Proteasome inhibitor MG-132 enhances histone deacetylase inhibitor SAHA-induced cell death of chronic myeloid leukemia cells by an ROS-mediated mechanism and downregulation of the Bcr-Abl fusion protein

Abstract

Recently, there has been progress in the treatment of chronic myeloid leukemia (CML). However, novel therapeutic strategies are required in order to address the emerging problem of imatinib resistance. Histone deacetylase inhibitors (HDACi) and proteasome inhibitors are promising alternatives, and may be amenable to integration with current therapeutic approaches. However, the mechanisms underlying the interaction between these two agents remain unclear. The present study assessed the cytotoxic effect of the HDACi, suberoylanilide hydroxamic acid (SAHA), in combination with the proteasome inhibitor, MG-132, in imatinib-sensitive K562 and imatinib-resistant K562G cells, and investigated the mechanism underlying this effect. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and protein expression levels were determined by western blotting. Reactive oxygen species (ROS) generation levels were observed under a fluorescence microscope The results indicated that SAHA and MG-132 act in a synergistic manner to induce cell death in K562 and K562G cells. This effect was associated with Bcr-Abl downregulation and the production of ROS. Notably, the ROS scavenger, N-acetyl-L-cysteine, almost fully reversed the cell death and Bcr-Abl downregulation that was induced by the combination of SAHA and MG-132. By contrast, the pan-caspase inhibitor, z-VAD-fmk, only partially reversed the cell death induced by these two drugs in CML cells. These results indicated that increased intracellular ROS levels are important in the induction of cell death and the downregulation of Bcr-Abl. In conclusion, the present results suggested that combined SAHA and MG-132 may be a promising treatment for CML.

Keywords: Bcr-Abl; MG-132; chronic myeloid leukemia; reactive oxygen species; suberoylanilide hydroxamic acid.

Figure 1.

Effects of SAHA on the K562 and K562G CML cell lines. (A) K562 and K562G cells were treated with SAHA (0.5–8 µM) for 48 h. Cell viability was measured using an MTT assay. Data are presented as the mean ± standard deviation of three independent experiments. (B) K562 cells were treated with SAHA (1–4 µM) for 24 h. Western blot analysis was performed, using antibodies against caspase-9, caspase-3, PARP and Ac-histone H3. β-actin was used as a loading control. A representative blot from three independent experiments is shown. (C) K562 cells were treated with SAHA (1–4 µM) for 24 h (upper panel) or with 4 µM SAHA for 3 h, 6 h or 12 h (lower panel). Western blot analysis for Bcr-Abl and p-Bcr-Abl was performed. β-actin was used as a loading control. A representative blot from three independent experiments is shown. SAHA, suberoylanilide hydroxamic acid; CML, chronic myeloid leukemia; Ac-histone, acetylated histone; PARP, poly adenosine diphosphate-ribose polymerase.

Figure 2.

Synergistic effect of SAHA and MG-132 in the K562 and K562G CML cell lines. (A) K562 cells and K562G cells were treated with SAHA (1 µM) or SAHA plus MG-132 (400 nM or 800 nM) for 24 h. Cell viability was measured using an MTT assay. *P<0.0001 vs. SAHA alone. (B) K562 cells were treated with SAHA (2 µM), MG-132 (200 nM) or SAHA plus MG-132 for 7 days. Colony formation was observed using a light microscope. Representative images are shown. (C) K562 cells were treated with SAHA (1 µM or 2 µM), MG-132 (400 nM) or SAHA plus MG-132 (400 nM) for 24 h. Western blot analysis was performed, using antibodies against Bcr-Abl, Ac-histone H3, caspase-3 and PARP. β-actin was used as a loading control. (D) K562G cells were treated with SAHA (1 µM or 2 µM), MG-132 (200 nM) or SAHA plus MG-132 (200 nM) for 24 h. Western blot analysis was performed, using antibodies against Bcr-Abl, Ac-histone H3, caspase-3 and PARP. β-actin was used as a loading control. Representative blots from three independent experiments are shown. (E) K562 cells and K562G cells were treated with SAHA (1 µM), MG-132 (200 nM) alone, or SAHA plus MG-132 for 24 h, prior to which cells were cultured in the presence or absence of z-VAD-fmk (20 µM) for 1 h. Cell viability was measured using an MTT assay. *P<0.05. Data are presented as the mean ± standard deviation of three independent experiments. SAHA, suberoylanilide hydroxamic acid; CML, chronic myeloid leukemia; Ac-histone, acetylated histone; PARP, poly adenosine diphosphate-ribose polymerase.

Figure 3.

ROS scavenger, NAC, reversed the effects of the combination of SAHA and MG-132 in the K562 and K562G CML cell lines. (A) K562 cells were treated with SAHA (1 µM) plus MG-132 (200 nM) for 24 h, with or without 1 h pretreatment with NAC (2 mM), Rosup (50 mg/ml) was used as a positive control. ROS generation was examined using a fluorescence microscope. Representative images from three independent experiments are shown. (B) K562 cells and K562G cells were treated with SAHA (1 µM) or MG-132 (200 nM) alone, or with SAHA plus MG-132 for 48 h, prior to which cells were cultured in the presence or absence of NAC (2 mM) for 1 h. Cell viability was measured using MTT assay. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.0001. (C) K562 cells were cultured with SAHA (1 µM) plus MG-132 (200 nM) for 6 h or 24 h, with or without pretreatment with NAC (2 mM) for 1 h. Western blot analysis was performed, using antibodies against Bcr-Abl and PARP. β-actin was used as a loading control. A representative blot from three independent experiments is shown. SAHA, suberoylanilide hydroxamic acid; CML, chronic myeloid leukemia; Ac-histone, acetylated histone; PARP, poly adenosine diphosphate-ribose polymerase; NAC, N-acetyl-L-cysteine.