Novel Histone Deacetilase (HDAC) Inhibitors: In Vitro Effects on Leukemic Cells

Abstract 4946

Histone deacetylase inhibitors (HDAC-I) are a class of agents with the capacity to induce acetylation of histone and non-histone proteins. These molecules 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 to clinically available HDAC-I have been obtained only in a proportion of patients, prompting further studies aimed at identifying more active compounds and at defining the molecular mechanisms of response to this class of agents. Acetyl-L-carnitine (ALCAR) is a metabolic intermediate that facilitates the influx and efflux of acetyl groups across the mitochondrial inner membrane, thereby contributing to the regulation of energy production and metabolism. ALCAR activity as a modulator of cellular stress response has prompted its use to protect against chemotherapy-induced neurotoxicity. However, ALCAR effects on neoplastic cells are still not defined, especially in combination with chemotherapy. Here we investigated the effects of MS-275, a HDAC-I, on cell proliferation and apoptosis in cell line models of acute myeloid leukemia (AML) acute lymphoblastic leukemia (ALL), and multiple myeloma (MM), in comparison with vorinostat also known as SAHA (suberoyl anilid hydroxamic acid), the most widely used HDAC-I in clinical setting. HDAC-I were tested at doses ranging from 5 to 5000nM. In addition, the effects of simultaneous exposure to 10 mM of ALCAR and selected sub-toxic concentration of HDAC-I were analyzed. The cytotoxic effect of the treatment was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (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 indicated that the tested compound MS-275 significantly inhibited cell growth, as assessed by MTT assay, when used at of 5000nM. Comparative analysis of the efficacy of the two different HDAC-I compounds indicated that MS-275 was the more effective agent and the only one with 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), U937 and HL60 myeloid cell lines, the lymphoid cell line Jurkat and the MM cell line ARH-77 showed intermediate sensitivity (IC50: < 1000 nM), while the lymphoid cell line CEM R was resistant (IC50 > 10 uM). 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% reduction compared to the control). Nevertheless, the combination of 500 nM SAHA with 10mM ALCAR reveled a synergistic interaction, with a 46% reduction in absorbance. We then analyzed the effects on apoptosis induction, as determined by the percentage of cells with a sub-G1 DNA content. 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 of 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. In summary, our results show that the HDAC-I MS-275 is a potent inhibitor of leukemic cell growth, capable of inducing apoptosis particularly in cell lines derived from myeloid leukemia and MM. Preliminary studies exploring the combined use of ALCAR with the SAHA support a potential anti-neoplastic synergism in selected hematological malignancies.