Tubacin suppresses proliferation and induces apoptosis of acute lymphoblastic leukemia cells

Abstract

Over the past decade, histone deacetylase inhibitors have increasingly been used to treat various malignancies. Tubacin (tubulin acetylation inducer) is a small molecule that inhibits histone deacetylase 6 (HDAC6) and induces acetylation of α-tubulin. We observed a higher antiproliferative effect of tubacin in acute lymphoblastic leukemia (ALL) cells than in normal hematopoietic cells. Treatment with tubacin led to the induction of apoptotic pathways in both pre-B and T cell ALL cells at a 50% inhibitory concentration (IC(50)) of low micromolar concentrations. Acetylation of α-tubulin increases within the first 30 min following treatment of ALL cells with tubacin. We also observed an accumulation of polyubiquitinated proteins and poly(ADP-ribose) polymerase (PARP) cleavage. Furthermore, the signaling pathways activated by tubacin appear to be distinct from those observed in multiple myeloma. In this article, we demonstrate that tubacin enhances the effects of chemotherapy to treat primary ALL cells in vitro and in vivo. These results suggest that targeting HDAC6 alone or in combination with chemotherapy could provide a novel approach to treat ALL.

Figure 1

Tubacin inhibits cell growth in ALL cells. Chemical structures of (A) tubacin and (B) its inactive carboxylic acid analog, niltubacin. (C) Effects of tubacin treatment for 72 h on proliferation of lymphoblastic leukemia cell lines. IC₅₀ = 2 μM for Jurkat cells, IC₅₀ = 1.2 μM for Loucy cells, IC₅₀ = 1.2 μM for Nalm-6 cells, and IC₅₀ = 1.4 μM for REH cells. Proliferation measurement was performed by the MTT method. Absorbance at 540 nm correlates with living cells. The bars in the graphs show the mean values of a single experiment performed in triplicate, representative of at least three independent experiments.

Figure 2

(A) Sensitivity of normal lymphocytes and bone marrow hematopoietic stem cells to tubacin treatments. MTT assay of normal T lymphocytes (2 ×10⁶ cells/mL) treated with DMSO or increasing concentrations of tubacin for 0 and 72 h. IC₅₀ = 16 μM for normal lymphocytes and IC₅₀ = 20 μM for colony formation in bone marrow hematopoietic stem cells. (B) Comparison of the effect in proliferation by tubacin and niltubacin treatments. Proliferation measurement was performed by the MTT method. Absorbance at 540 nm correlates with living cells. The bars in the graphs show the mean values of a single experiment performed in triplicate, representative of at least three independent experiments.

Figure 3

Tubacin treatment induces apoptosis specifically in leukemia cells. (A) Western blot of PARP protein cleavage after treating Jurkat and Nalm-6 with 2.5 μM of tubacin for 24 h. (B) Comparison of cell cycle profile by flow cytometry, corresponding to Jurkat and Nalm-6 cells treated with 2.4 μM tubacin for 24 h. Cells were stained with propidium iodide, showing the 2n and 4n DNA picks. Apoptosis is indicated by a black arrow as a sub-2n population. The histograms show a single experiment, representative of at least three independent experiments with similar findings. Induction of acetylated tubulin and polyubiquitinated protein accumulation by tubacin treatments in leukemia cell lines. (C) Western blot of acetylated α-tubulin protein levels after treating Jurkat, Loucy, and Nalm-6 cells with 2.5 μM of tubacin for the indicated times. (D) Western blot of polyubiquitinated protein levels after treating Jurkat and Loucy cells with 2.5 μM of tubacin for the indicated times. These experiments were repeated three times.

Figure 4

Polyubiquitinated protein accumulation by tubacin treatments in leukemia cells induces apoptosis through a SAPK/JNK-independent signaling pathway. (A) Western blots of acetylated α-tubulin, β-tubulin, polyubiquitinated protein, PARP cleavage, phospho-SAPK/JNK, total SAPK/JNK, 63Ser phospho-Jun, 73Ser phospho-Jun, and c-Jun protein levels after treating Jurkat with the indicated compounds and concentrations for 14 h. (B) Western blot of PARP protein cleavage after treating Jurkat cells with 2.5 μM of tubacin for the indicated times.

Figure 5

Accumulation of acetylated α-tubulin in Jurkat cells by tubacin correlates in time with an inhibition of cellular K⁺/Na⁺ transportation, an increase of cytosolic concentration of calcium and apoptosis. (A) Effect of increasing concentrations of tubacin in K⁺/Na⁺ transportation in whole cells. (B) Effect of increasing concentrations of tubacin assayed directly on K⁺/Na⁺-ATPase activity. (C) Densitometry corresponding to a Western blot of acetylated α-tubulin protein levels after treating Jurkat and Loucy with increasing concentrations of tubacin for 30 min. (D) Western blot of PARP protein cleavage after treating Jurkat with increasing concentrations of tubacin for 14 h. (E) Levels of cytoplasmic calcium in tubacin-treated Jurkat and Nalm-6 cells.

Figure 6

Tubacin enhances the effect of chemotherapy and small molecules in vitro and in vivo. Tubacin increases apoptosis of ALL cells with vincristine and bortezomib (Velcade). Nalm-6 human pre-B ALL cells were treated with either (A) vincristine (V) or (B) Velcade; tubacin alone; or both together at the indicated concentrations for 72 h. We observed a significant decrease in viable cells with both together compared to each drug alone. MTT absorbance of 0.2 is maximal inhibition within the linear range. Experiments were performed in triplicate. (C) Primary ALL cells were intravenously injected into sub-lethally irradiated NOD/SCID mice (5 ×10⁴ cells/mouse). Recipient mice were treated for 4 weeks intraperitoneally with saline (n = 3), 50 mg/kg/day tubacin only (n = 3), vincristine, dexamethasone, and _L-asparaginase (VDL) (n = 7), or VDL + tubacin (n = 7). Log-rank (Mantel–Cox) test for survival analysis was performed (p = 0.0171).

Figure 7

‘Domino effect’ triggered by tubacin treatment. Tubacin treatment leads to an accumulation of acetylated α-tubulin. Effects on membranes potentially reduce the activity of K⁺/Na⁺-ATPase and the lack of K⁺/Na⁺-ATPase activity would lead to an increment of intracellular Na⁺ and osmotic stress. This osmotic stress would lead to an intracellular calcium release that could subsequently trigger apoptosis.