Apoptosis induction of U937 human leukemia cells by diallyl trisulfide induces through generation of reactive oxygen species
© Park and Choi; licensee BioMed Central Ltd 2012
Received: 21 January 2012
Accepted: 11 May 2012
Published: 11 May 2012
Diallyl trisulfide (DATS) is one of the major constituents in garlic oil and has demonstrated various pharmacological activities, including antimicrobial, antihyperlipidemic, antithrombotic, and anticancer effects. However, the mechanisms of antiproliferative activity in leukemia cells are not fully understood. In this study, the apoptotic effects of DATS were investigated in human leukemia cells.
Results of this study indicated that treatment with DATS resulted in significantly inhibited leukemia cell growth in a concentration- and time-dependent manner by induction of apoptosis. In U937 cells, DATS-induced apoptosis was correlated with down-regulation of Bcl-2, XIAP, and cIAP-1 protein levels, cleavage of Bid proteins, activation of caspases, and collapse of mitochondrial membrane potential. The data further demonstrated that DATS increased intracellular reactive oxygen species (ROS) generation, which was attenuated by pretreatment with antioxidant N-acetyl-l-cysteine (NAC), a scavenger of ROS. In addition, administration of NAC resulted in significant inhibition of DATS-induced apoptosis by inhibiting activation of caspases.
The present study reveals that the cytotoxicity caused by DATS is mediated by generation of ROS and subsequent activation of the ROS-dependent caspase pathway in U937 leukemia cells.
Reactive oxygen species (ROS), such as hydrogen peroxides (H2O2), hydroxyl radicals (OH·), and superoxide anions (·O2-) are commonly generated as a consequence of aerobic metabolism in mitochondria [1, 2]. In normal physiological states, ROS are produced through electron leakage when molecular oxygen is used for ATP production in the mitochondrial electron transport chain [3–5]. As well as ROS production by mitochondria, these are also generated by various exogenous sources, such as chemicals and radiation [6–8]. Excessive accumulation of ROS results in severely demolished cellular macromolecules, such as DNA, and induces G2/M phase arrest by DNA damage [9–11]. Increased intracellular ROS also triggers apoptosis by activation of the intrinsic apoptotic pathway through induction of mitochondrial permeability transition and release of cytochrome c from mitochondria to the cytosol [4, 12–14].
Garlic (Allium sativum) is a common plant used mainly as food and has recently been reported to have medicinal properties [15–17]. Many researchers have recently demonstrated that sulfur-containing compounds, such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), which are major components of garlic, may be associated with reduced risk of certain cancers [18, 19]. These compounds are known to inhibit cell proliferation and increase apoptosis in various cancer cell lines [20–22]. In particular, DATS containing three sulfur atoms against DAS and DADS have been known to have stronger biological activity, such as anti-cancer and anti-inflammatory effects [23–26]. Recently, Das et al.  have shown that DATS induces apoptosis through activation of ROS-dependent caspase activation in human glioblastoma cells. Others studies have also supported the notion that DATS induces dramatic ROS generation in cancer cells through a mitochondrial pathway [26, 28, 29]. However, the cellular and molecular mechanisms underlying the compound have yet not been completely elucidated.
In the present study, we hypothesized that DATS would also induce functional changes in mitochondria in association with ROS generation in the course of apoptosis induction in human leukemia cells. To test this hypothesis, we evaluated the effects of DATS on mitochondrial membrane potential (MMP) values, ROS generation, and apoptosis using the human leukemia U937 cell line. Our results indicated the requirement of ROS generation in apoptosis induced by DATS in U937 cells.
Reagents and antibodies
DATS was purchased from LKT Laboratories (St Paul, MN). 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl-tetrazolium (MTT), propidium iodide (PI), 5,5′, 6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), 4,6-Diamidino-2-phenyllindile (DAPI), 5-(and 6)-carboxy-2’7’-dichlorodihydrofluorescein diacetate (DCFDA), and N-acetyl-l-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) and caspase activity assay kits were obtained from GIBCO-BRL (Gaithersburg, MD) and R&D Systems (Minneapolis, MN), respectively. DNA staining kit (CycleTEST™ PLUS Kit) and enhanced chemiluminescence (ECL) kit were purchased from Becton Dickinson (San Jose, CA) and Amersham (Arlington Heights, IL), respectively. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture and MTT assay
The human leukemia cell lines used in our studies included human monocytic leukemia (U937 and THP-1), human acute myeloblastic leukemia (HL60) and human erythroid chronic myeloid leukemia (K562) were purchased from the American Type Culture Collection (Rockville, MD). They were maintained at 37°C in humidified 95 % air and 5 % CO2 in RPMI1640 supplemented with 10 % heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. DATS was dissolved in dimethyl sulfoxide (DMSO) as a stock solution at a 100 mM concentration, and the stock solution was then diluted with the medium to the desired concentration prior to use. For the cell viability study, cells were grown to 70 % confluence and treated with DATS. Control cells were supplemented with complete media containing 0.1 % DMSO (vehicle control). Following treatment, cell viability was determined by use of the MTT assay, which is based on the conversion of MTT to MTT-formazan by mitochondrial enzymes . The effect of DATS on inhibition of cell growth was assessed as the percentage of cell viability, where vehicle-treated cells were considered 100 % viable.
Nuclear staining with DAPI
For DAPI staining, cells were washed with phosphate-buffered saline (PBS) and fixed with 3.7 % paraformaldehyde (Sigma-Aldrich) in PBS for 10 min at room temperature. Fixed cells were washed with PBS and stained with 2.5 μg/ml DAPI solution for 10 min at room temperature. Cells were then washed twice with PBS and analyzed using a fluorescence microscope (Carl Zeiss, Germany).
DNA fragmentation assay
Following DATS treatment, cells were lysed in a buffer containing 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.5 % Triton X-100 for 1 h at room temperature. Lysates were vortexed and cleared by centrifugation at 19,000 g for 30 min at 4°C. A 25:24:1 (v/v/v) equal volume of neutral phenol : chloroform : isoamyl alcohol (Sigma-Aldrich) was used for extraction of DNA in the supernatant, followed by electrophoretic analysis on 1.0 % agarose gels containing 0.1 μg/ml ethidium bromide (EtBr, Sigma-Aldrich) .
Measurement of cell cycle, mitochondrial membrane potential (MMP) values and ROS generation by a flow cytometer
For analysis of the cell cycle, cells were collected, washed with cold PBS, and fixed in 75 % ethanol at 4°C or 30 min. A DNA staining kit was used according to the manufacturer’s instructions for measurement of the DNA content of the cells . Flow cytometric analyses were carried out using a flow cytometer and the relative DNA content was determined using CellQuest software based on the presence of red fluorescence. MMP (ΔΨm) was determined using the dual-emission potential-sensitive probe, JC-1. Cells were collected and incubated with 10 μM JC-1 for 20 min at 37°C in the dark. Cells were then washed once with PBS and analyzed using a flow cytometer . For measurement of ROS generation, cells were treated with DATS for various periods and the medium was discarded; cells were then incubated with new culture medium containing 10 μM of DCFDA at 37°C in the dark for 20 min. Cell lysates were used for evaluation of ROS generation using a flow cytometer .
Protein extraction and Western blotting
Cells were harvested and washed twice in PBS at 4°C. Total cells lysates were lysed in lysis buffer (40 mM Tris (pH 8.0), 120 mM, NaCl, 0.5 % NP-40, 0.1 mM sodium orthovanadate, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 100 μg/ml phenymethylsulfonyl fluoride). Supernatants were collected and protein concentrations were then measured using protein assay reagents (Pierce, Rockford, IL). Equal amounts of protein extracts were denatured by boiling at 95°C for 5 min in sample buffer (0.5 M Tris–HCl, pH 6.8, 4 % SDS, 20 % glycerol, 0.1 % bromophenol blue, 10 % β-mercaptoethanol) at a ratio of 1:1, subjected to SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, NH) by electroblotting. Membranes were blocked with 5 % non-fat dry milk in PBS with Tween 20 buffer (PBS-T) (20 mM Tris, 100 mM NaCl, pH 7.5, and 0.1 % Tween 20) for 1 h at room temperature. Membranes were then incubated overnight at 4°C with the primary antibodies, probed with enzyme-linked secondary antibodies, and visualized using an ECL kit, according to the manufacturer's instructions.
Caspase activity assay
Activities of caspases were determined by use of colorimetric assay kits, which utilize synthetic tetrapeptides (Asp-Glu-Val-Asp (DEAD) for caspase-3; Ile-Glu-Thr-Asp (IETD) for caspase-8; Leu-Glu-His-Asp (LEHD) for caspase-9, respectively) labeled with p-nitroaniline (pNA). Briefly, DATS-treated and untreated cells were lysed in the supplied lysis buffer. Supernatants were collected and incubated with the supplied reaction buffer containing DTT and DEAD-pNA, IETD-pNA, or LEHD-pNA as substrates at 37°C. The reactions were measured by changes in absorbance at 405 nm using the VERSAmax tunable microplate reader.
Unless otherwise indicated, each result is expressed as the mean ± SD of data obtained from triplicate experiments. Statistical analysis was performed using a paired Student t-test. Differences at p < 0.05 were considered statistically significant.
Inhibition of cell viability by DATS in human leukemia cells
Induction of apoptosis in human leukemia cells by DATS
Activation of caspases by DATS in U937 cells
Effects of DATS on expression of Bcl-2 and IAP family proteins in U937 cells
Loss of MMP values and increase of ROS generation by DATS in U937 cells
DATS-induced apoptosis was associated with generation of ROS in U937 cells
Although an increasing amount of data indicate that DATS can suppress the growth of cultured cancer cells by causing cell cycle arrest at G2/M phase and generation of apoptosis [26–29, 35–37], little is known about the effects of this compound on the growth of human leukemia cells. In the present study, we demonstrated that DATS-induced anti-proliferative effects in four leukemia cell lines (U937, THP-1, HL60 and K562) were related to induction of apoptosis, as confirmed by measurement of chromatin condensation of nuclei, DNA fragmentation, and induction of sub-G1 phase. Our data also indicated that DATS induced apoptosis of U937 cells through generation of ROS and mitochondrial dysfunction, suggesting that ROS act as upstream signaling molecules for initiation of cell death.
Mounting evidence suggests that damaged mitochondria stimulate increased ROS production, subsequently resulting in activation of the signaling pathways that control cancer cell growth. However, loss of MMP as a result of mitochondrial depolarization in association with apoptosis appears to be more common. The mechanisms by which ROS cause or regulate apoptosis typically include caspase activation and modulation of Bcl-2 family protein expression [38, 39]. Caspases, a family of cystein-containing aspartate-specific proteases, are known to play key roles during apoptosis and to lead to initiation and execution of apoptosis. Activation of initiator caspases, such as caspase-8 and −9, resulted in downstream activation of effector caspases, such as caspase-3 and −7 [40, 41]. The decrease in MMP causes disruption of the outer mitochondrial membrane and contributes to release of cytochrome c. Release of cytochrome c has been reported to contribute to activation of caspase-9, which in turn causes activation of caspase-3. In particular, activation of capase-3 is responsible for proteolytic degradation of many key proteins, including PARP and β-catenin, finally leading to apoptosis [42, 43]. Modulation of anti- and pro-apoptotic proteins of the Bcl-2 family also controls mitochondrial function. In mammals, members of the Bcl-2 family can be divided into two subfamilies; the anti-apoptotic protein family, including Bcl-2, and the pro-apoptotic protein family, including Bax. Balance between anti-apoptotic and pro-apoptotic members also determines the fate of the cell through mitochondrial dysfunction [44, 45]. In addition, activation of caspase-8 by apoptotic stimuli converts Bid to truncated Bid (tBid), leading to conformational changes in Bax, mitochondrial depolarization, and release of cytochrome c from mitochondria. This leads finally to activation of caspase-3 and induction of apoptosis via a complex of apoptotic protease activating factor-1 (Apaf-1), procaspase-9, and cytochrome c after translocation of tBid to the mitochondria [41, 44–46]. Furthermore, members of the IAP family, which includes XIAP, cIAP-1, and cIAP-2, have been reported to exert their anti-apoptotic effects due to their function as direct inhibitors of activated caspases. Therefore, down-regulation of IAPs relieves the triggering block of proapoptotic signaling and execution of caspases, thus activating cell death [47, 48].
In this study, our data indicated an association of DATS-induced apoptosis of U937 cells with increased generation of ROS and enzymatic activity of both the extrinsic and intrinsic caspase cascades, including caspase-8 and −9. Although the truncated form of Bid fwas not detected, the levels of intact Bid proteins were gradually down-regulated by DATS in a concentration-dependent manner. DATS also caused a significant reduction in MMP values, which was connected with activation of caspase-3 and concomitant degradation of PARP and β-catenin. In addition, down-regulation of anti-apoptotic Bcl-2 and IAP family proteins, including Bcl-2, XIAP, and cIAP-1, was observed in U937 cells exposed to DATS, as compared with control cells. Thus, the results indicated that caspase-8 activation by DATS appeared to trigger mitochondrial apoptotic events by inducing conformational changes in apoptotic proteins.
ROS-mediated caspase activation and mitochondrial dysfunction have been suggested as critical for DATS-induced apoptosis in several cancer cell lines [26–29]; however, the current role of mitochondrial functional changes associated with ROS generation in the response of human leukemia cells to DATS has not yet been explored. Therefore, we next investigated the question of whether these observations and apoptosis by DATS in U937 cells were associated with generation of ROS. The results revealed that activation of caspase-9 and −3, degradation of PARP, and inhibition of Bcl-2 in DATS-treated cells were ROS-dependent and that co-culture with NAC, a commonly used ROS scavenger, effectively blocked DATS-induced apoptosis in U937 cells. Findings from the present study also indicated that activation of caspase-8 in DATS-treated cells is ROS-dependent, which suggests that ROS may act upstream of caspase-8 activation in U937 cells. Therefore, it is reasonable to assume that the initial signal for activation of caspase-8 after treatment with DATS is also derived from ROS. In addition, blocking of ROS generation prevented DATS-induced down-regulation of XIAP and cIAP-1. Because IAP family proteins are also substrates of caspase-3 [49, 50], the observed decrease in XIAP and cIAP-1 expression may be due to caspase-3-mediated processing following DATS treatment. Thus, our data indicate that ROS production and mitochondrial dysfunction are possible contributing factors to DATS toxicity.
In summary, the present study demonstrated that human leukemia cells undergo apoptosis in response to treatment with DATS, and that this occurs through a mitochondria-mediated pathway that requires ROS generation upstream for disruption of the MMP, which leads to subsequent activation of caspases. Although in this paper we only performed a rough assessment for determination of whether ROS was involved in the apoptosis induced by DATS, our data emphasize the key role of ROS in apoptosis induced by DATS in U937 cells, and indicate that a positive correlation exists between ROS and mitochondrial events leading to apoptosis, and may aid in understanding of the mechanisms for the anti-cancer activity of DATS.
This work was supported by a grant (Code #7–19-42) from Rural Development Administration, Republic of Korea
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