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Department of Nutrition and Food Sciences, Fu-Jen University, Hsin-Chuang, Taiwan, ROC
2To whom correspondence should be addressed. E-mail: rweifen{at}mails.fju.edu.tw.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • homocysteine thiolactone • antioxidant • folate • apoptosis • HL-60 cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
) and serum (3
). Increased levels of homocysteine as well as HcyT are considered to be atherosclerotic and carcinogenic (4
–6
). The molecular mechanisms of these homocysteine derivatives to cause cellular injury have been investigated extensively. It was suggested that both homocysteine and thiolactone derivatives could elicit oxidative stress in cells and animals (7
–10
). The resulting oxidative damage is evidenced by enhanced lipid peroxidation (11
–13
), elevated 8-hydroxy-2'-deoxyguanosine levels in DNA (14
) and impaired antioxidant enzymatic function (15
). Homocysteine-induced oxidative injury can be prevented by a variety of antioxidants, suggesting that reactive oxygen species (ROS) act as mediators (16
,17
).
Apoptosis, a programmed cell death, can be activated in response to oxidative stress conditions such as during ROS generation (18
,19
). Apoptosis in cells is characterized by chromatin condensation, nucleosomal DNA fragmentation and membrane disruption due to phosphatidylserine (PS) exposure (20
–22
). As cells are stimulated to undergo intra- or extranuclear apoptotic events, the common signaling pathways for initiation and execution of apoptosis are involved in the activation of caspases, a family of aspartate-specific cysteine proteases (23
,24
). One of the signals for apoptosis execution involves caspase-3, which is an important apoptotic effector for DNA damage or membrane disorder (25
–27
). Although the mechanisms by which ROS induce apoptosis are not fully defined, it has been suggested that the activation of caspases is involved. Evidence has shown that apoptosis induced by ROS was accompanied by caspase-3 activation, whereas inhibition of caspase-3 blocked apoptosis (28
). After hydrogen peroxide (H2O2) treatment, caspase-3 was activated during apoptosis in HL-60 cells (29
).
HcyT was shown previously to induce apoptotic damage in vascular endothelial cells (30
). We demonstrated that HcyT-induced apoptosis is mediated by H2O2 generation and caspase 3-activation in HL-60 cells (31
). Because oxidative apoptotic damage may play a pivotal role in the etiology of malignancies and diseases (32
), it is of interest to study the protective effects of some antioxidants on HcyT-induced apoptosis. N-Acetylcysteine (NAC) is a precursor of reduced glutathione that has the ability to quench hydroperoxides. Vitamin C (Vit C) is an effective scavenger of hydroxyl radicals, and vitamin E (Vit E) is a lipid radical chain breaker that scavenges oxygen radicals and alkyl radicals (33
). The ability of NAC, Vit C or Vit E to reduce cell damage elicited by various apoptotic stimuli has also been well studied (34
–37
). Folate has recently been proposed to scavenge peroxyl radicals, azide radicals and hydroxyl radicals in an in vitro radical reaction model system (38
). However, the antioxidant action of folate against apoptotic damage has not been defined. To determine whether and how these antioxidants might be able to counteract HcyT-induced apoptosis, effects of NAC, Vit C, Vit E and folate, along with catalase, were investigated using HL-60 cells, a promyeloid-like cell line with specific myeloid characteristics, which is one of the most commonly used cell lines in the study of apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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L-HcyT (homocysteine thiolactone hydrochloride salt), Vit C (L-ascorbic acid sodium salt), Vit E (
-tocopherol acetate), NAC (N-acetyl-L-cysteine), folate (pteroylmonoglutamic acid), catalase, RNase, 2',7'-dichlorofluorescein diacetate (DCFH-DA), hydroethidine (HE), propidium iodide (PI) and other reagents were purchased from Sigma Chemical (St. Louis, MO). The caspase-3 colorimetric substrate Ac-benzyloxycarbonyl aspartyl glutamylvalylaspartic acid (DEVD)-para-nitroaniline (pNA) was purchased from R & D Systems (Minneapolis, MN). The Annexin-V-Fluos reagent was from Boehringer Mannheim Biochemicals (Mannheim, Germany). Fetal bovine serum (FBS) was from HyClone Laboratories (Logan, UT). RPMI-1640 medium, penicillin, streptomycin, Fungizone, trypsin and trypan blue were from GIBCO Laboratories (Grand Island, NY).
Cell culture and viability.
HL-60, a human promyelocytic cell line, were cultured in RPMI-1640 medium supplemented with 10% heat inactivated FBS and antibiotics (2000 U/L penicillin and 20 mg/L streptomycin). Cell cultures were maintained in a humidified 5% CO2 atmosphere at 37°C. Cell viability was determined by trypan blue exclusion.
Antioxidant preparation.
Stock solutions of Vit C and NAC were prepared at 5 and 200 mmol/L in the culture medium, respectively. A stock solution of Vit E was prepared at 30 mmol/L in ethanol. The ethanol concentration in the culture medium has no effect on cell viability. A stock solution of folate was prepared at 10 mmol/L in bicarbonate solution. The catalase solution was prepared at the final concentration of 106 U/L. All the antioxidant stocks and the catalase solution were freshly prepared before experiments.
Induction of apoptosis and effect of antioxidant supplementation.
Apoptosis was induced with the HcyT treatment (31
). Confluent cells (1 x 109 cells/L) were treated either with or without 1 mmol/L HcyT for 3, 6, 12 or 24 h and harvested for apoptosis assay. For antioxidant supplementation, the concentrations and the time of antioxidant treatment to suppress HcyT-induced cytotoxicity were previously tested (39
,40
). The concentration selected to work in this study was 5 mmol/L for NAC, 100 µmol/L for both Vit C and Vit E, and 10 µmol/L for folate. Cells (1 x 109 cells/L) were preincubated with NAC, Vit C, Vit E or catalase for 2 h, or preincubated with folate for 3 d before HcyT treatment. Cells grew normally under our preincubation conditions. Further incubation with antioxidants or catalase alone did not affect cellular viability (39
). After 3 or 6 h of incubation, the indicated time points, HcyT-untreated (Control), HcyT-treated only (HcyT) and HcyT-treated cells with antioxidant preincubation were harvested for the following assays.
Analysis of apoptotic cells with membrane PS exposure.
The annexin-V-Fluos kit was used to measure the apoptotic cells with PS exposure. Cells (1 x 106) were suspended in incubation buffer (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, 5 mmol/L CaCl2) containing 1 mg/L PI and a 1:50 dilution of Annexin-V-Fluos labeling solution (catalog number, 1828681). The mixed solution was incubated for 15 min on ice. Incubation buffer (400 µL) was added, and green (annexin-V stain: apoptotic cells) and red (PI stain: necrotic cells) fluorescence intensities were analyzed on a Coulter EPICS XL-MCL flow cytometer (Coulter, Miami, FL) using 488 nm excitation, a 515 nm bandpass filter and a filter >560 nm for PI detection. Cells with Annexin-V positive and PI negative fluorescence were defined as apoptotic cells.
Analysis of apoptotic cells with hypodiploid DNA contents.
Briefly, cells were fixed in ice-cold 100% ethanol. RNase A (500 mg/L) and 0.5% Triton were added to samples at 37°C for further incubation for 60 min. Cells were then incubated with PI (50 mg/L) for 20 min at 37°C. After centrifugation (300 x g, 5 min), cellular DNA (red fluorescence) in 10,000 cells was analyzed on a Coulter EPICS XL-MCL flow cytometer with the System II software program (Coulter). The percentage of cells in different cell cycle phases was estimated from PI histograms using the WinMDI 2.8 program (Coulter). Hypodiploid cells, i.e., with sub-G0/G1 DNA contents, were defined as apoptotic cells as described by Endresen et al. (41
).
Agarose electrophoresis of DNA fragmentation.
As previously described by Huang et al. (42
), 3 x 106 cells at each indicated time point were harvested and suspended in ice-cold lysis buffer (100 mmol/L NaCl, 10 mmol/L Tris HCl, 25 mmol/L EDTA, 5 g/L SDS, and 0.3 g/L proteinase K) for 15 h in a 50°C water bath. The standard phenol/chloroform/isoamyl alcohol method (25:24:1) was used to remove protein and extract DNA. RNA was digested with 1 mg/L ribonuclease A for 1 h at 37°C. DNA fragments were electrophoresed on a 2% agarose minigel at 100 V for 40 min and visualized with ethidium bromide staining under UV illumination. Multimers of 100 base pair DNA were used as DNA markers (Pharmacia, Piscataway, NJ).
Determination of intracellular ROS levels.
Intracellular ROS were assayed using the fluorescent dyes of DCFH-DA and HE (43
). DCFH-DA readily diffuses into cells, and the acetate groups of the molecule are cleaved by intracellular esterase to yield DCFH. As DCFH is trapped within cells, H2O2 or nitric oxide oxidize DCFH to the highly fluorescent compound 2',7'-dichlorofluorescein (DCF). HE oxidation is particularly sensitive to superoxide anions, hydroxyl radicals and peroxynitrite. The intracellular fluorescence intensity of DCF or HE is proportional to the amount of ROS produced by the cells. Thirty minutes before HcyT treatment was terminated, DCFH-DA (5 µmol/L) or HE (5 µmol/L) was added into cultured cells. At each indicated time point, cells were harvested, washed and the fluorescence intensity of intracellular DCF (excitation 488 nm, emission 530 nm) or HE (excitation, 488 nm; emission, 585 nm) was monitored on a Coulter EPICS XL-MCL flow cytometer. PI (10 mg/L) was added to each tube 10 min before flow analysis to ensure that only living and early apoptotic cells were analyzed.
Caspase-3 enzymatic activity.
Caspase-3 activity was assayed by a method modified from DiPietrantonio et al. (29
). Cells (1 x 106) were incubated with 25 µL cold cell lysis buffer for 10 min. Cell lysate containing 50 µg protein was added to 148 µL reaction buffer (100 mmol/L HEPES, pH 7.5, 20% glycerol, 0.5 mmol/L EDTA, and 5 mmol/L dithiothreitol) and 2 µL caspase-3 colorimetric substrate DEVD-pNA. Samples were incubated at 37°C for 6 h in a 96-well flat-bottomed microplate. The fluorescence was read using an MRX model ELISA reader (Dynatech Laboratories, West Sussex, UK) at 405 nm wavelength.
Statistics.
All data were presented as means ± SD. One-way ANOVA and Duncan’s test were used for comparisons among groups. Significant difference was indicated when the P-value was <0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Figure 1
shows the kinetic profile of apoptotic progress and ROS generation in HcyT-treated cells during a 24-h incubation. Compared with the HcyT untreated control cells at the corresponding time, the proportion of apoptotic cells (measured by membrane PS exposure) increased by 50% after 3 h of HcyT incubation. It increased to 4.3-fold of control at 6 h, and declined to 1.3-fold at 12 and 24 h of HcyT incubation. Maximal apoptotic damage occurred at 6 h HcyT treatment. Caspase-3 activity was elevated 3.7-fold after 3 h HcyT treatment compared with control values and this persisted for 6–12 h. Activity was suppressed at 24 h of HcyT treatment. The intracellular peroxide level measured by DCF fluorescence after 3 and 6 h of HcyT treatment was 3.9-fold the control values. Levels gradually diminished after 6 h. Changes in HE fluorescent intensity induced by HcyT resembled the kinetic profile of DCF intensity, although the magnitude of changes was substantially lower for HE intensity.
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Preincubation of cells with catalase (106 U/L) for 2 h completely diminished the rise of intracellular DCF intensity induced by 3- and 6-h HcyT treatment, suggesting that the ROS in these cells were mostly H2O2 species (Table 1
). Preincubation of cells with NAC for 2 h significantly reduced the increased H2O2 generation at 3 and 6 h of HcyT treatment. Vit C and Vit E also were potent scavengers of intracellular H2O2 in HcyT-treated cells, but their effect was not evident until after 6 h of HcyT treatment. In contrast, preincubation of HcyT-treated cells with folate did not reduce the intracellular H2O2 levels after either 3 or 6 h HcyT treatment.
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. At 6 h of HcyT treatment, NAC preincubation did not affect HE intensity levels of HcyT-treated cells, whereas folate, Vit C and Vit E significantly reduced the rise of HE intensity in HcyT-treated cells. HE fluorescent intensity in HcyT-treated cells preincubated with folate was reduced to 73% of HcyT-untreated controls. Effects of antioxidants on caspase-3 enzymatic activity during HcyT-induced apoptosis.
Catalase pretreatment completely inhibited caspase-3 activation at 3 and 6 h HcyT incubation, indicating that H2O2 scavenging played a crucial role in caspase-3 activation (Table 1)
. At 3 h HcyT incubation, all antioxidants tested, NAC, Vit C, Vit E and folate, partially suppressed caspase-3 activity. The inhibitory effect of NAC, Vit C and Vit E on caspase-3 activation persisted at 6 h HcyT treatment. In contrast, folate did not suppress caspase-3 activation in HcyT-treated cells at 6 h.
Effects of antioxidants on the apoptosis reduction in HcyT-treated cells.
Because our experiments revealed that the percentage of apoptotic cells was maximal after 6 h of HcyT treatment (Fig. 1)
, this time point was chosen to examine the effects of antioxidants on apoptosis reduction. Preincubation of HL-60 cells with NAC, Vit C or Vit E significantly reduced the percentage of cells with membrane PS exposure treated with HcyT for 6 h (Fig. 2
). Preincubation with folate did not protect cells from apoptotic membrane damage, whereas catalase preincubation completely protected HcyT-treated cells.
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). These two antioxidants markedly reduced the percentage of cells with hypodiploid DNA content to nearly half of that of HcyT-treated cells. Preincubation of cells with NAC and folate did not protect HcyT-treated cells from DNA breakage. Catalase preincubation completely protected HcyT-treated cells from chromosome breakage.
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). Preincubation with Vit C or Vit E inhibited the DNA fragmentation induced by 6 h HcyT treatment (lanes 2, 5). NAC or folate did not diminish HcyT-induced DNA laddering (lanes 1, 3), whereas catalase (lane 4) or the combination of NAC, Vit C and Vit E pretreatment (lane 6) completely protected HcyT-treated cells from DNA fragmentation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Catalase pretreatment fully quenched the rise of HcyT-induced DCF intensity (Table 1)
and completely prevented HL-60 cells from caspase-3 activation as well as apoptotic damage (Table 1
, Figs. 2
3
4
). In contrast, the increase of HE intensity in HL-60 cells was substantially lower than the rise of DCF during HcyT treatment (Fig. 1
, Table 1
), and the addition of superoxide dismutase did not protect cells from caspase-3 activation or from apoptotic DNA damage (31
). These observations suggest that the generation of superoxide anions is not the mediator of apoptosis induced by HcyT, whereas H2O2 generation serves as a necessary messenger in the signaling transmission of HcyT-induced apoptosis. Similarly, the ROS-elicited damage due to homocysteine treatment was associated with DNA strand breaks and apoptosis in rat hippocampal neurons (44
). The prevention of homocysteine-induced toxicity by catalase indicates that H2O2 acts as the mediator of oxidative injury (6
,14
,16
, 17
). Our findings with those of others suggest that ROS generation, particularly H2O2, plays a key role in the apoptotic damage induced by homocysteine derivatives.
Our results demonstrated that the NAC, Vit C or Vit E pretreatment significantly inhibited HcyT-induced apoptosis. The strong antioxidant effect on H2O2 scavenging may largely account for their inhibition of HcyT-mediated cytotoxicity. The capacity of Vit C and Vit E to suppress caspase-3 activation coincided with their efficiency in inhibiting HcyT-induced apoptotic events including membrane PS exposure, DNA loss and DNA fragmentation. It was reported that VitE can restore mitochondria function to inhibit cytochrome c release (45
), which in turn modulates caspase-3 activation to affect apoptotic end point damage (46
). NAC was also a potent inhibitor of hydrogen peroxide production and caspase-3 activation in HcyT-treated cells. However, NAC pretreatment reduced only HcyT-promoted apoptotic membrane PS exposure, not DNA breakage. The reasons are presently unknown. The antioxidant activity of NAC completely prevented the induction of various DNA alterations in rat lung cells (47
). Conversely, it was reported that NAC treatment induced DNA damage in HL-60 cells (48
). The authors proposed that radicals other than hydrogen peroxide might be elicited during NAC treatment in the presence of metal ions, which might promote additional DNA damage (48
). This possibility is further supported by our finding that preincubation of HcyT-treated cells with the combination of NAC, Vit C and Vit E can offset the DNA-damaging effect of NAC under HcyT-treated conditions, and completely prevent cells from apoptotic DNA fragmentation (Fig. 4)
.
In the present study, preincubation of HcyT-treated cells with folate did not ameliorate the apoptotic damage. The failure of folate to protect cells from apoptotic DNA fragmentation or membrane dysfunction may result from its inability to scavenge HcyT-elicited intracellular H2O2 and subsequently to suppress caspase-3 activation (Figs. 2
3
4)
. This finding is in agreement with that of Duthie and Hawdon (49
) who showed that the folate supply had no effect on the reduction of oxidative DNA damage as indicated by oxidized pyrimidine levels in lymphocytes. Although folate had no H2O2 scavenging capacity and did not suppress caspase-3 activity, it demonstrated antioxidant behavior. Our data provided the direct evidence to support the hypothesis that folic acid is an effective free radical scavenger, as demonstrated by Joshie et al. (38
) in an in vitro radical reaction system. Our data also confirmed the finding of Doshi et al. (50
) that folate exhibited an antioxidant effect and reduced intracellular superoxide. The concentrations of folate necessary to reduce intracellular ROS was reported by Doshi et al. (50
) to be in the 500 micromolar range; we reported 10 µmol/L in this present study, whereas normal human plasma folate levels are at the nanomolar range (12
,51
). At micromolar concentrations, folate (10 µmol/L), Vit C (100 µmol/L) and Vit E (100 µmol/L) had similar antioxidant capacities to scavenge superoxide, hydroxyl radicals and peroxynitrite. To our knowledge, this phenomenon has not been observed previously in intact cells. In circumstances in which ROS such as superoxide anions, hydroxyl radicals or peroxynitrite are the major contributors toward the etiology of diseases, folate supplementation may be protective. Further studies are required to elucidate folate’s antioxidant capabilities under various physiologic and pathologic conditions.
In conclusion, the present work demonstrated that preincubation of HL-60 cells with NAC, Vit C or Vit E significantly attenuated HcyT-induced apoptotic damage. The capacity of NAC, Vit C and Vit E to scavenge HcyT-induced hydrogen peroxide and to suppress caspase-3 activation correlates well with their efficiency to protect against HcyT-promoted apoptotic damage. Folate had no hydrogen peroxide-scavenging capacity and did not counteract HcyT-induced apoptosis, although it did exhibit antioxidant behavior toward superoxide anions, hydroxyl radicals and peroxynitrite.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: DCF, 2',7'-dichlorofluorescein, DCFH-DA, 2',7'-dichlorofluorescein diacetate; DEVD, benzyloxycarbonyl aspartyl glutamylvalylaspartic acid; FBS, fetal bovine serum; HcyT, homocysteine thiolactone; HE, hydroethidine; NAC, N-acetylcysteine; PI, propidium iodide; pNA, para-nitroaniline; PS, phosphatidylserine; ROS, reactive oxygen species; Vit C, vitamin C; Vit E, vitamin E.
Manuscript received 20 November 2001. Initial review completed 8 February 2002. Revision accepted 15 April 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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