Histone Deacetylase Inhibitors and Retinoic Acids Inhibit Growth of Human Neuroblastoma In Vitro Dennis C. Coffey, MD, 1,2 Martha C. Kutko, MD, 2 Richard D. Glick, MD, 3 Steven L. Swendeman, PhD, 4 Lisa Butler, PhD, 5 Richard Rifkind, MD, 5 Paul A. Marks, MD, 5 Victoria M. Richon, PhD, 5 and Michael P. LaQuaglia, MD 4 * Background. Neuroblastoma is a common childhood cancer with a poor overall progno- sis. Retinoic acids (RAs) have been studied as a potential therapy, showing promise in recurrent disease. The histone deacetylase inhibitor (HDACI) M-carboxycinnamic acid bishydrox- amide (CBHA) is another potential therapy, which we recently described. Combinations of RAs and HDACIs currently under investigation display synergy in certain neoplasms. In this study, we evaluate the effect of combinations of RAs and HDACIs on human neuroblastoma cells. Procedure. Established cell lines were cultured in increasing concentrations of HDA- CIs, RAs, and combinations thereof. Following exposure, viable cell number was quantified by trypan blue dye exclusion on a hemacytom- eter. Cell cycle analysis was performed by propidium iodide staining and FACS. Results. All assayed HDACIs and RAs decreased viable cell number. Lower concentrations of each agent were effective when the two were com- bined. The primary reason for decreased cell number appears to be apoptosis following HDACI exposure and G1 arrest following RA exposure. Both effects are seen with cotreat- ment. Caspase inhibition abrogates the apop- totic response. Conclusions. CBHA causes ap- optosis of human neuroblastoma in vitro, an effect that can add to the effects of RA. HDACIs and RAs inhibit neuroblastoma in significantly lower concentrations when used together than when used individually. Combination therapy may improve the ultimate efficacy while reduc- ing the side effects of these agents in clinical use. Med. Pediatr. Oncol. 35:577–581, 2000. © 2000 Wiley-Liss, Inc. Key words: histone deacetylase inhibitors; retinoic acids; neuroblastoma INTRODUCTION Neuroblastoma (NB) arises in the sympathetic ner- vous system and is the most common extracranial solid tumor of childhood. Overall survival of high-risk pa- tients, despite multimodality treatment, is approximately 40% and in some series as low as 12% [1]. This dismal outlook has spurred active investigation of new agents with therapeutic potential. We recently described the ef- fect of one such agent by demonstrating apoptosis in NB cell lines treated with CBHA [2]. CBHA belongs to a class of agents that inhibit the enzyme histone deacetylase (HDAC) [3], thereby alter- ing chromatin structure and changing gene expression patterns (for reviews see Zhou et al. [4] and Kornberg and Lorch [5]). Known HDAC inhibitors (HDACIs) in- clude the butyrates [6], trichostatin A [7], and a group of hybrid polar compounds that includes CBHA and several others currently under investigation [8]. These agents in- duce accumulation of acetylated histones in a variety of cell types, presumably activating genes that affect cellu- lar growth, death, and differentiation. These effects have been observed in several types of transformed cells, in- cluding murine erythroleukemia, human bladder, my- eloma, and breast and rat prostate [6–9]. Recently, combinations of HDACIs with other known therapies, including retinoic acids (RAs), have been in- vestigated. RAs are well-studied in neuroblastoma, in- ducing growth inhibition and/or differentiation in vitro [10,11] and showing promise clinically as well [12]. RAs exert their effects via a nuclear receptor complex that interacts with the promoters of RA-responsive genes. An HDAC subunit is an integral part of this corepressor complex, which is involved in transcriptional silencing in the absence of ligand [13]. This association provides a rationale for combining HDACIs and RAs therapeuti- cally. Yu and colleagues [14] recently showed an en- 1 Department of Pediatrics, Sloan-Kettering Institute and Memorial Sloan-Kettering Cancer Center, New York, New York 2 Department of Pediatrics, Joan and Sanford I. Weill Graduate School of Medical Sciences of Cornell University, New York, New York 3 Department of Surgery, Joan and Sanford I. Weill Graduate School of Medical Sciences of Cornell University, New York, New York 4 Department of Surgery (Pediatric Surgery), Sloan-Kettering Institute and Memorial Sloan-Kettering Cancer Center, New York, New York 5 Department of Cell Biology, Sloan-Kettering Institute and Memorial Sloan-Kettering Cancer Center, New York, New York *Correspondence to: Michael P. LaQuaglia, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail: laquaglm@mskcc.org Medical and Pediatric Oncology 35:577–581 (2000) © 2000 Wiley-Liss, Inc.