Abstract 4872: Synergistic induction of cell death and apoptosis by the histone deacetylase inhibitor vorinostat and the demethylating agent 5-aza-2′-deoxycytidine in multiple myeloma
Authors: Jost Edgar , Schubert Claudia , Brümmendorf Tim H. , Galm Oliver 
Published: February 2011

Introduction: Hypermethylation of CpG islands in the promoter region of genes is an epigenetic modification associated with transcriptional silencing. In addition, acetylation of core histones is necessary for the maintenance of transcriptional activity of genes. DNA methylation and histone deacetylation are reversible and can be influenced by DNA methyltransferase (DNMT) inhibitors such as 5-aza-2′-deoxycytidine (DAC) and histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA, vorinostat), respectively.

Material and Methods: To investigate the in vitro effects of SAHA on the multiple myeloma (MM) cell lines U266, LP-1, RPMI-8226 or OPM-2 and assess possible interactions with DNMT inhibitors, cells were first incubated with DAC at a final concentration of 0.1 or 0.2 μM for 72 hours. After exposure to DAC, cells were incubated for 72 or 96 hours with SAHA at final concentrations between 0.1 and 20 μM. The toxic effects of the treatment were assessed by MTT assay. Induction of apoptosis was analyzed by flow cytometry with annexin V-binding. In addition, expression of the epigenetically silenced tumor suppressor genes SFRP-2 and DAB2 was determined by real-time RT-PCR.

Results: In LP-1 and U-266 cells, no relevant enhancement of the cytotoxic effect of SAHA was observed after previous exposure to DAC. In contrast, in OPM-2 and RPMI-8226 cells, a significant increase in cytotoxicity of SAHA was observed, when the cells were first incubated with DAC with a decrease of the IC50 from 6.5 μM to 2.43 μM and 10.37 μM to 4.5 μM, respectively. After sequential exposure to DAC and SAHA for 72 hours each, no change in the apoptotic cell fraction was observed for the cell lines OPM-2 and RPMI-8226. However, for U266 and LP-1, a significant increase in apoptotic cells was observed after incubation with SAHA, when the cells were previously exposed to DAC with an increase in the apoptotic cell fraction from 39.5 % to 55.4 % and 2.5 % to 14.4 %, respectively. Corresponding transcriptional silencing for SFRP-2 and DAB2 was demonstrated in untreated cells, and exposure of cell lines to DAC and SAHA resulted in reexpression. A synergism for the induction of reexpression of these genes was observed when cells were incubated with DAC and SAHA sequentially.

Discussion: After treatment with SAHA, we observed a dose-dependent induction of cell death and apoptosis as assessed by MTT and annexin V assay, respectively. In the different MM cell lines, we observed a synergism between SAHA and DAC for cytotoxic effects and induction of apoptosis. A synergism was also observed for the reexpression of epigenetically silenced genes after exposure to DAC and SAHA. These data can be considered as a basis for further in vitro studies and preclinical models with SAHA in combination with demethylating agents in order to improve therapeutic options in MM patients.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4872.