[CANCER RESEARCH 64, 689 – 695, January 15, 2004] Selective Growth Inhibition of Tumor Cells by a Novel Histone Deacetylase Inhibitor, NVP-LAQ824 Peter Atadja, Lin Gao, Paul Kwon, Nancy Trogani, Heather Walker, Meier Hsu, Lakshmi Yeleswarapu, Nagarajan Chandramouli, Larry Perez, Richard Versace, Arthur Wu, Lidia Sambucetti, Peter Lassota, Dalia Cohen, Kenneth Bair, Alexander Wood, and Stacy Remiszewski Department of Oncology, Novartis Institutes for Biomedical Research, East Hanover, New Jersey ABSTRACT We have synthesized a histone deacetylase inhibitor, NVP-LAQ824, a cinnamic hydroxamic acid, that inhibited in vitro enzymatic activities and transcriptionally activated the p21 promoter in reporter gene assays. NVP-LAQ824 selectively inhibited growth of cancer cell lines at submi- cromolar levels after 48 –72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. Flow cytometry studies revealed that both tumor and normal cells arrested in the G 2 -M phase of the cell cycle after compound treatment. However, an increased sub-G 1 population at 48 h (reminiscent of apoptotic cells) was observed only in the cancer cell line. Annexin V staining data supported our hypothesis that NVP-LAQ824 induced apoptosis in tumor and transformed cells but not in normal cells. Western blotting experiments showed an increased histone H3 and H4 acetylation level in NVP-LAQ824-treated cancer cells, suggesting that the likely in vivo target of NVP-LAQ824 was histone deacetylase(s). Finally, NVP-LAQ824 exhibited antitumor effects in a xenograft animal model. Together, our data indicated that the activity of NVP-LAQ824 was con- sistent with its intended mechanism of action. This novel histone deacety- lase inhibitor is currently in clinical trials as an anticancer agent. INTRODUCTION Coordinated and precise regulation of gene expression is critical for maintenance of normal cell growth and differentiation (reviewed in Ref. 1). In eukaryotic cells, DNA is packaged into chromatin, and to alter gene expression, a dynamic process is required: local remodeling of nucleosomes (1). Reversible acetylation is one such process facil- itated by histone acetyltransferases and histone deacetylases (HDACs). Transcriptionally active chromatin regions, such as the euchromatin, are associated with hyperacetylated histones, whereas transcriptionally silent heterochromatin regions are generally hy- poacetylated (reviewed in Ref. 1). Both histone acetyltransferases and HDACs are recruited to target genes in complexes with sequence- specific factors and cofactors to regulate gene expression and ulti- mately cell function. Inappropriate expression of genes required for cell proliferation, differentiation, or tumor suppression has been linked to cancer. Sev- eral lines of evidence suggest that aberrant recruitment of HDACs and the resulting chromatin modifications may lead to changes in gene expression seen in transformed cells: (a) silencing of tumor suppres- sor genes at the chromatin level (2– 8); (b) interaction of HDAC- containing complexes with proteins involved in tumorigenesis (9 –11); (c) reports of HDAC inhibitors (HDACIs) having significant antipro- liferative effects, such as promoting differentiation, cell cycle arrest, or apoptosis (12–17); and (d) induction of key mediators of G 1 cell cycle arrest and differentiation, such as p21 with HDACIs (12, 18 – 21). Studies of hematological malignancies also support involvement of HDACs in cancer development and/or maintenance. Aberrant re- cruitment of HDAC complexes is necessary for the carcinogenic properties of the fusion proteins PLZF-RAR, PML-RAR, and AML1/ETO in acute leukemia (reviewed in Ref. 22). In t(8;21) acute myelocytic leukemia (AML), HDAC-mediated repression of target genes such as AML1 blocked differentiation of hematopoietic precur- sors (23, 24). In acute promyelocytic leukemia with t(11;17)/PLZF- RAR translocation, a combination of all-trans retinoic acid with the HDACI trichostatin resulted in differentiation of all-trans retinoic acid-resistant acute promyelocytic leukemia cells (25). Furthermore, treatment of leukemic blasts obtained from non-acute promyelocytic leukemia AML patients with a combination of retinoic acid and trichostatin induced terminal myeloid differentiation (26). Preclinical experiments using small molecule inhibitors of HDACs, such as MS-275 and suberoylanilide hydroxamic acid (SAHA), ex- hibited efficacy against several human tumor xenografts in athymic mice (19, 27). In addition, the natural product HDACIs of the trapoxin class and trichostatin were shown to activate the p21 promoter, increase p21 protein levels, inhibit cyclin-dependent kinase 2 kinase activity, reduce retinoblastoma (Rb) phosphorylation, and cause cell cycle arrest or apoptosis in three human tumor cell lines (21, 28, 29). NVP-LAQ824, our novel synthetic HDACI, showed similar proper- ties at submicromolar concentrations. Our studies also indicated that NVP-LAQ824 induced apoptosis in tumor cells but cell cycle arrest in normal fibroblasts. Moreover, increased histone acetylation in NVP- LAQ824-treated cancer cells confirmed the activity of our compound against HDACs. We therefore conclude that the antitumor effect seen was, in fact, attributable to specific inhibition of HDAC. MATERIALS AND METHODS Materials. NVP-LAQ824 was prepared in-house as the lactate salt and dissolved in DMSO. MS-275 was prepared as described in Suzuki et al. (29) as a free base and was dissolved in DMSO. Suberoylanilide hydroxamic acid (SAHA) was prepared as described in Richon et al. (30) and was dissolved in DMSO. The ECF Western blotting reagent pack for mouse or rabbit was purchased from Amersham Pharmacia Biotech Inc. (Piscataway, NJ). Pre-cast NuPAGE gels were from Invitrogen Life Technologies, Inc. (Carlsbad, CA). The CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay was from Promega (Madison, WI). The ApoAlert Annexin V Apoptosis kit was from Clontech Laboratories, Inc. (Palo Alto, CA). The Hybond-P, polyvinylidene difluoride, membrane was purchased from Amersham Biosciences (Piscat- away, NJ). Cell Culture. H1299, HCT116, A549, DU145, PC3, and MDA435 cells were obtained from American Type Culture Collection (Rockville, MD) and were maintained according to the supplier’s instructions. Normal dermal human fibroblast (NDHF) and normal human bronchial epithelial (NHBE) cells were obtained from Clonetics (San Diego, CA) and were maintained in DMEM supplemented with 15% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, and BEGM BulletKit (Clonetics, San Diego, CA) supplemented with retinoic acid. hTERT/SV40 T antigen-transformed NHBE (hTERT/SV40 NHBE) cells were a kind gift from Dr. Barrett Rollins (Dana- Farber Cancer Institute, Harvard Medical Center, Boston, MA) and were maintained in BEGF BulletKit supplemented with retinoic acid. Received 7/9/03; revised 11/7/03; accepted 11/10/03. 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. Requests for reprints: Peter Atadja, Oncology Molecular and Cellular Biology Unit, Novartis Institutes for Biomedical Research, East Hanover, NJ 07936. Phone: (862) 778- 0435; Fax: (973) 781-7578; E-mail: peter.atadja@pharma.novartis.com. 689 Downloaded from http://aacrjournals.org/cancerres/article-pdf/64/2/689/2518989/zch00204000689.pdf by guest on 29 June 2023