AntitumorActivity of Suberoylanilide Hydroxamic Acid against Thyroid Cancer Cell Lines In vitro and In vivo

Purpose: The histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), has multiple antitumor effects against a variety of human cancers. Experimental design:We treated several anaplastic and papillary thyroid cancer cell lines with SAHA to determine if it could inhibit the growth of these cells in vitro and in vivo. Results: SAHA effectively inhibited 50% clonal growth of the anaplastic thyroid cancer cell lines, ARO and FRO, and the papillary thyroid cancer cell line, BHP 7-13, at 1.3 10 7 to 5 10 7 mol/L, doses that are achievable in patients. In concert with growth inhibition, SAHA down-regulated the expression of cyclin D1and up-regulated levels of p21. AnnexinV and cleavage of poly(ADP)ribose polymerase were both increased by exposure of the thyroid cancer cells to SAHA. Expression of the death receptor 5 (DR5) gene was also increased by SAHA, but the combination of the DR5 ligand, tumor necrosis factor ^ related apoptosis-inducing ligand (TRAIL), with SAHAhad little effect comparedwith SAHA alone. Of note, the combination of paclitaxel, doxorubicin, or paraplatin with SAHA enhanced cell killing of the thyroid cancer cells. In addition, murine studies showed that SAHA administered daily by i.p. injection at 100 mg/kg inhibited the growth of human thyroid tumor cells. Conclusion:Our data indicate that SAHA is a plausible adjuvant therapy for thyroid cancers. Histone acetyltransferase and histone deacetylases (HDAC) have antagonistic actions on histones depending on the state of the cells (1, 2). Often, DNA methylation and histone deacetylation of tumor suppressor genes occur in many human cancers, leading to suppression of function of these genes and thereby conferring a growth advantage for the tumor cells (3, 4). HDAC inhibitors (HDACI), such as suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), can directly interact with HDAC enzymes at the catalytic site and inhibit their functions (5, 6). This leads to acetylation of histones, which ‘‘opens up’’ the chromatin structure, allowing transcription of antigrowth and proapoptotic genes. In addition, the open chromatin structures provide additional target sites for DNA-damaging anticancer drugs, such as cisplatin and paraplatin. In thyroid cancers, current therapy uses surgery to remove all, or nearly all, of the thyroid gland. Usually, surgery is followed by I therapy to ablate any remaining thyroid cells. Chemotherapy and external beam radiotherapy have had limited use (7, 8). In this study, we show that SAHA has antitumor activities against thyroid cancers both in vitro as well as those growing in immunodeficient mice. Furthermore, SAHA, when combined with chemotherapy agents, e.g., paclitaxel, doxorubicin, or paraplatin, had enhanced anticancer activity. Materials andMethods Cell culture and treatments. Papillary thyroid cancer cell lines BHP 2-7, BHP 7-13, BHP 10-3, and BHP 18-21 (9), and the anaplastic thyroid cancer cell lines, ARO and FRO (obtained from Dr. Guy Juillard, University of California at Los Angeles, Los Angeles, CA), were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (Invitrogen), 100 units/mL penicillin, and 100 Ag/mL streptomycin. Cultures were incubated at 37jC with 5% CO2. Cultured cells were treated with the following agents either alone or in combinations as described in the text: SAHA (100 nmol/L-5 Amol/L), VPA (50 Amol/L-5 mmol/L), tumor necrosis factor – related apoptosis-inducing ligand (TRAIL; 10-40 ng/mL), paraplatin (10-100 ng/mL), doxorubicin (10-100 ng/mL), and paclitaxel (0.5-5 ng/mL). All treatments were done for the period of time indicated in the text. Clonogenic soft agar assay. Cells were plated into 24-well, flatbottomed plates using a two-layer soft agar system with 1 10 per well in a volume of 400 AL/well as previously described (10). After 14 days Cancer Therapy: Preclinical Authors’ Affiliations: Department of Medicine and the Samuel Oschin Comprehensive Cancer Center, Cedars-Sinai Medical Center, University of California at Los Angeles School of Medicine and Division of Endocrinology and Diabetes, Department of Medicine, David Geffen School of Medicine at University of California at Los Angeles and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California Received 2/15/06; revised 6/1/06; accepted 7/6/06. Grant support: Inger Foundation and the C & HKoeffler Fund. 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 with18 U.S.C. Section1734 solely to indicate this fact. Note: H.P. Koeffler is holder of the Mark Goodson Chair in Oncology Research at Cedars-Sinai Medical Center and is a member of the Jonsson Cancer Center and TheMolecular Biology Institute at University of California at Los Angeles. Current address for Q.T. Luong: Department of Hematology/Oncology, Department of Medicine, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA. Requests for reprints: QuangT. Luong, Division of Hematology/Oncology, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Davis Building, Room 5022, Los Angeles, CA 90048. Phone: 310-423-7739; Fax: 310-423-0225; E-mail: trong.luong@gmail.com. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-0367 www.aacrjournals. org Clin Cancer Res 2006;12(18) September 15, 2006 5570 Research. on January 6, 2018. © 2006 American Association for Cancer clincancerres.aacrjournals. org Downloaded from of incubation, the colonies were counted. All of the experiments were done in triplicate wells on each plate and in triplicate plates per