(CANCER RESEARCH57. 5272-5276. December 1. l997J Advances in Brief Galectin-3: A Novel Antiapoptotic Molecule with A Functional BH1 (NWGR) Domain of Bcl-2 Family' Shiro Akahani, Pratima Nangia-Makker, Hidenori Inohara,2 Hyeong-Reh Choi Kim, and Avraham Raz3 Tumor Progression and Metastasis Program (S. A., P. N.M., H. 1., A. RI, Karmanos Cancer institute, and Departments of Pathology (H-R. C. K.] and Radiation Oncology. Wayne State University School of Medicine IA. RI, Detroit, Michigan 48201 Abstract Galectin-3, a @-galactoside-bindingprotein, has been shown to be involved in tumor progression and metastasis. Here, we demonstrate that expression of galectin-3 in human breast carcinoma BT549 cells inhibits cis-dlamminedichloroplatinum (cisplatin)-induced poly(ADP-ribose) po lymerase degradation and apoptosis, without altering Bcl-2, Bcl-XL, or Bax expressions. Galectin-3 contains the NWGR amino acid sequence highly conserved in the BH1 domain of the bcl-2 gene family, and a substitution of glycine to alanine in this motif abrogated its antiapoptotic activity. Our findings demonstrate that galectin-3 inhibits apoptosis through a cysteine protease pathway and highlight the functional signif icance of the NWGR motif in apoptosis resistance of a non-Bcl-2 protein. Introduction A number of antineoplastic agents have been developed for the eradication of malignancies, but successful chemotherapy still de pends on the control of multidrug resistance, because failure may lead to mortality. For example, CDDP,4 a potent anticancer compound, which functions through interstrand DNA cross-links and the induc tion of apoptosis (1), has improved the outcome of many cancer patients, but the mechanism(s) of CDDP resistance remains to be defined. Expression of galectin-3, a Mr 31,000 carbohydrate-binding protein (2), correlates with neoplastic progression in head and neck cancer (3), thyroid cancer (4), gastric cancer (5), and colon cancer (6). In several experimental tumor systems, galectin-3 expression is re lated to the metastatic potential (7, 8). Recently, it has been suggested that galectin-3 may inhibit apoptosis through interactions with com plementary carbohydrates (9) or with the antiapoptotic protein Bcl-2 (10). Galectin-l and -9 have been reported to induce apoptosis (11— 13), suggesting that some members of the galectin family are involved in the regulation of apoptosis. Moreover, a domain in the COOH terminus of galectin-3 was found to have a significant sequence similarity with the BH1 domain of the Bcl-2 family of proteins containing the NWGR motif (10), which is responsible for the anti apoptotic activity of Bcl-2. Further investigation was thus prompted to establish a possible role of galectin-3 in the mechanism of drug induced apoptosis. To this end, we have used the recently identified galectin-3-null cells, i.e., the human breast carcinoma BT549 cells. In this study, we demonstrate that expression of galectin-3 in human Received 8/28/97; accepted 10/16/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ads'ertisemen: in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported in part by United States Army Grant DAMD 17-96.1-6181, NIH Grants R29-CA64139 (to H-R. C. K.) and ROl-CA46120 (to A. R.), The Henry Ford Health Sciences Center and NIH Grant ROl-CA69480 (to A. R.), and the Paul Zuckerman Support Foundation for Cancer Research (to A. R.). 2 Present address: Department of Otolaryngology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. 3 To whom requests for reprints should be addressed, at Tumor Progression and Metastasis, Karmanos Cancer Institute, I 10 East Warren Avenue, Detroit, MI 48201. Phone:(313)833-0960; Fax:(313)831-7518. 4 The abbreviations used are: CDDP, cis-diamminedichloroplatinum (cisplatin); CMF PBS, calcium-magnesium-free PBS; PARP, poly(ADP-ribose) polymerase. breast carcinoma BT549 cells inhibits CDDP-induced PARP degra dation and apoptosis, and that the NWGR motif of galectin-3 is required for its antiapoptotic activity, as determined by analysis of a mutagenesis study showing that an amino acid substitution of glycine to alanine at position 182 abrogates its function. Materials and Methods Cells and Culture Conditions. The human breast cancer cell line BT549 was obtained from Dr. E. W. Thompson, Vincent T. Lombardi Cancer Re search Center, Georgetown University Medical Center (Washington, D. C.). BT549 transfectants with plasmid DNA accompanying inserts in either the sense (11811, 11913, and 11914) or the antisense (41421) orientation encoding the human galectin-3 cDNA were established as reported previously (8). These cell lines were cultured in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, 2 nmi glutamine, nonessential amino acids, and antibiotics (Life Technologies, Inc.). The cul tures were maintained at 37°C in a humidified atmosphere of7% CO2 and 93% air. Cultures were used for the experiments within three passages after recov cry from frozen stocks. Drugs. CDDP (Sigma Chemical Co., St. Louis, MO) was dissolved in the filtrated normal saline and stored at 4°Cuntil use. Galectin-3 and Bcl-2 Expressions in Untreated BT549 Cells and Tram fectants. Cells (5X 106) were pelleted by centrifugation, washed with CMF PBS (pH 7.4), and subsequently lysed in 1 ml of reducing SDS-PAGE sample buffer containing 2% SDS, 62.5 mr@i Tris-HC1(pH 6.8), 10% glycerol, and 5% f3-mercaptoethanol. Then, cell lysates were agitated overnight at room tem perature and stored at —70°Cuntil use. For Western blot analyses, approxi mately 20 @xg of total proteins were boiled for S mm, separated on reducing 15% SDS-PAGE, and blotted onto a nitrocellulose membrane. Blots were probed with rat anti-galectin-3 (American Type Culture Collection, Rockville, MD) or mouse anti-Bcl-2 antibody (DAKO Corp., Carpinteria, CA) according to the enhanced chemiluminescence (Amersham, Buckinghamshire, England). Western blot procedure. Cell Viability. Cell viability was assessed by a trypanblue dye exclusion test. Cells were cultured with or without CDDP and were collected as de scribed by Huddart et a!. (14). Briefly, at the indicated times, cells were trypsinized and centrifuged at 2000 rpm for S mm. Viability was evaluated on the hemacytometer (Baxter, McGaw Park, IL) following the addition of 0.4% trypan blue (Matheson, Coleman, and Bell, Norwood, OH) dissolved in 0.85% saline. DNA Degradation. DNA fragmentation inducedby CDDP was detectedas an increased fluorescence in the hypoploid DNA region of a flow cytometric cell cycle histogram according to the method described by Telford et a!. (15). Briefly, cells with or without CDDP treatment were fixed with 80% ethanol at 4°Cfor 30 mm, washed with CMF-PBS (pH 7.4), and resuspended with 50 ;xgbml propidium iodide (Coulter Immunology, Hialeah, FL) diluted with CMF-PBS (pH 7.4) containing 0.1% Triton X-l00, 0.1 mistEDTA (pH 8.0), and 50 i.tg/ml RNase A. Then, samples were agitated at 4°C for not less than 15 mm prior to analysis with flow cytometry. Cell Morphology. The change of cell morphologywith CDDP treatment was assessed by fluorescence microscopy using the DNA-binding fluoro chrome bis-(benzimide)-trihydrochloride (Hoechst 33258; Sigma) as described previously by Oberhammer et a!. (16). Briefly, 1X 106 CDDP-treated cell pellets were collected by centrifugation at 2000 rpm for S mm and fixed with 3% paraformaldehyde at room temperature for 10 mm. Then, cells were 5272 on July 10, 2015. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from