Novel Histone Deacetilase (HDAC) inhibitors: in vitro effects on leukemic cells.

Introduction. Histone deacetylase inhibitors (HDAC-I) are a class of agents that have been intensively investigated in a variety of malignancies, because of their ability to inhibit proliferation, induce differentiation and apoptosis in tumor cells. However, clinical response have been obtained only in a proportion of patients, prompting further studies aimed at identifying more active compounds. Since acetyl-l-carnitine (ALCAR) is a metabolic intermediate that facilitates the influx/efflux of acetyl groups across the mitochondrial inner membrane, studies are ongoing to evaluate its activity as modulator of cellular stress response, in combination with chemotherapy. Here we investigated the effects of two HDAC-I, MS-275 and vorinostat (SAHA), alone or in combination with ALCAR, on cell proliferation and apoptosis in cell line models of haematological malignancies: acute myeloid leukemia (AML) acute lymphoblastic leukemia (ALL), and multiple myeloma (MM). Methods. HDAC-I were tested at doses ranging from 5 to 5000nM. In addition, the effects of simultaneous exposure to 10 mM of ALCAR and sub-toxic concentrations of HDAC-I were analyzed. The cytotoxic effect of the treatment was assessed by MTT assay. The drug concentration inducing 50% cell killing (IC-50) was calculated from the dose-response curve. Cell cycle inhibition and induction of apoptosis were analyzed by flow cytometry using the Acridine-Orange (AO) technique. Results. Comparative analysis of the efficacy of the two different HDAC-I indicated that MS-275 was the more effective agent with a clear dose-dependent activity, while SAHA displayed a flat dose-response curve, which dropped only at the highest concentration. In particular, the myeloid cell line Molm-13 was strikingly sensitive to MS-275 (IC50: < 15 nM), while the lymphoid cell line CEM R was resistant (IC50 > 10 uM). Other different cell lines exhibited intermediate sensitivity (IC50: < 1000 nM). SAHA showed no activity in U937 cells when used at concentrations ranging from 100 to 1000 nM, with a dramatic reduction of absorbance at 5000 nM (>80%). Nevertheless, the combination of 500 nM SAHA with 10mM ALCAR revealed a synergistic interaction, with a 46% reduction in absorbance. We then analyzed the effects on apoptosis induction. MS-275 dose-dependently induced apoptosis in HL-60 cells (4.2%, 17.1%, 60.8%, and 87.5% in the presence of 100, 500, 1000, 5000 nM MS-275, respectively). Conversely, SAHA induced minimal apoptosis (< 10%) at concentration ranging from 100 to 1000 nM, although > 75% of cells became apoptotic after treatment with the compound at 5000 nM. Conclusions. Our results show that the HDAC-I MS-275 is a potent inhibitor of leukemic cell growth, capable of inducing apoptosis particularly in AML and MM cell lines. Preliminary studies exploring the combined use of ALCAR with the SAHA support a potential anti-neoplastic synergism in hematological malignancies.