The new low-toxic histone deacetylase inhibitor S-(2) induces apoptosis in various acute myeloid leukaemia cells

Abstract

Histone deacetylase inhibitors (HDACi) induce tumour cell cycle arrest and/or apoptosis, and some of them are currently used in cancer therapy. Recently, we described a series of powerful HDACi characterized by a 1,4-benzodiazepine (BDZ) ring hybridized with a linear alkyl chain bearing a hydroxamate function as Zn(++)--chelating group. Here, we explored the anti-leukaemic properties of three novel hybrids, namely the chiral compounds (S)-2 and (R)-2, and their non-chiral analogue 4, which were first comparatively tested in promyelocytic NB4 cells. (S)-2 and partially 4--but not (R)-2--caused G0/G1 cell-cycle arrest by up-regulating cyclin G2 and p21 expression and down-regulating cyclin D2 expression, and also apoptosis as assessed by cell morphology and cytofluorimetric assay, histone H2AX phosphorylation and PARP cleavage. Notably, these events were partly prevented by an anti-oxidant. Moreover, novel HDACi prompted p53 and α-tubulin acetylation and, consistently, inhibited HDAC1 and 6 activity. The rank order of potency was (S)-2 > 4 > (R)-2, reflecting that of other biological assays and addressing (S)-2 as the most effective compound capable of triggering apoptosis in various acute myeloid leukaemia (AML) cell lines and blasts from patients with different AML subtypes. Importantly, (S)-2 was safe in mice (up to 150 mg/kg/week) as determined by liver, spleen, kidney and bone marrow histopathology; and displayed negligible affinity for peripheral/central BDZ-receptors. Overall, the BDZ-hydroxamate (S)-2 showed to be a low-toxic HDACi with powerful anti-proliferative and pro-apototic activities towards different cultured and primary AML cells, and therefore of clinical interest to support conventional anti-leukaemic therapy.

Fig 1

Chemical structures of compounds used in this work. The numbers labelling the compounds are the same as in ref. [11].

Fig 2

Biological effects of (S)-2, (R)-2 and 4 on NB4 cells. (A) Growth curves: NB4 cells (1×10⁻⁵/ml) were incubated up to 96 hrs without/with increasing concentrations of either (S)-2, (R)-2 or 4. Cells densities were evaluated daily with the aid of a Bürker chamber and reported as results of a typical experiment out of three. (B) Cell cycle analysis was performed on propidium iodide (PI)-stained NB4 cells treated without/with 2 μM drugs for 24 hrs, by flow cytometry (PI labelling: X-axis; total events: Y-axis). (C) Cyclin D2, cyclin G2 and p21 mRNA levels from cells treated without/with 2 μM drugs for 6, 15 and 24 hrs were measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR; S1). Columns and bars were the means ± SD of three separate experiments and asterisks indicated the significant differences between treated and untreated cells (***P < 0.001; *P < 0.05).

Fig 3

(A-top) Cytofluorimetric analysis of apoptosis. NB4 cells were treated without/with 2.5 μM drugs for 48 hrs and incubated with AnnexinV-Fluos in a HEPES buffer containing PI for 15 min.; the number of apoptotic cells was then measured by flow cytometry (FACScan equipment). X axis: % of apoptotic cells; Y axis: conditions of stimulation; bars were the means ± SD of three separate experiments. (A, bottom) Cell morphology. Cytosmears of untreated and drug-treated NB4 cells as above were stained with May-Grünwald/Giemsa. Pictures (magnification: ×200) were from a typical experiment out of three. Drug-induced PARP cleavage (at 15 hrs, B) to yield a fragment of approximately 89 kD, and H2AX phosphorylation (at 24 hrs, C) were observed by Western blot and immunostaining; α-tubulin was used as the reference protein.

Fig 4

Drug-induced acetylation of histone and nonhistone proteins in NB4 cells. (A) Cells were incubated for 6 hrs without/with 2 μM (S)-2 or (R)-2 or 4, and then processed by Western blot and immunostained for acetylated H3/H4; H3/H4 were the loading controls. Cell extracts were also analysed by immunoblotting for (B) acetylated forms of nonhistone proteins α-tubulin and p53 after 6 and 24 hrs of treatment, respectively; α-tubulin and p53 were used as the reference proteins. (C) Drug-induced inhibition of human recombinant HDAC1 and HDAC6 activity was determined by a fluorimetric cell-free assay (S2); IC₅₀ values were from a typical experiment out of three.

Fig 5

(S)-2 triggers apoptosis in different cultured and primary AML cells. (A) Different AML cell lines were treated for 2 days without/with increasing (S)-2 concentrations and then submitted to the Annexinn V/PI assay to determine the amount of apoptotic cells within the population. IC₅₀ values represent the drug concentration (μM) needed for inducing 50% of cell population toward apoptosis relative to control; values were the means ± SD of three separate experiments. (B) Pro-apoptotic effects of (S)-2 in NB4 cells developed through the drug-mediated generation of ROS. Activation of apoptosis was revealed at 15 hrs, by the cleavage of PARP and phosphorylation of H2AX protein; and these events were efficiently contrasted by 15 mM N-Acetyl Cysteine (NAC) applied 2 hrs before drug addition; α-tubulin was used as the reference protein. (C) Ex vivo experiments on human different primary AML cells. May-Grünwald/Giemsa stained cytosmears of blasts (magnification: ×200) from peripheral blood of four newly diag nosed AML patients with M1, M2, M3 and M4 subtypes after 48 hrs of treatment without/with 1 and 2 μM drug. Peripheral blood mononuclear cells (PBMCs) from normal donors have also been isolated and treated as above after a 12-hr stimulation with PHA (125 μg/ml) and IL-2 (1 ng/ml).

Fig 6

(A) (S)-2 is well tolerated in normal CD-1 mice which have been used as the model for acute toxicity experiments. Animals were injected i.p. only once with increasing (S)-2 concentrations dissolved in 0.1 ml DMSO and sacrificed after a week (S3). Pictures (magnification: ×200) reported in this panel concern the histopathology of organs explanted from mice which were treated with the higher drug dosage, i.e. 150 mg/kg, corresponding to about 5 mg/mouse. No specific drug-induced tissue alteration in treated versus mice treated with the vehicle alone was observed. Consecutive 2.5–5 μm sections of samples were stained with May-Grünwald/Giemsa and examined under a bright field microscope (Nikon Eclipse, mod. 50i) equipped with a digital camera (DS-5M USB2; Nikon Instruments, Florence, Italy). (B) Binding experiments in tissue extracts. The affinity of novel HDACi, at the two concentrations used (10 and 100 μM, respectively) for CBR was assayed by using membranes from rat cerebral cortex that were prepared as described by Mehta and Shank [17], while for PBR, rat kidney mitochondrial membrane preparations as described by Miccoli et al. were employed [18]. Bars were the means ± SE of three separate experiments; for details see S4.