The organic arsenic derivative GMZ27 induces PML-RARα-independent apoptosis in myeloid leukemia cells

Abstract

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against acute promyelocytic leukemia. However, organic arsenic derivatives (OAD) have a more favorable toxicity profile than ATO. We herein characterized dipropil-S-glycerol arsenic (GMZ27), a novel OAD. GMZ27 had potent antiproliferative activity against human acute myeloid leukemia (AML) cell lines that was higher than that of ATO. In contrast to ATO, GMZ27 only marginally induced maturation of leukemia cells and had no effect on the cell cycle. The anti-leukemia activity of GMZ27 against AML cells was independent of the presence of the PML-RARα fusion protein. GMZ27 dissipates mitochondrial transmembrane potential, and induces cleavage of caspase 9 and activation of caspase 3 without altering the expression levels of (BCL-2), BAX and BCL-xl. GMZ27 induces the formation of intracellular superoxide, a reactive oxygen species (ROS) which plays a major role in the antileukemia activity of this OAD. In addition to ROS generation, GMZ27 concomitantly reduces intracellular glutathione which markedly weakens the cellular antioxidant capacity, thus enhancing the detrimental intracellular effects of ROS production. These results indicate that GMZ27 induces apoptosis in AML cells in a PML-RARα-independent fashion, through the induction of ROS production. This activity provides the rationale for the testing of GMZ27 in patients with AML.

Figure 1

Activity of GMZ27 towards different leukemia cell lines. Summary of GMZ27-induced cell growth inhibition in 72-h MTS assays performed with HL60 (A) and NB4 (B) cells. Growth inhibition by GMZ27 of the HL60, NB4, U937, and KBM-5 leukemia cell lines in 72-h MTS assays (Table I). Viability of the HL60, NB4, U937, and KBM-5 leukemia cell lines after GMZ27 treatment in 72-h Trypan blue assays (C). Data represent results obtained in three independent experiments.

Figure 2

The antileukemia effect of GMZ27 is PML–RARα independent. NB4 cells were treated with GMZ27 for 24 hours and dose-dependent PML–RARα protein degradation was assayed by western blot using a rabbit anti-PML-RARα antibody (A). NB4 and U937 cells were incubated with GMZ27 for 72 hours and subjected to MTS assay to evaluate cell proliferation. U937 was sensitive to GMZ27 but not to arsenic trioxide (ATO) (B). After 72 hours of incubation with GMZ27 or ATO, NB4 cells were incubated with phycoerythrin-conjugated anti-CD11b monoclonal antibody (dilution 1:10) and then subjected to flow cytometry analysis using CellQuest software. GMZ27 treatment induces marginal myeloid maturation compared to ATO (C).

Figure 3

GMZ27 does not induce cell cycle arrest. HL60 cells were treated with GMZ27 for 24 and 48 hours. Cell cycle analysis showed no arrest. The percentage of Sub G₁ phase of cells after treatment with of GMZ27 is also presented (B).

Figure 4

GMZ27 induces apoptosis of HL60 cells in a dose- and time-dependent manner. HL60 cells were treated with ATO or GMZ27 for 24 h and 48 h and apoptosis was evaluated by flow cytometry. Different aspects of the apoptotic process were analyzed. (A) Compromised cell membrane integrity (annexin V/PI staining). (B) Caspase activation (PhiPhiLux/PI assay). (C) Dissipation of mitochondrial transmembrane potential (CMXRos/MTGreen staining). Representative data are shown from experiments performed in triplicate.

Figure 5

GMZ27 induces apoptosis in HL60 cells through activation of caspase-9. After incubation with GMZ27 for 24 hours, whole-cell lysate was subjected to western blot to detect pro-caspase-8, pro-caspase-9 and their active, cleaved fragments. PARP cleavage was also detected by western blotting. GMZ27-mediated apoptosis occurs preferentially through the intrinsic apoptosis pathway (A). GMZ27 does not alter the expression levels of Bcl-2, BAX and BCL-xl. Expressions of BCL family proteins were detected by anti-BCL-2, anti-BCL-xl and anti-BAX antibodies. β-Actin expression was detected by the anti-β-Actin antibody and was used as loading control (B).

Figure 6

The antileukemia activity of GMZ27 is mediated through modulation of intracellular oxidative status and production of reactive oxygen species (ROS). HL60 cells were pre-incubated with or without 100 μM L-buthionine-[S,R]-sulfoximine (L-BSO) (A) or 10 mM N-acetyl-L-cysteine (NAC) (B) for 24 hours, and were then treated with GMZ27 at different concentrations. After 72 hours MTS assay was performed in order to evaluate cell proliferation. GMZ27 treatment results in production of superoxide. HL60 cells were treated with GMZ27 for 2, 4, 6, 8 hours. Superoxide production was assessed using a dihydroethidium probe. Each experimental point represents the mean±SD of three independent experiments (C, D).

Figure 7

GMZ27 treatment depletes intracellular GSH content in NB4 cells. After 2, 8, 14, 24 hours of GMZ27 treatment, 20×10⁶ NB4 cells were harvested and the intracellular GSH was measured with 4-chloro-1-methyl-7-trifluromethyl-quinolinium methylsulfate as per the manufacturer’s instructions. The GSH content is expressed as μmol/μg protein.