUNEL method, is illustrated in Fig. 4. The Bxpc3 cell the one hand, and the differentiation state of cell lines on the line, which had 71.5% apoptotic cells, was the most affected, other hand (Table 4). The immunocytochemical results of p53 whereas Aspc1 cells with an apoptotic rate of 5.5% were the staining correlate with the reported data (21, 22). There were least responsive. The response correlated closely with the cell also no differences in the immunoreactivity between the cells death rate of the cell line (Fig. 2). Agarose gel electrophoresis before and after TRAIL treatment. showed that TRAIL induced DNA fragmentation (Fig. 5) in all The expression of all four TRAIL receptors (DR4, DR5,

Table 4 Immunocytochemical findings in human pancreatic cancer cells treated with TRAIL Highly sensitive Moderately sensitive Slightly sensitive Bxpc3 Panc89 Miapaca2 SW979 HPAF Panc1 Aspc1 Differentiation Mod.a Mod. Undif. Mod. Well-Mod. Undif. Undif. % apoptosis 71.5 15.8 24.9 13.9 38.0 16.1 5.3 p53b ϩϩ ϩ Ϫ ϩ ϩ Ϫ a Mod., moderately differentiated; Undif., undifferentiated; Well, well-differentiated. b The immunoreactivity correlated with the published data (21, 22).

Fig. 7 Expression of TRAIL receptors in the pancreatic cancer cell lines. The expression of DR4, DR5, DcR1, and DcR2 mRNA was assessed by RT-PCR analysis. The expected sizes for the DR4, DR5, DcR1, and DcR2 PCR products are 216, 151, 139, and 399 bp. Lane 1, Bxpc3; Lane 2, Panc89; Lane 3, Panc1; Lane 4, SW979; Lane 5, Miapaca2; Lane 6, HPAF; Lane 7, Aspc1; Lane 8, H79; Lane 9,H2O. Fig. 8 Expression of TRAIL receptor proteins in pancreatic cancer cell lines. Four TRAIL receptors (DR4, DR5, DcR1, and DcR2) were detected by Western blots on highly TRAIL-sensitive Bxpc3 (Lane 1), moderately sensitive HPAF (Lane 2), and TRAIL-resistant Aspc1 cells (Lane 3). DcR1, and DcR2) was detected by RT-PCR analysis. The glioma cell line H79, which was used as a positive control, expressed only receptors DR5 and DcR1 (Fig. 7). The TRAIL receptors were also detected by Western blot in all four cell lines expression of DcR1 has been shown to protect mammalian cells (Fig. 8). from TRAIL-induced apoptosis (28). In some aspects, the effects of TRAIL differ from other TNF family members. Besides being nontoxic to normal cells, it DISCUSSION also induces apoptosis in FasL-resistant tumor cells (14) and Previous studies have shown the presence of high affinity does not have a graft-versus-host effect but has a favorable receptors to recombinant human TNF-␣ and IFN-␥ in pancreatic graft-versus-tumor effect (4, 6). The resistance of human pan- cancer cells, which showed no, little, or profound sensitivity to creatic cancer cells to Fas-mediated apoptosis, despite the ex- these cytokines (15). Similar growth-inhibitory effects were pression of Fas and FasL in these cells (14), clearly distin- obtained with the natural human TNF-␣ and IFN-␣ (23). TRAIL guishes the effect of TRAIL from FasL. TRAIL activity in (also called APO2 ligand), a new and novel member of the TNF hematological malignancies was not reduced by the overexpres- superfamily, has induced apoptosis in many transformed cell sion of the multidrug-resistant protein and was not enhanced by lines by binding to its death-signaling receptors, DR4 and DR5, 9-cis retinoids, which down-regulate the bcl-2 protein (6). and initiating a cascade of events leading to apoptosis in a Therefore, TRAIL has been considered a therapeutic drug and caspase-dependent fashion (24, 25). Although its death domain- has shown growth-inhibitory effects on a variety of mesenchy- containing receptors are expressed in both normal and cancer mal and epithelial cancer cell lines (6–11). Although bcl-2, bax cells (3, 26), TRAIL is not cytotoxic to normal cells, although caspases, and the overexpression of caspase-1, which causes some toxicity to normal human hepatocytes was reported re- apoptosis in rat fibroblasts (30), have been identified in pancre- cently (12). This protection has been thought to be due to the atic cancer cells (31, 32), little information is available on the presence of the antagonist DcRs DcR1 (or TRID) and DcR2 that effect of TRAIL on these cells. Based on the unavailability of inhibit TRAIL signaling in normal cells but are absent in most any therapeutic modalities for pancreatic cancer, its high mor- tumor cells (27–29). The DcR1 receptor is a distinct gene tality, and lack of early diagnostic possibilities, TRAIL could product with an extracellular TRAIL-binding and a transmem- provide a unique drug to control this dismal disease. brane domain but no intracellular signaling domain. Ectopic Realizing the cell heterogeneity of solid tumors including

pancreatic cancer, which may determine the cell response to complex process. Nevertheless, the results promise an effective therapeutic agents, we tested the effect of TRAIL on heteroge- therapeutic modality for the treatment of pancreatic cancer re- nous human pancreatic cancer cell lines of different origin and sistant to TRAIL. differentiation to examine whether the effect of TRAIL is dependent or independent from the cancer cell type. We used the leucin zipper form of human TRAIL because it causes cell death REFERENCES in a TRAIL-sensitive human adenocarcinoma cell line (8). Our 1. Nagata, S. Apoptosis by death factor. Cell, 88: 355–365, 1997. initial scope was to assess the short-term and long-term effect of 2. Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., TRAIL on cell survival. and Ashkenazi, A. Induction of apoptosis by Apo-2 ligand, a new TRAIL was effective in reducing the cell number and member of the tumor necrosis factor cytokine family. J. Biol. 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Hosei Matsuzaki, Bruno M. Schmied, Alexis Ulrich, et al.

Clin Cancer Res 2001;7:407-414.

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