Mechanisms underlying hydroxamic acid analogue (HA) histone deacetylase (HDAC) inhibitors (HDIs)-induced apoptosis: New role of HDAC6 inhibition, acetylation and inhibition of hsp90 and depletion of pro-growth and pro-survival oncoproteins Free

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In the present studies we demonstrate that Hydroxamic acid analogue (HA) histone deacetylase (HDAC) inhibitors (HDIs), e.g. SAHA (suberoylanilide hydroxamic acid) and LAQ824 (cinnamic acid hydroxamate), increase the intracellular levels of p21 and p27, induce cell cycle arrest and apoptosis of human AML (cultured: HL-60, MV4-11 and U937, and primary AML), CML-BC (cultured; K562 and LAMA84, and primary CML-BC) and cultured breast cancer BT474 and LAMA84 cells. In p21−/− and p27−/− mouse embryonic fibroblasts, HA-HDI-induced cell cycle G1 arrest but not apoptosis was blocked. HA-HDIs induced mitochondrial signaling of apoptosis, which was associated with increased Bak, Bax and Bim levels, but attenuation of Bcl-2, Bcl-x_L and XIAP levels. As compared to the colon cancer HCT116 cells, HA-HDI induced significantly less apoptosis of HCT116 cells without p21, Bax, Puma and p53 expression (p<0.01); HCT116/Bax−/− cells displayed the greatest resistance to apoptosis. Conversely, HA-HDIs induced significantly more apoptosis of HCT116/XIAP−/− cells. Although HA-HDI treatment increased the levels of reactive oxygen species (ROS), this occurred after the cytosolic accumulation of pro-death proteins (e.g., cytochrome c, Smac and Omi) and caspase 3 activation. This makes it unlikely that HA-HDI-mediated ROS induction is mechanistically responsible for HA-HDI-induced apoptosis. Treatment with HA-HDIs, which inhibit HDAC class I, IIA and IIB (including HDAC6 and 10), not only induced histone H3 & H4 and α tubulin acetylation but also induced hyperacetylation of the heat shock protein (hsp) 90. This was associated with inhibition of ATP binding and chaperone function of hsp90, promoting proteasomal degradation of hsp90 client proteins, including Bcr-Abl (wild type and Gleevec-refractory mutant) and mutant FLT-3 in acute leukemia cells, Her-2 in breast cancer cells, as well as depleted AKT and c-Raf levels in both cell types. HA-HDI-mediated hsp90 acetylation was not seen with treatment with sodium butyrate and trapoxin (which does not inhibit HDAC6). Significantly, HDAC6 could be co-immunoprecipitated with hsp90. A 90% attenuation of HDAC6 by RNAi to HDAC6 induced hyperacetylation of hsp90 and α tubulin. Conversely, ectopic overexpression of HDAC6 in K562 cells inhibited HA-HDI-induced α tubulin and hsp90 acetylation. Taken together, these findings indicate that HA-HDIs induce apoptosis by multiple mechanisms: a. by inducing pro-death and depleting pro-survival proteins; b. by causing hyperacetylation of hsp90 and depleting the levels of those hsp90 client proteins to which leukemia and breast cancers may be addicted for their growth and survival.