Arsenic trioxide induces apoptosis in cells of MOLT-4 and its daunorubicin-resistant cell line via depletion of intracellular glutathione, disruption of mitochondrial membrane potential and activation of caspase-3
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- Hu, X., Hirano, T. & Oka, K. Cancer Chemother Pharmacol (2003) 52: 47. doi:10.1007/s00280-003-0629-5
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To demonstrate that arsenic trioxide (As2O3) induces apoptosis via a mitochondrial pathway in both parent T lymphoblastoid leukemia MOLT-4 cells and cells of its daunorubicin-resistant subline, MOLT-4/DNR, expressing functional P-gp.
Cell growth was measured using an MTT assay. Cell viability was determined using a dye exclusion test. Intracellular glutathione (GSH) was measured using a glutathione assay kit. Mitochondrial membrane potential (MMP) was assessed by rhodamine 123 (Rh123) staining intensity on flow cytometry. Caspase-3 activity was evaluated using a commercially available assay kit on flow cytometry. The percentage of cells undergoing apoptosis was estimated in terms of caspase+/PI− cells on flow cytometry after assessment for activation of caspase-3 by adding PI.
MOLT-4 cells and MOLT-4/DNR cells were similarly sensitive to the apoptosis-inducing effect of As2O3. Buthionine sulfoxide (BSO) and ascorbic acid (AA) rendered these cells more sensitive to As2O3, whereas N-acetylcysteine (NAC) reduced this sensitivity. BSO and AA decreased, but NAC increased, the intracellular GSH contents of both MOLT-4 and MOLT-4/DNR cells. Decreasing GSH with BSO potentiated As2O3-mediated growth inhibition, disruption of MMP, activation of caspase-3 and apoptosis of cells. Clinically relevant doses of AA enhanced the anticancer effects of As2O3 via the disruption of MMP, activation of caspase-3, and induction of apoptosis. In contrast, increase GSH levels with NAC attenuated all of these As2O3-mediated actions.
The sensitivity of MOLT-4 and MOLT-4/DNR cells to As2O3 was associated with the intracellular GSH content. As2O3 induced apoptosis in parent MOLT-4 cells and MOLT-4/DNR cells expressing functional P-gp via depletion of intracellular GSH, and subsequent disruption of MMP and activation of caspase-3.
KeywordsArsenic trioxideApoptosisGlutathioneMitochondrial membrane potentialCaspase-3
In response to the observation that patients with chemorefractory acute promyelocytic leukemia (APL) still respond to arsenic trioxide (As2O3) therapy [14, 45, 47] and in vitro studies of the effects of As2O3 on drug-resistant APL cell lines , investigations have been initiated to evaluate the therapeutic potential of As2O3 in several malignant diseases [44, 56, 57]. Most of the reports suggest that As2O3 suppresses growth and induces apoptosis in malignant cells including drug-resistant cells [5, 11, 44, 56, 57].
Some data indicate that As2O3 is not sensitive to drug efflux pump mechanisms of resistance [12, 37]. We have established a cell line resistant to daunorubicin (DNR) from T lymphoblastoid leukemia MOLT-4 cells . This resistant cell line, MOLT-4/DNR, has been revealed to overexpress functional P-glycoprotein (P-gp) . Our previous study showed that As2O3 is effective in the suppression of growth of MOLT-4 and MOLT-4/DNR cells . Moreover, we found that As2O3 induces apoptosis in both MOLT-4 and MOLT-4/DNR cells . MOLT-4/DNR cells have been revealed to be more than ten times more resistant to DNR than the parent MOLT-4 cells, as assessed by an MTT assay . As2O3 inhibits growth and induces apoptosis in MOLT-4/DNR cells without having an influence on their P-gp expression and function .
Thus, in the present study, we examined the apoptosis-inducing mechanisms of As2O3 in MOLT-4 and MOLT-4/DNR cell lines from the viewpoint of modification of cellular GSH levels, mitochondrial membrane potential (MMP), and caspase-3.
Materials and methods
RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, N.Y.). Cell proliferation kits I and II (MTT) were purchased from Roche Diagnostics (Indianapolis, Ind.). Ascorbic acid (AA), N-acetylcysteine (NAC), l-buthionine-[S,R]-sulfoxide (BSO), rhodamine 123 (Rh123) and As2O3 were obtained from Sigma Chemical Company (St. Louis, Mo.). All of the agents were dissolved in phosphate-buffered saline (PBS) and diluted to a working concentration before use. A GSH assay kit was obtained from Cayman Chemical Company (Ann Arbor, Mich.). Metaphosphoric acid (MPA) and triethanolamine (TEAM) were purchased from Aldrich Chemical Company (Milwaukee, Wis.). A PhiPhiLux-G1D2 kit was from MBL (OncoImmunin, Gaithersburg, Md.). Propidium iodide (PI) was from BD PharMingen.
MOLT-4 and MOLT-4/DNR cells were maintained in RPMI-1640 medium containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. The leukemia cells were washed and resuspended with the above medium to 5×105 cells/ml, then 196 μl cell suspension was placed in each well of a 96-well flat-bottom plate. PBS solutions (4 μl) containing As2O3, AA, BSO, and NAC alone or in combination with As2O3 with AA, BSO or NAC were added to yield the final indicated concentrations (see Results). PBS (4 μl) was added to the control wells. The cells were incubated for 72 h in an atmosphere comprising 5% CO2/air at 37°C in a humidified chamber.
After the incubation period, 20 μl of the MTT labeling reagent was added to each well to yield a final concentration of 0.5 mg/ml and the plate was mixed on a microshaker for 10 s. The cells were further incubated for 4 h in a humidified atmosphere. Subsequently, 100 μl solubilization solution (Roche) were added to each well, and the plate was mixed on a microshaker for 10 s and allowed to stand overnight in an incubator in a humidified atmosphere. The spectrophotometric absorbance of the sample was measured on a microplate reader (Corona MT P-32; Corona Company, Ibaragi, Japan) at 570 nm. A dose response curve was plotted for each drug, and the concentrations that yielded 50% inhibition of cell growth (IC50) were calculated.
Viability of cells
MOLT-4 and MOLT-4/DNR cell lines were cultured by seeding 5×105 cells/ml of fresh medium in the presence or absence of As2O3 alone or in combination with As2O3 with AA, BSO and NAC for 24–96 h in an atmosphere comprising 5% CO2/air at 37°C in a humidified chamber. The number or percentage of viable cells was determined by staining cell populations with trypan blue. On the day of determination, four parts of 0.2% trypan blue (w/v in water) were mixed with one part of 5× saline (4.25% NaCl, w/v in water), then one part of the trypan blue saline solution was added to one part of the cell suspension. Subsequently, the cells were loaded into a hemocytometer, and the unstained (viable) cells and the stained (dead) cells were counted separately within 3 min of being stained with trypan blue.
Measurement of intracellular GSH
Cells (1×107) were treated for 48 h with various agents and collected by centrifugation at 1300 g for 10 min at 4°C. The cells were resuspended in 500 μl 50 mM cold MES buffer, and then homogenized with a Handy pestle (Toyobo Company, Osaka, Japan) or sonicated with a Handy sonic (Tomy Seiko Company, Tokyo, Japan). After centrifugation at 10,000 g for 15 min at 4°C, the supernatant was removed and stored on ice. The supernatant was deproteinated with 5% MPA at room temperature for 5 min and centrifuged at 3000 g for 4 min. Subsequently, 50 μl 4 M TEAM reagent per ml of the supernatant was added, and the solution was mixed immediately with a vortex mixer. The standard and samples were combined with fresh assay cocktail according to the manufacturer's instructions. Samples were incubated in the dark on an orbital shaker before measurement. The absorbance in the wells was measured at 415 nm using a microplate reader. The total GSH levels were determined by the End Point method.
Evaluation of MMP
MOLT-4 and MOLT-4/DNR cells (1×106/ml) were treated with As2O3 alone or in combination with AA, BSO, or NAC. The cells were washed twice in a cold PBS (pH 7.2) and then incubated with 10 μg/ml Rh123 for 15 min. The cells were then washed twice in cold PBS, and were analyzed by flow cytometry. Living cells concentrate Rh123 in the mitochondria, while the MMP in cells undergoing apoptosis is disrupted and the mitochondria in such cells release Rh123 . Therefore, the disruption of MMP and subsequent cell apoptosis can be detected by the decrease in the Rh123 fluorescence intensity in the cells. A total of 30,000 non-gated cells were analyzed using a FACSCalibur analyzer (Becton Dickinson, San Jose, Calif.) to obtain dot data. These data were further analyzed using CellQuest Software (Becton Dickinson).
Assessment of caspase-3 activity
Cells (1×106/ml) were treated with As2O3 alone or in combination with AA, BSO, or NAC. After incubation, an aliquot of cells was placed into a 1.5-ml microcentrifuge tube, and the cells were then centrifuged and the entire culture medium was removed. Subsequently, 50 μl of a substrate solution containing 10 μM caspase-3 substrate was added to the cell pellet, and the suspension was mixed by flicking the tubes with the fingertip. The substrate molecule contains a peptide homodoubly labeled with a fluorophore. The cleaved substrate has specific fluorescence peak characteristics (λex 505 nm and λem 530 nm), which can be detected by flow cytometry. After incubation of the tubes in 5% CO2/air at 37°C in a humidified chamber for 60 min, the cells were washed once by adding 1 ml ice-cold flow cytometry dilution buffer. The cells were resuspended in 1 ml fresh dilution buffer for analysis by flow cytometry. All samples were analyzed within 6 min of the end of the 37°C incubation.
After collecting data for caspase-3 activity, 5 μl 50 μg/ml PI was added and samples were reanalyzed flow cytometrically according to the manufacturer's instructions. The data were reanalyzed within 5 min of PI addition, and the caspase+/PI− cells were considered apoptotic cells.
Morphological changes of cells
MOLT-4 and MOLT-4/DNR cells (5×105/ml) were cultured in the absence or presence of As2O3 alone or in combination with AA, BSO, or NAC for 72 h in an atmosphere comprising 5% CO2/air at 37°C in a humidified chamber. Pictures were taken under a microscope (Olympus Optical Company, Tokyo, Japan).
Student's t-test was used to compare the data between two groups, and the Bonferroni/Dun multiple comparison test was used to compare the data among multiple (more than three) groups. In each case, P values less than 0.05 were considered to be significant.
Effects of BSO, AA, or NAC on the growth-inhibitory action of As2O3 in MOLT-4 and MOLT-4/DNR cells
IC50 values(μM) of As2O3 alone or in combination with BSO, AA, and NAC. Cells were treated with 100 μmol/l BSO, 125 μmol/l AA, and 10 mmol/l NAC for 72 h, and cell growth was determined by an MTT assay. Values are the means±SD of three independent experiments
As2O3+AA (125 μM)
As2O3+BSO (100 μM)
As2O3+NAC (10 mM)
Intracellular GSH content modulated by BSO, AA, or NAC in MOLT-4 and MOLT-4/DNR cells
The GSH contents of MOLT-4 and MOLT-4/DNR cells were 105.0±1.6 and 105.6±2.4 μM/107 cells, respectively, when the two cell lines were incubated in the absence of modulating agents for 48 h. Thus, the intracellular GSH contents of MOLT-4 and MOLT-4/DNR cells were at the same level.
Disruption of MMP in MOLT-4 and MOLT-4/DNR cells
Activation of caspase-3 in MOLT-4 and MOLT-4/DNR cells
Modulation of As2O3-induced apoptosis by AA, BSO, or NAC in MOLT-4 and MOLT-4/DNR cells
GSH content had a decisive effect on As2O3-mediated cytotoxicity
Our previous work has shown that the effects of As2O3 on the growth of MOLT-4/DNR cells are the same as its effects on parental MOLT-4 cells, and the actions of As2O3 do not influence their P-gp expression or function . The findings of this study indicate that the intracellular GSH levels in parental MOLT-4 and MOLT-4/DNR cells are almost the same. A reduction in GSH levels following incubation with BSO and AA enhanced the As2O3-mediated inhibition of cell growth, disruption of MMP, activation of caspase-3 and apoptosis in MOLT-4 cells and MOLT-4/DNR cells. Whereas, NAC elevated the intracellular GSH levels and protected the cells from the cytotoxicity of As2O3. These findings led us to conclude that the sensitivity of both MOLT-4 and MOLT-4/DNR cells to As2O3 is closely correlated with intracellular GSH levels.
GSH is a low molecular weight, non-protein, sulfhydryl compound and its role in the protection of cells from oxidative injury has been documented in a study that demonstrated the enhancement of damage by depletion of cellular GSH . GSH exerts antioxidant effects and it can conjugate with and thereby inactivate molecules that generate free radicals. Trivalent arsenic has been reported to form a complex with GSH, forming a transient As(GS)3 molecule , which is easily excreted by the MRP2/cMOAT transporter . Thus, GSH maintains an optimum cellular redox potential, and depletion, physical efflux from the cell, or intracellular redistribution of GSH are associated with the onset of apoptosis . BSO is a selective inhibitor of γ-glutamylcysteine synthetase, the rate-limiting enzyme in the synthesis of GSH. Cells incubated with BSO are depleted of GSH as a result of the inhibition of enzymatic GSH production .
AA has been shown to have pro-oxidant properties [3, 40]. Auto-oxidation of AA to dehydroascorbate results in the production of H2O2 [42, 50]. Dehydroascorbate is then rapidly reduced back to AA by glutaredoxin in a GSH-dependent manner. This reduction of dehydroascorbate to AA results in a decrease in intracellular GSH [51, 53]. Clinically relevant doses of AA act as oxidizing agents decreasing the GSH content of the cells, and synergizes with the growth-inhibitory and apoptosis-inducing effects of As2O3. This potentiating effect of AA is due to its capacity to undergo auto-oxidation resulting in the formation of H2O2, which enhances the effects of As2O3 . Indeed, it has been found that AA significantly synergizes with As2O3 in treating patients with acute myeloid leukemia (AML) . NAC is an antioxidant since it donates a cysteine to the de novo synthesis of GSH. As2O3 has been reported to bind to vicinal thiol groups , while NAC contains two such thiol groups  and gives GSH. Taking these observations into consideration, the decrease in GSH levels following incubation with BSO and AA could abate As2O3 efflux, thereby causing an intracellular accumulation of As2O3 and enhancing its cytotoxicity. In contrast, the increase in intracellular GSH levels following incubation with NAC could enhance As2O3 efflux, and thereby attenuate the toxic effects of As2O3.
As2O3-induced apoptosis via depletion of GSH, loss of MMP and activation of caspase-3
Intracellular GSH depletion results in morphological and functional changes to mitochondria . It has been found that the changes to mitochondria after treatment with As2O3 can be divided into three stages . In the early stage, mitochondria appear to undergo an adaptive proliferation. In the middle stage, a degenerative change can be observed. In the late stage, the mitochondria swell and this is followed by damage to the outer membrane and cell death which exhibits apoptotic changes to the nucleus. Mitochondria are known to play a major role in apoptosis triggered by many stimuli . In the first stage of apoptosis, signal transduction cascades or damage pathways are activated. Subsequently, the mitochondrial membrane function is lost, and then proteins released from mitochondria cause the activation of catabolic proteases and nucleases . Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates . Caspase-3 is considered a primary executioner of apoptosis .
In the present study, the As2O3-mediated growth inhibition in parental MOLT-4 and MOLT-4/DNR cells is similar to that observed in our previous work . These suppressive effects of As2O3 can be potentiated by BSO, which inhibits synthesis of GSH, and by AA, which reduces GSH-dependent dehydroascorbate. In contrast, the effect of As2O3 was attenuated by NAC, which promotes synthesis of GSH in both MOLT-4 and MOLT-4/DNR cells. Furthermore, the disruption of MMP and activation of caspase-3 are not only caused by As2O3 itself, but are also enhanced by AA and BSO and blocked by NAC in both MOLT-4 and MOLT-4/DNR cells. The extent of MMP disruption and caspase-3 activation are closely associated with intracellular GSH levels and occur in a time-dependent manner. After 72 h of treatment with As2O3 alone or in combination with BSO or AA, the percentages of cells with decreased MMP and activated caspase-3 were higher than the percentage of cells that underwent apoptosis. These results suggest that MMP collapse and caspase-3 activation occur prior to apoptosis.
Recent reports suggest that sensitivity to As2O3 correlates with intracellular GSH levels in cancer cells . The GSH content modulates the growth-inhibitory and apoptosis-inducing effects of arsenicals [7, 19, 33, 34, 41]. Cells expressing higher levels of GSH or GSH-associated enzymes are less sensitive to As2O3 than cells expressing lower levels of these molecules [19, 25]. Arsenic-resistant cells are also reported to contain higher levels of GSH [22, 25, 29]. Moreover, cells with increased GSH levels can be sensitized to As2O3 by agents that deplete intracellular GSH [7, 22]. The cytotoxic effects of As2O3 may be influenced by modulators of GSH [12, 13, 23, 32], especially in drug-resistant cell lines [10, 38].
A decrease in GSH levels in cells acts as a potent early activator of apoptosis signaling . Depletion of GSH, especially mitochondrial GSH, is believed to induce the loss of MMP . Mitochondrial permeability transition resulting from intracellular thiol depletion is known to be a critical event in apoptosis [26, 54]. Mitochondria undergo major changes in membrane integrity before classical signs of apoptosis become manifest, and these changes lead to disruption of the MMP . These alterations in mitochondrial activity can be distinguished and monitored by the fluorescent dye Rh123 on flow cytometry [42, 50], which specifically stains mitochondria depending on the MMP [24, 35, 39]. Mitochondrial depolarization precedes caspase-3 activation and apoptosis , and apoptotic cells express high caspase-3 activity [16, 58]. Caspase-3 is considered to be a primary executioner of apoptosis . Caspase-3 activation has been causally related to the release of mitochondrial cytochrome c in the cytoplasm as a result of the collapse of the MMP .
As2O3 is known to act at several points in apoptosis induced through mitochondrial pathways , which includes the forming of reversible bonds with thiol groups  and the depletion of GSH [7, 22, 34, 55], loss of MMP , and activation of caspase-3 [10, 17, 18, 30, 36]. From these points of view, we examined the apoptosis-inducing effect of As2O3 in the presence of GSH modulators in MOLT-4 and MOLT-4/DNR cells.
We conclude that As2O3 induces apoptosis in MOLT-4 and MOLT-4/DNR cells via the depletion of intracellular GSH, and subsequent MMP disruption and caspase-3 activation.