trichostatin-a and Retinoblastoma

Retinoblastoma, an intraocular malignant tumor of childhood, is caused by a mutation in the retinoblastoma tumor-suppressor gene RB. Retinoblastoma cells are thought to be resistant to transforming growth factor-beta (TGF-beta) because they do not express the TGF-beta type II receptor (TbetaR-II). In several tumor cell lines, trichostatin A (TSA), a potent inhibitor of histone deacetylase, induces expression of the TbetaR-II gene. The objective of the present study was to determine the effects of TSA on TbetaR-II gene expression in retinoblastoma cells.. Four retinoblastoma cell lines were transfected with a TbetaR-II promoter-luciferase reporter construct and analyzed for the effect of TSA on TbetaR-II mRNA expression, TbetaR-II promoter activity, transforming growth factor (TGF)-beta-related signal transduction, and cell growth using RT-PCR, Western blot analysis, chromatin immunoprecipitation, luciferase activity assay, and cell viability assays.. TSA treatment induced the expression of TbetaR-II mRNA, activated the TbetaR-II promoter, and inhibited cell growth in the examined retinoblastoma cell lines. It did not restore TGF-beta-related signaling, however.. These data show that TSA induces the expression of TbetaR-II mRNA and activates the TbetaR-II promoter in retinoblastoma cells. However, TSA treatment alone was insufficient to restore TGF-beta signaling in these cell lines. The inhibitory effect of TSA on cell growth may be unrelated to its effect on TbetaR-II expression.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; DNA Primers; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Retinal Neoplasms; Retinoblastoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad Proteins; Transfection

To evaluate the potential utility of histone deacetylase inhibitors (HDACi) for treatment of retinoblastoma (RB).. Growth-inhibitory effects of HDACi [trichostatin A (TSA), suberoylanilide 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 distribution 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 acetyl-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 21 or 13 days, respectively.. 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 G1 arrest. Cytotoxicity of MS-275 was dependent on increased reactive oxygen species levels and was reversed by antioxidant pretreatment. Intraocular administration of 1 microL of 10 micromol/L MS-275 did not alter ocular tissue morphology. Increased acetyl-histone levels confirmed MS-275 delivery to retinal tissue after systemic administration. MS-275 significantly reduced tumor burden in both mouse and rat models of RB.. HDACi should be considered for clinical trials in children with RB.

Topics: Animals; Annexin A5; Antineoplastic Agents; Apoptosis; Benzamides; Blotting, Western; Caspase 3; Caspase 7; Cell Proliferation; Cell Survival; Enzyme Inhibitors; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; In Vitro Techniques; Mice; Mice, Transgenic; Polymerase Chain Reaction; Pyridines; Rats; Reactive Oxygen Species; Retinal Neoplasms; Retinoblastoma; Vorinostat; Xenograft Model Antitumor Assays