Evaluation of the In vitro and In vivo Antitumor Activity of Histone Deacetylase Inhibitors for the Therapy of Retinoblastoma Clifton Lee Dalgard, Kurtis R.Van Quill, and Joan M. O’Brien Abstract Purpose: To evaluate the potential utility of histone deacetylase inhibitors (HDACi) for treatment of retinoblastoma (RB). Experimental Design: Growth-inhibitory effects of HDACi [trichostatin A (TSA), suberoylani- lide hydroxamic acid (SAHA), or MS-275] were assessed in human and transgenic murine RB cells. Effects of TSA and MS-275 were also assessed in combination with standard therapeutic agents for RB. Proapoptotic effects of MS-275 and TSA were evaluated by caspase-3/7 activity, Annexin V translocation, and/or Bim expression analyses. Effects of MS-275 on cell cycle distri- bution and reactive oxygen species levels were determined by flow cytometry. Retinal tissue morphology was evaluated in mice after local administration of MS-275. Analysis of retinal ace- tyl-histone levels was used to assess MS-275 delivery after systemic administration. Therapeutic effects of MS-275 were determined in transgenic mouse and rat ocular xenograft models of RB after i.p. injection of 20 mg/kg every other day for 21or 13 days, respectively. Results: TSA, SAHA, and MS-275 dose dependently reduced RB cell survival. TSA and MS-275 showed additive growth-inhibitory effects in combination with carboplatin, etoposide, or vincristine. TSA and MS-275 increased caspase-3/7 activity. MS-275 increased Annexin V membrane translocation and induced G 1 arrest. Cytotoxicity of MS-275 was dependent on increased reactive oxygen species levels and was reversed by antioxidant pretreatment. Intra- ocular administration of 1 A L of 10 Amol/L MS-275 did not alter ocular tissue morphology. Increased acetyl-histone levels confirmed MS-275 delivery to retinal tissue after systemic admin- istration. MS-275 significantly reduced tumor burden in both mouse and rat models of RB. Conclusions: HDACi should be considered for clinical trials in children with RB. Cancer is a genetic disease. Tumorigenesis arises from a stepwise accumulation of DNA alterations resulting in gain of function of oncogenes or loss of function of tumor suppressor genes (1). Epigenetic changes, such as DNA methylation and histone acetylation/deacetylation, also contribute to tumor development by regulating the transcriptional activity of genes, including those important for cellular proliferation, differ- entiation, and survival (2 – 4). Histone acetylases and histone deacetylases (HDACs) regulate transcription through the transfer and removal of acetyl groups on lysine residues at the NH 2 terminus of histones, the major protein component of chromatin (5). In general, acetylated histones force chromatin into an open conformational state that promotes gene tran- scription, whereas deacetylated histones maintain chromatin in a condensed state that silences transcription. Histone acetylases and HDACs target many other proteins in addition to histones (6), and deregulation of these interactions can also be tumo- rigenic (7, 8). Nonhistone targets of histone acetylases and HDACs include numerous proteins implicated in tumor initia- tion or progression, including p53, c-MYC, nuclear factor-nB, and E2F. Retinoblastoma (RB) affects 1 in 15,000 children and accounts for 12% of infant cancers (9, 10). Standard treatment options for this disease are limited and associated with signi- ficant toxicities. First-line therapy at most centers includes combination chemotherapy with adjuvant focal therapy (laser therapy, cryotherapy, or brachytherapy). The standard regimen is carboplatin and etoposide or teniposide, with or without vincristine (11). Carboplatin is associated with nephrotoxicity as well as ototoxicity (12), a particularly undesirable outcome in children with this blinding eye disease. Whereas two recent studies have found no association between carboplatin treat- ment and hearing loss in these patients (13, 14), a 5.6% rate of carboplatin ototoxicity has been reported in children treated at other centers (11). This discrepancy may be attributable to variations in schedule and dosing. Etoposide and teniposide are topoisomerase II inhibitors, which have been associated with the induction of secondary leukemias (15). A recent survey Cancer Therapy: Preclinical Authors’ Affiliation: Ocular Oncology Unit, Department of Ophthalmology, University of California at San Francisco, San Francisco, California Received 12/17/07; revised 1/14/08; accepted 2/20/08. Grant support: National Eye Institute RO1Grant EY13812 and Core Grant EY02162, Wayne and Gladys Valley Foundation, Knights Templar Eye Foundation postdoctoral fellowship (C.L. Dalgard), Research to Prevent Blindness, and That Man May See Foundation. 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: Joan M. O’Brien, Ocular Oncology Unit, Department of Ophthalmology, University of California at San Francisco, 10 Koret Way, Box 0730, San Francisco, CA 94143. Phone: 415-502-1867; Fax: 415-476-0336; E-mail: obrienj@vision.ucsf.edu. F 2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-4836 www.aacrjournals.org Clin Cancer Res 2008;14(10) May 15, 2008 3113 Research. on June 13, 2020. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from