The Therapeutic Potential of AN-7, a Novel Histone Deacetylase Inhibitor, for Treatment of Mycosis Fungoides/Sezary Syndrome Alone or with Doxorubicin.

The 2 histone deacetylase inhibitors (HDACIs) approved for the treatment of cutaneous T-cell lymphoma (CTCL) including mycosis fungoides/sezary syndrome (MF/SS), suberoylanilide hydroxamic acid (SAHA) and romidepsin, are associated with low rates of overall response and high rates of adverse effects. Data regarding combination treatments with HDACIs is sparse. Butyroyloxymethyl diethylphosphate (AN-7) is a novel HDACI, which was found to have selective anticancer activity in several cell lines and animal models. The aim of this study was to compare the anticancer effects of AN-7 and SAHA, either alone or combined with doxorubicin, on MF/SS cell lines and peripheral blood lymphocytes (PBL) from patients with Sezary syndrome (SPBL). MyLa cells, Hut78 cells, SPBL, and PBL from healthy normal individuals (NPBL) were exposed to the test drugs, and the findings were analyzed by a viability assay, an apoptosis assay, and Western blot. AN-7 was more selectively toxic to MyLa cells, Hut78 cells, and SPBL (relative to NPBL) than SAHA and also acted more rapidly. Both drugs induced apoptosis in MF/SS cell lines, SAHA had a greater effect on MyLa cell line, while AN-7 induced greater apoptosis in SPBL; both caused an accumulation of acetylated histone H3, but AN-7 was associated with earlier kinetics; and both caused a downregulation of the HDAC1 protein in MF/SS cell lines. AN-7 acted synergistically with doxorubicin in both MF/SS cell lines and SPBL, and antagonistically with doxorubicin in NPBL. By contrast, SAHA acted antagonistically with doxorubicin on MF/SS cell lines, SPBL, and NPBL, leaving <50% viable cells. In conclusion, AN-7 holds promise as a therapeutic agent in MF/SS and has several advantages over SAHA. Our data provide a rationale for combining AN-7, but not SAHA, with doxorubicin to induce the cell death in MF/SS

Effect of SAHA and AN-7 on the viability of MF/SS cell lines, SPBL and NPBL.

<p>Viability curves based on the MTT assay of MyLa cells, Hut78 cells, (a,b), and SPBL (n = 3) (c,d) compared to NPBL (n = 8) following treatment with SAHA (a,c) and AN-7 (b,d) for 72 h. Also shown are the IC₅₀ and SI values of SAHA and AN-7 in MF/SS cell lines and SPBL and NPBL based on viability curves a-d, and their p values (e).</p

Apoptosis induction of AN-7 and SAHA in SPBL.

<p>PBL from 2 SS patients were plated at a concentration of 0.5x10⁶ cells/mL, and were then treated with SAHA 4 μM or AN-7 200 μM for 48 h. The cells were then stained with annexin V and PI. FACS plots are shown with percent of cells in each quadruplet, and the percent of cells in apoptotic cells (early + late apoptosis) are shown also in column.</p

Toxic and apoptotic effect of SAHA and AN-7 on MF/SS cell lines as a function of exposure time.

<p>Viability curves based on trypan blue staining of MyLa and Hut78 cells following short or long exposure to SAHA (a, c) or AN-7 (b, d). IC₅₀ values of short and long exposure to SAHA and AN-7 in MF/SS cell lines based on viability curves a-d (e). Apoptosis curves based on FACS analysis of annexin V and PI staining (f-i). Percent of apoptotic MyLa cells (early + late apoptosis) after short or long exposure to SAHA (f) or AN-7 (g), and apoptotic Hut78 cells after short or continuous exposure to SAHA (h) or AN-7 (i).</p

Effect of SAHA and AN-7 on specific protein expression and modification in MF/SS cell lines.

<p>Immunoblot of apoptotic and proapoptotic proteins in MyLa and Hut78 cells treated with SAHA 10 μM or AN-7 300 μM for the indicated periods (a). Basal HDAC1 protein expression in NPBL and MF/SS cell lines (b) and in MF/SS cell lines treated with SAHA 10 μM or AN-7 300 μM (c). Acetylated H3 in the nuclear lysate of MF/SS cell lines treated with and the same concentrations of SAHA and AN-7 for the indicated periods (d).</p