[CANCER RESEARCH 62, 1178 –1183, February 15, 2002] GATA-6 Transcriptional Regulation of 15-Lipoxygenase-1 during NSAID-induced Apoptosis in Colorectal Cancer Cells 1 Imad Shureiqi, 2 Wei Jiang, Susan M. Fischer, Xiaochun Xu, Dongning Chen, J. Jack Lee, Reuben Lotan, and Scott M. Lippman Departments of Clinical Cancer Prevention [I. S., W. J., X. X., D. C., S. M. L.], Gastrointestinal Medical Oncology [I. S.], Carcinogenesis [S. M. F.], Biostatistics [J. J. L.], and Division of Cancer Prevention [R. L.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 ABSTRACT The mechanisms by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in colorectal cancer cells are undergoing in- tensive investigation. We found previously that NSAIDs induce apoptosis in these cells by restoring 15-lipoxygenase-1 (15-LOX-1) expression. The present study examined the NSAID mechanism for up-regulating 15- LOX-1 in two colorectal cancer cell lines (RKO and DLD-1). We found that NSAID effects on 15-LOX-1 occurred at the level of transcriptional regulation. We then studied NSAID effects on GATA-6, a transcription factor that suppresses 15-LOX-1 expression. Beginning within 4 h, NSAIDs progressively down-regulated GATA-6 expression. Ectopic GATA-6 expression blocked NSAID induction of 15-LOX-1 and apoptosis. NSAIDs down-regulate GATA-6 to transcriptionally up-regulate 15- LOX-1 and induce apoptosis in colorectal cancer cells. INTRODUCTION Apoptosis is important for regulating and maintaining homeostasis in normal colon epithelia (1). The loss of the propensity to undergo apoptosis is a critical event in the development of human colorectal (2– 4) and other (5) cancers, and restoring apoptosis is thought to be an important cancer therapeutic and preventive mechanism (5, 6). We have shown that 15-LOX 3 -1 is down-regulated and 13-S-HODE, a 15-LOX-1 product of linoleic acid, restores apoptosis in colorectal cancer cells (7). We showed recently that the up-regulation of 15- LOX-1 expression and 13-S-HODE production in colorectal cancer cells plays an important independent role in the apoptotic effects of NSAIDs, a very promising class of agents for the chemoprevention of colorectal cancers (8, 9). Various levels of the regulation of 15-LOX-1 expression have been shown (10, 11), but the mechanisms for the up-regulation of 15-LOX-1 by NSAIDs have not been reported previously. GATA is a family of six transcriptional regulation proteins that play important roles in regulating cell differentiation during vertebrate embryogenesis (12). GATA-6 expression increases during prolifera- tion and decreases during differentiation of intestinal cells (13). GATA-6 is expressed in undifferentiated cancer cells, especially colon cancer cells (13–15), in which GATA-6 suppresses the tran- scription of 15-LOX-1 (11). There are no previous reports on the relationship between GATA-6 and NSAID effects on 15-LOX-1. We designed our current study to test the hypothesis that the mechanism of NSAID-induced apoptosis in colorectal cancer cells involves GATA-6 transcriptional regulation of 15-LOX-1 expression. MATERIALS AND METHODS Materials. We obtained DLD-1 (human colon carcinoma) cells from the American Type Culture Collection (Manassas, VA) and RKO (human rectal carcinoma) cells from Dr. Michael Brattain (The University of Texas, San Antonio, TX). We purchased sulindac from Sigma Chemical Co. (St. Louis, MO), sulindac sulfone from LKT Laboratories, Inc. (St. Paul, MN), NS-398 from Cayman Chemical, Inc. (Ann Arbor, MI), and antihuman GATA-6 and histone antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Human GATA-6 wild-type cDNA subcloned into pcDNA1/Amp vector and a control pcDNA1/Amp vector that carries human GATA-6 cDNA with a deletion mutation in the zinc finger domains were gifts from Dr. Kenneth Walsh (Tufts University, Boston, MA) (16). Rabbit polyclonal antiserum to human recombinant 15-LOX-1 and standards of recombinant 15-LOX-1 were gifts from Drs. Mary Mulkins and Elliot Sigal (Roche Bioscience, Palo Alto, CA). Other reagents, molecular grade solvents, and chemicals were obtained from regular commercial manufacturers or as specified below. Cell Culture. DLD-1 and RKO cells were grown in RPMI 1640 supple- mented with 10% fetal bovine serum, penicillin, and streptomycin (Life Technologies, Inc., Grand Island, NY). Cells were treated with NSAIDs at 60 – 80% confluence, cultured, and harvested for each assay as described below. The selected NSAID concentrations to induce apoptosis [NS-398 (120 M), sulindac sulfone (300 M), or sulindac (150 M)] were based on prior studies in colorectal cancer cells and apply to all of the experiments described below (8, 17). NSAIDs were dissolved in DMSO. DMSO final concentrations were 0.5% (Ref. 8; DMSO had no independent effect on cell growth (data not shown)]. Northern Blot Analysis of 15-LOX-1 and GATA-6 RNA Expressions. Total RNA was isolated by TRI reagent (Sigma Chemical Co.), and Northern blot analyses were performed as described previously (18). RNA was separated (20 g/lane) on 0.7% denaturing agarose gels containing 1.8% (v/v) formal- dehyde and transferred onto nylon membranes (Bio-Rad). The membranes were hybridized overnight at 42°C with a 32 P-labeled cDNA probe in a solution containing 50% (v/v) formamide, 5 SSC, 50 mM sodium phosphate buffer (pH 6.5), 250 g/ml sheared salmon sperm DNA, 10 Denhart’s solution, and 10% dextran sulfate. The radiolabeled ( 32 P) cDNA probe for human 15-LOX-1 was 952 bp that were generated by reverse transcription- PCR of human 15-LOX-1 with the primers 5'-GAGTTGACTTTGAGG- TTTCGC-3' and 5'-GCCCGTCTGTCTTATAGTGG-3'. A 644-bp human GATA-6 probe was generated by the primers 5'-TTGGATTGTCCTGT- GCCAACT-3' and 5'-GGTTCACCCTCGGCGTTT-3'. The human GAPDH probe, used as control, was a 600-bp PCR piece made with 5'-CCACCCAT- GGCAAATTCCATGGCA-3' primer and 5'-TCTAGACGGCAGGTCAG- GTCCACCACC-3' primer. Amplified cDNA was cloned using the pGEM-T vector (Promega). After hybridization and washes, the blots were autoradio- graphed by exposure to hyperfilm-MP films (Amersham Corp). Nuclear Run-On Assay. DLD-1 and RKO cells were cultured, treated with either sulindac, NS-398 or sulindac sulfone, and harvested 48 h later (19). Nuclei (5  10 7 cell/sample) were isolated by using NP-40 lysis buffer and stored at -80°C in storage buffer. Nuclei were thawed and incubated in 100 l of 2 reaction buffer and 100 Ci of [- 32 P]UTP at 30°C for 30 min. RNase-free DNase and CaCl 2 were added to remove template DNA after transcription, and the mixture was incubated at 26°C for 30 min. To digest protein, 25 l of 10 SET, 2 l of 100 mg/ml proteinase K, and 5 l of tRNA (Roche Molecular Biochemicals, Indianapolis, IN) were added for 30-min Received 7/23/01; accepted 12/11/01. 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 This work was supported in part by Grants 1KO7 CA86970 and CA16672 from the National Cancer Institute, NIH, Department of Health and Human Services. S. M. L. holds the Anderson Clinical Faculty Chair for Cancer Treatment and Research. 2 To whom requests for reprints should be addressed, at Department of Clinical Cancer Prevention, The University of Texas M. D. Anderson Cancer Center, Box 236, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: (713) 745-4928; Fax: (713) 792- 0628; E-mail: ishureiqi@mdanderson.org. 3 The abbreviations used are: LOX, lipoxygenase; GAPDH, glyceraldehyde-3-phos- phate dehydrogenase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP- biotin end labeling; DAPI, 4',6-diamidino-2-phenylindole; HODE, hydroxyoctadecadi- enoic acid; NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase. 1178 Research. on November 30, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from