Human acute promyelocytic leukemia NB4 cells are sensitive to esculetin through induction of an apoptotic mechanism

Highlights

• Esculetin produced toxic and apoptosis effects on human leukemia NB4 cells.

• Esculetin reduced Bcl2/Bax ratio and activated caspases 3 and 9 in NB4 cells.

• Esculetin altered mitochondrial membrane potential with release of cytochrome c.

• It increased peroxides and superoxide levels in NB4 cells treated for long times.

• Esculetin also increased p-Erk levels in leukemia NB4 cells.

Abstract

Acute promyelocytic leukemia (APL) is a type of cancer, in which immature cells called promyelocytes proliferate abnormally. Human NB4 cell line appears to be a suitable in vitro model to express the characteristics of APL. In this work, we have investigated the effects of esculetin, a coumarin derivative with antioxidant properties, on the viability, the induction of apoptosis and the expression of apoptotic factors in NB4 cells. Cells treated with esculetin at several concentrations (20–500 μM) and for different times (5–24 h) showed a concentration- and time-dependent viability decrease with increased subdiploid DNA production. Esculetin inhibited cell cycle progression and induced DNA fragmentation. Moreover, annexin-V-FITC cytometry assays suggested that increased toxicity is due to both early and late apoptosis. This apoptosis process is be mediated by activation of caspase-3 and caspase-9. Treatments with progressively increasing concentrations (from 100 μM to 500 μM) of esculetin produced a reduction of Bcl2/Bax ratio in NB4 cells at 19 h, without affecting p53 levels. Proapoptotic action of esculetin involves the ERK MAP kinase cascade since increased levels of phosphorylated ERK were observed after those treatments. Increments in the levels of phosphorylated-Akt were also observed. Additionally, esculetin induced the loss of mitochondrial membrane potential with a release of cytochrome c into the cytosol which starts at 6 h of treatment with esculetin and increases up to 24 h. Esculetin induced an increase in superoxide anion at long times of treatment and a reduction of peroxides at short times (1 h) with an observed increase at 2–4 h of treatment. No significant changes in NO production was observed. Esculetin reduced the GSH levels in a time-dependent manner.

In summary, the present work shows the cytotoxic action of esculetin as an efficient tool to study apoptosis mechanism induction on NB4 cell line used as a relevant model of APL disease.

Introduction

Acute promyelocytic leukemia (APL) is a malignant disorder of the white blood cells. In this disease, a rapid growth of immature cells (promyelocytes) is observed with their accumulation in peripheral blood and bone marrow. APL is considered as a subtype of acute myeloid leukemia (AML) characterized by chromosomal translocation t (15; 17) [1] that occurs between the promyelocytic leukemia (PML) gene and the retinoic acid receptor-α (RARα) gene, resulting in the generation of a PML–RARα fusion protein [2]. NB4 cell line appears to be the most suitable in vitro model to express the characteristics of APL. This cell line shows the unique property of carrying this chromosomal translocation, expressing different levels of some enzymatic markers and of different differentiation behavior as compared to other APL-derived cell lines, such as HL-60 [3], [4]. NB4 cell line is known to show resistance to several chemotherapeutic drugs [5] such as all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) [6], [7], [8]. The efficiency of the antitumor action depends on the cell death induction to eliminate malignant cells. To this respect, we have recently shown the induction of apoptosis in NB4 cells by novel reagents such as dequalinium [9], [10], active compounds present in Ganoderma lucidum [11], [12] and antimitotic compounds such as paclitaxel or vinblastine that induce cytotoxicity in the NB4 cell line by regulation of signal transduction kinases and apoptotic factors [13].

Esculetin (6,7-dihydroxycoumarin) is a coumarin, produced by many plants traditionally used as natural medicines. Coumarins have a diversity of biological and pharmacological activities, such as antioxidative, anti-inflammatory and antiviral effects [14]. Esculetin inhibits lipoxygenase and tyrosinase [15], [16] and displays cytoprotective properties against oxidative stress-induced cell damage [17], [18], [19]. It shows anti-inflammatory activities [20] and induces apoptosis in some cell lines such as adipocyte 3T3-L1 cells [21], human leukemia U937 cells [22], [23], HL-60 cells [24] and in human oral cancer SAS cells [25].

Some intracellular kinases are key proteins in the regulation of cellular functions. MAPKs and PI3K/Akt are both activated in response to various stimuli leading to activation of genes involved in cell progression, differentiation, proliferation and apoptosis. At least three MAPK families have been characterized: ERK, JNK and p38MAPK. ERK is directly downstream of the ERK pathway and some reports indicate that ERK is constitutively activated in several human leukemias [26] while JNK activation is associated with induction of apoptosis [27].

Apoptosis may proceed through either the intrinsic (or mitochondrial) pathway or the extrinsic (death receptor) pathway. Stimulation of these pathways leads to the activation of caspases which begins the process of apoptosis [28], [29]. Activation of the intrinsic pathway can produce mitochondrial damage with release of cytochrome c. The intrinsic pathway is regulated through proteins of the Bcl-2 family: the subfamily of antiapoptotic proteins like Bcl-2 that inhibit apoptosis and the subfamily of proapoptotic proteins like Bax that promotes apoptosis. Some of them can be activated by tumor suppressor protein (p53) when there is DNA damage [30], [31].

The role of these signaling proteins may be particularly relevant in order to know the mechanism by which esculetin could eliminate leukemia human cells. From different studies, it appears that the involvement of such proteins is variable and depends on the cellular model. Esculetin-induced apoptosis may proceed, without alteration in the expression of Bcl2 [25] or may require down-regulation of Bcl2 [24]. Similarly, an increase in the levels of Bax [32] may be needed in esculetin induced apoptosis. In contrast, a translocation of this protein from the cytosol to mitochondria could be observed in some cellular models [22]. Thus, the mechanism of induction of apoptosis by esculetin is dependent on the cell type and lineage. Differences may even exist among different lineages of the same leukemia disease model.

The activation of ERK in esculetin-induced apoptosis seems also controversial [22], [23], [32]. Some authors claim the inhibition of ERK for induction of apoptosis by esculetin in hepatoma HepG2 cells [32], while ERK activation seems to be relevant in U937 leukemia cells [22], [23]. The use of new unstudied cell lines or the discovery of new relationships between pathways may explain some differences on the activation in this kinase [32].

Esculetin could produce changes in ROS species and imbalance the redox equilibrium producing changes in the levels of reduced glutathione what might play a role in the cytotoxic effect on leukemia cells. Esculetin could act as a scavenger compound changing the redox balance in NB4 leukemia cell line [10]. Thus, the study of superoxide anion and peroxide levels could be relevant to know their role in the apoptosis induced by esculetin in this leukemia cell line that shows a high level of oxidative stress [10]. Additionally, intracellular GSH depletion is an early hallmark in the progression of cell death in response to different apoptotic stimuli including cytotoxic chemotherapeutic agents inducing oxidative stress. Since leukemia cells, as other cancer cells, are under an increased oxidative stress, ROS overproduction has been proposed as a useful mechanism for killing these malignant cells [33].

The effect of esculetin in NB4 leukemia cell model has not yet been reported, so the purpose of our study was to investigate the effect of esculetin on cell viability, induction of apoptosis and expression of apoptotic factors as well as the relationship between cell death and intracellular kinases in this cell model. Furthermore, we aimed to know if redox imbalance in NB4 leukemia cells could be relevant in the cytotoxicity of esculetin in these cells. We also intended to elucidate the roles of superoxide anion, of peroxides and reduced glutathione in this process. Further investigation can establish whether the changes in ROS levels are the result of the induced apoptosis process or they are important effector of the observed cell death.