Molecular mechanisms of underlying synergistic effect of combinations of histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) with docetaxel and trastuzumab against human breast cancer cells

560 Background: Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor (HDI), which inhibits class I and II HDACs, induces hyperacetylation of the core nucleosomal histones, increases p21WAF1, and induces growth arrest and apoptosis of human breast cancer cells. In the present studies, we determined the effects of SAHA on Her-2, AKT and c-Raf (all hsp90 client proteins) levels, as well as on the cytotoxic effects of docetaxel and trastuzumab on human breast cancer BT-474 and SKBR-3 cells containing Her-2 amplification. Methods: The cells were treated with SAHA (1.0 to 5.0 μM) and/or trastuzumab (10 to 20 μg/ml) or docetaxel (5 to 20 nM) for 24 to 48 hours. Following this, % of apoptotic cells was determined or Western blot analyses of p21, p27, acetylated histones H3 and H4, AKT, c-Raf, and Her-2, as well as of Bcl-2, Bcl-xL, Bak, Bax, Bim, XIAP and survivin were performed. Attenuation of HDAC6 levels was achieved by siRNA to HDAC6 and estimated by immunoblot analysis. Synergistic interaction between drugs was evaluated by median dose-effect and isobologram analysis. Results: Treatment with SAHA upregulated p21 and p27 levels, increased the % of cells in G2M phase of the cell cycle, as well as induced apoptosis of the cells. This was associated with upregulation of pro-death Bak and Bim, as well as attenuation of the levels of pro-survival XIAP, survivin, Bcl-2, Bcl-xL and Her-2 proteins. SAHA treatment induced acetylation of hsp90. Attenuation of HDAC6 levels by siRNA to HDAC6 also caused acetylation of hsp90, indicating the mechanistic role of HDAC6 in SAHA-mediated acetylation of hsp90. This reduced the chaperone association of Her-2 with hsp90, which promoted the degradation of Her-2 by the proteasome. Co-treatment with SAHA and docetaxel or trastuzumab exerted synergistic apoptotic effects. Co-treatment with SAHA and trastuzumab caused greater attenuation of Her-2, p-AKT, as well as greater induction of p27 and Bim. Conclusions: These pre-clinical findings support the development of SAHA in combination with docetaxel and/or trastuzumab against Her-2 amplified breast cancer. No significant financial relationships to disclose.