153 DIFFERENT MOLECULAR MECHANISMS FOR HISTONE DEACETYLASE INHIBITOR-INDUCED APOPTOSIS IN DOG FIBROBLASTS AND MESENCHYMAL STEM CELLS

Abstract

Histone deacetylase (HDAC) inhibitors have been applied to cancer research for a therapeutic purpose or somatic cell nuclear transfer for improvement of embryonic reprogramming. Considering the ubiquitous expression of HDAC in normal cells, effects of HDAC inhibitors on normal cells need to be evaluated in detail. Therefore, we aimed to investigate molecular mechanisms of HDAC inhibitor-induced apoptosis in mesenchymal stem cells and compare with the results from fibroblasts. Beagle skin fibroblasts (BF) and adipose-derived mesenchymal stem cell (MSC) lines were established from a 7-year-old beagle. Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and RCMEp (K-stem Cell Ltd, Seoul, Korea) were used as culture media for BF and MSC, respectively. A Food and Drug Administration-approved HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), was dissolved in dimethyl sulfoxide (DMSO). On passage 4, cells were subcultured to reach 50% of confluency, and cultured with 5 μM of SAHA. Culture medium containing the same volume of DMSO used in the SAHA group was used for the control. After 24 h, all cells were harvested, RNAs were extracted, and cDNA were synthesised. Transcript expression of anti-apoptotic genes (BFL, MCL, BCLxl, BCL2) and pro-apoptotic genes (BAX, BID, BIM) were analysed using RT-PCR. Two-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) with Bonferroni post-tests was performed for statistical analysis. Expression of MCL and BCLxl was significantly decreased in both groups. However, although BFL expression was remarkably increased only in BF (16.7-fold), BCL2 was significantly increased in MSC (7.2-fold) after SAHA treatment. Also, transcript of BAX was significantly increased in MSC (1.5-fold), and BID was significantly decreased in BF (0.3-fold). These results would be helpful to understand different molecular mechanisms of apoptosis induced by HDAC inhibitor on fibroblasts and mesenchymal stem cells.

This study was supported by RDA (#PJ010928032015), IPET (#311062-04-3SB010), NRF (#2014R1A1A2059928), Research Institute for Veterinary Science, Nestle Purina PetCare, and the BK21 plus program.