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444 Edgar L. Rhodes Center for Animal and Dairy Science, University of Georgia, Athens, GA 30602-2771. E-mail: email@example.com
Objective: Determine the biochemical pathways involved in induction of apoptosis by ajoene, an organosulfur compound from garlic.
Research Methods and Procedures: Mature 3T3-L1 adipocytes were incubated with ajoene at concentrations up to 200 μM. Viability and apoptosis were quantified using an MTS-based cell viability assay and an enzyme-linked immunosorbent assay for single-stranded DNA (ssDNA), respectively. Intracellular reactive oxygen species (ROS) production was measured based on production of the fluorescent dye, dichlorofluorescein. Activation of the mitogen-activated protein kinases extracellular signal-regulating kinase 1/2 (ERK) and c-Jun-N-terminal kinase (JNK) was shown by Western blot. Western blot was also used to show activation of caspase-3, translocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus, and cleavage of 116-kDa poly(ADP-ribose) polymerase (PARP)-1.
Results: Ajoene induced apoptosis of 3T3-L1 adipocytes in a dose- and time-dependent manner. Ajoene treatment resulted in activation of JNK and ERK, translocation of AIF from mitochondria to nucleus, and cleavage of 116-kDa PARP-1 in a caspase-independent manner. Ajoene treatment also induced an increase in intracellular ROS level. Furthermore, the antioxidant N-acetyl-l-cysteine effectively blocked ajoene-mediated ROS generation, activation of JNK and ERK, translocation of AIF, and degradation of PARP-1.
Discussion: These results indicate that ajoene-induced apoptosis in 3T3-L1 adipocytes is initiated by the generation of hydrogen peroxide, which leads to activation of mitogen-activated protein kinases, degradation of PARP-1, translocation of AIF, and fragmentation of DNA. Ajoene can, thus, influence the regulation of fat cell number through the induction of apoptosis and may be a new therapeutic agent for the treatment of obesity.
Obesity is a chronic and costly condition that is increasing rapidly throughout the world. Obesity is considered a major risk factor for type 2 diabetes (1) and has also been linked to cancer and immune dysfunction (2). Adipose tissue mass is determined by processes governing adipocyte size and number (3). Reduction of adipocyte number can result from preadipocyte and adipocyte apoptosis, as well as adipocyte dedifferentiation (4). Therefore, apoptosis may be an important mechanism regulating adipose tissue mass.
Several organosulfur compounds from garlic, including allicin and its derivatives, have inhibited the proliferation and induced apoptosis of human mammary, bladder, and skin tumor cell lines (5, 6, 7). Ajoene is a garlic-derived compound and has a greater chemical stability than allicin. Ajoene has been shown to induce apoptosis in human promyeloleukemic cells through the generation of reactive oxygen species (ROSs)1 and activation of nuclear factor-κB (8).
ROSs are recognized to play a key role in cell signaling. At the cellular level, oxidant injury elicits a broad spectrum of responses ranging from proliferation to growth arrest, to senescence, to cell death (9). Activation of mitogen-activated protein kinases (MAPK) is considered to be a pivotal step in ROS-induced signaling pathways. It has been shown that increased ROS production in leukemic cells leads to the activation of MAPK and cell death (10, 11, 12, 13). The MAPK pathways consist of three parallel kinase modules, that is, the extracellular signal-regulating kinase (ERK1/2), the Jun-N-terminal kinase (JNK), and the p38 MAPK pathways. In general, JNK and p38 MAPK activation is associated with apoptosis induction, whereas ERK activation is cytoprotective (14).
DNA single-strand breaks, resulting from free radical and oxidant cell injury, can trigger the activation of the nuclear enzyme, Poly(ADP ribose) polymerase (PARP), which contributes to the pathogenesis of various diseases (15, 16). PARP is a highly conserved, 113-kDa nuclear enzyme, which, when activated, generates a cleaved 85-kDa PARP product.
Apoptosis-inducing factor (AIF) was more recently cloned and identified as a mitochondrial intermembrane space protein. In response to apoptotic stimuli, AIF is released from mitochondria and translocates to the nucleus, then participates in the induction of chromatin condensation, the exposure of phosphatidyl-serine in the outer leaf of the plasma membrane, and the dissipation of the mitochondrial transmembrane potential, ultimately resulting in apoptosis (17). Recently, the importance of AIF in oxidant-induced cell injury in neurons was shown (18). Furthermore, the garlic compound allicin was shown to induce a caspase-independent apoptotic pathway mediated by mitochondrial release of AIF in gastric epithelial cells (19). Currently, the apoptotic pathway in the response of 3T3-L1 adipocytes to agents such as ajoene has not yet been explored nor have the relationships among components of this pathway.
In this study, using ajoene, we found that ROS generation plays a central role in these events and is responsible for activation of the MAPK pathways. Furthermore, we present evidence to support the translocation of AIF from the mitochondria to the nucleus and the role of PARP in regulating this process.
Research Methods and Procedures
Phosphate-buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) medium were purchased from GIBCO (BRL Life Technologies, Grand Island, NY). Ajoene was received as a gift from Rafael Apitz-Castro (Institute for Thrombosis Research, Caracao, Venezuela). ApoStrand ELISA Apoptosis Detection Kit was purchased from BIOMOL (Plymouth Meeting, PA). The viability assay kit (CellTiter 96 Aqueous one solution cell proliferation assay) and Caspase-Glo 3/7 assay kit were purchased from Promega (Madison, WI). β-Actin, catalase, and N-acetyl-l-cysteine (NAC) were purchased from Sigma (St. Louis, MO). Antibodies specific for polyclonal anti-AIF, caspase-3, and PARP-1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for polyclonal anti-phospho-p38 (Thr180/Tyr182), total p38, phospho-JNK (Thr183/Tyr185), total JNK, phospho-ERK1/2 (Thr202/Tyr204), and total ERK1/2 were from Cell Signaling Technology (Beverly, MA).
Cell Line and Cell Culture
3T3-L1 mouse embryo fibroblasts were obtained from American Type Culture Collection (Manassas, VA) and were cultured as described elsewhere (20). Briefly, cells were cultured in DMEM containing 10% bovine calf serum until confluent. Two days after postconfluence (D0), the cells were stimulated to differentiate with DMEM containing 10% fetal bovine serum (FBS), 167 nM insulin, 0.5 μM isobutylmethylxanthine (IBMX), and 1 μM dexamethasone for 2 days (D2). Cells were then maintained in 10% FBS/DMEM medium with 167 nM insulin for another 2 days (D4), followed by culturing with 10% FBS/DMEM medium for an additional 4 days (D8), at which time >90% of cells were mature adipocytes with accumulated fat droplets. All media contained 100 U/mL of penicillin, 100 μg/mL of streptomycin, and of 292 μg/mL glutamine (Invitrogen, Carlsbad, CA). Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere.
3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) Cell Viability Assay
Tests were performed in 96-well plates. For mature adipocytes, cells were seeded (5000 cells/well) and grown to maturation as described above. Adipocytes were incubated with either DMSO or increasing concentrations of ajoene for 6, 12, and 24 hours. The medium was changed and replaced with 100 μL fresh 10% FBS/DMEM medium and 20 μL MTS solution. Cells were returned to the incubator for an additional 2 h before 25 μL of 10% sodium dodecyl sulfate was added to stop the reaction. The absorbance was measured at 490 nm in a plate reader (μQuant Bio-Tek Instruments, Winooski, VT) to determine the formazan concentration, which is proportional to the number of live cells.
For the assessment of apoptosis, we used the ApoStrand ELISA Apoptosis Detection Kit (Biomol, Plymouth Meeting, PA) and caspase-Glo 3/7 assay kit (Promega, Madison, WI). The ApoStrand ELISA Apoptosis Detection Kit detects single-stranded DNA, which occurs in apoptotic cells but not in necrotic cells or in cells with DNA breaks in the absence of apoptosis (21, 22). Tests were performed in 96-well plates. For mature adipocytes, cells were seeded (5000 cells/well) and grown to maturation as described above. For ssDNA ELISA, adipocytes were incubated with either DMSO or increasing concentrations of ajoene for 3, 6, 12, and 24 hours. Thereafter, cells were fixed for 30 minutes and assayed according to the manufacturer's instructions. For the caspase activity assay, cells were incubated with either DMSO (carrier; 0.1%) or increasing concentrations of ajoene for 1, 3, 6, 12, and 24 hours. Caspase-3/7 activity was measured using the substrate DEVD-aminoluciferin from the caspase-Glo 3/7 assay kit according to the manufacturer's instructions.
Measurement of Intracellular ROS Generation
The determination of ROS was based on the oxidation of the non-fluorescent 2, 7-dichlorodihydroflourescein diacetate into a fluorescent dye, 2, 7-dichloroflourescein, by peroxide. Control cells and cells treated with 200 μM ajoene were analyzed for changes in fluorescence. Cells were washed twice with PBS and incubated for 30 minutes at 37 °C in the dark with the oxidation-sensitive probe, 2, 7-dichlorodihydroflourescein diacetate (Molecular Probes, Eugene, OR), at 2.5 μM. Production of ROS was measured by changes in fluorescence at an excitation wavelength of 495 nm and an emission wavelength of 525 nm.
Western Blot Analysis
To prepare the whole cell extract, cells were washed with PBS and suspended in a lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1mM EGTA, 1% Triton X-100, 2.5 mM Na pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 100 μg/mL phenylmethylsulfonyl fluoride). After 30 minutes of rocking at 4 °C, the mixtures were centrifuged (10, 000g) for 10 minutes, and the supernatants were collected as the whole cell extracts. The cytosolic protein concentration was determined by the method of Bradford (23) with bovine serum albumin as the standard. Western blot analysis was performed using the commercial NUPAGE system (Novex/Invitrogen), where a lithium dodecyl sulfate sample buffer (Tris/glycerol buffer, pH 8.5) was mixed with fresh dithiothreitol and added to samples. Samples were then heated to 70 °C for 10 minutes. All cell lysates were separated by 12% acrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline and incubated with primary antibodies overnight at 4 °C. After washing the membranes, an alkaline-phosphatase—conjugated secondary antibody was added. The target proteins became visible after the addition of 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium, a substrate of alkaline phosphatase. All experiments were repeated at least two times. Representative Western blots are shown along with the graphs of the quantitative data.
Quantitative Analysis of Western Blot Data
Measurement of signal intensity on PVDF membranes after Western blotting with various antibodies was performed using a FluorChem densitomer with the AlphaEaseFC image processing and analysis software (Alpha Innotech Corp.). In Figure 4, density values for the protein bands of interest are expressed as percentage of the control or percent 0 hours. In Figures 5A and 5B and 6A, density values are expressed as percentage of the highest value to show more clearly how the levels of each protein changed. All figures showing quantitative analysis include data from at least two independent experiments.
Preparation of Nuclear and Mitochondrial Fractions for Measurement of AIF by Western Blot
The cells were washed with ice-cold PBS, left on ice for 10 minutes, and resuspended in isotonic homogenization buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluoride, 10 mM HEPES-KOH, pH 7.4). After 80 strokes in a Dounce homogenizer, the unbroken cells were spun down at 30g for 5 minutes. The nuclei and heavy mitochondria fractions were fractionated at 750g for 10 minutes and 14, 000g for 20 minutes, respectively, from the supernatant. The nuclei fraction was washed three times with homogenization buffer containing 0.01% NP-40. AIF in mitochondrial and nuclear fractions was measured by Western blot as described above.
Detection of DNA Fragmentation by Gel Electrophoresis
Cell pellets (3 × 106 cells) were resuspended in 500 μL of lysis buffer (0.5% Triton X-100, 10 mM EDTA, and 10 mM Tris-HCl, pH 8.0) at room temperature for 15 minutes and centrifuged at 16, 000g for 10 minutes. DNA was extracted twice with phenol:chloroform (1:1), precipitated with ethanol, and resuspended in Tris/EDTA buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). DNA was analyzed after separation by gel electrophoresis (2% agarose).
ANOVA (GLM procedure, Statistica, verion 6.1; StatSoft) was used to determine significance of treatment effects and interactions. Fisher's post hoc least significant difference test was used to determine significance of differences among means. Statistically significant differences are defined at the 95% confidence index. Data shown are means ± standard error.
Ajoene Reduces Cell Viability
Adipocytes were treated with different concentrations (0, 50, 100, 200, and 400 μM) of ajoene for 6, 12, and 24 hours. After treatment, the number of live cells was determined by MTS assay. As shown in Figure 1, ajoene time- and dose-dependently reduced viability in adipocytes. Ajoene at 200 μM decreased cell viability ∼50% after 24 hours of treatment. These concentrations of ajoene were selected for subsequent analyses.
Ajoene Induces Apoptosis
Next, using ssDNA ELISA assay as a determinant of cellular apoptosis, we studied whether the reduction in cell number by ajoene was caused by apoptosis. As shown in Figure 2A, exposure of adipocytes to ajoene resulted in a time- and dose-dependent induction of cell death that was detected after 6 hours with 200 μM ajoene and higher. Longer (e.g., 24 hours) exposures to ajoene significantly increased apoptosis at 200 and 400 μM ajoene (p < 0.05). DNA extracts from adipocytes treated with ajoene displayed a characteristic apoptotic ladder pattern of discontinuous DNA fragments on agarose gel electrophoresis (Figure 2B).
Ajoene Triggers Apoptosis by Oxidative Stress
Several reports suggest an involvement of ROS in the signal transduction pathways leading to apoptosis (24). To determine the involvement of ROS in ajoene-induced apoptosis, ROS levels were determined in ajoene-treated adipocytes. As shown in Figure 3A, ajoene increased ROS production by 2.1-fold after 5 minutes, reaching a plateau after 20 minutes. There was a decrease in ROS production between 20 and 40 minutes after treatment. Furthermore, when adipocytes were pretreated with 10 mM NAC (a thiol-containing antioxidant) for 1 hour before incubation with ajoene, ajoene-induced apoptosis was effectively reduced (Figure 3B) to nearly the control level. However, pretreatment of adipocytes with 400 units of catalase (a scavenger for hydrogen peroxide) did not prevent the ajoene-induced apoptosis.
Ajoene Induces Activation of MAPKs
Results from our group and others suggest that the apoptotic effects of ajoene involve the generation of ROS (8). Furthermore, alterations in oxidative state are intimately connected to perturbations in MAPK pathways (9). Therefore, studies were undertaken to determine the relationship between ajoene-induced oxidative stress and changes in JNK and ERK1/2 activation. Incubation of adipocytes with ajoene (200 μM) led to phosphorylation of JNK (Figure 4A, top). Quantitative analysis (Figure 4A) shows that activation of JNK occurred as late as 60 minutes, with maximum activity at 180 minutes, after which JNK decreased gradually. Interestingly, ERK1/2, a kinase suggested to play a role in survival pathways, was also phosphorylated on exposure to ajoene. Figure 4B (top) shows the time-dependent phosphorylation of ERK1/2 in adipocytes treated with ajoene (200 μM). Maximum activation of ERK1/2 was observed 30 minutes after incubation with ajoene and quickly diminished after 60 minutes. Pretreatment with the antioxidant NAC (10 mM) completely blocked both ajoene-mediated ERK1/2 activation and JNK activation (Figure 4C and 4D).
Ajoene Induces PARP-1 Cleavage and AIF-mediated Cell Death in a Caspase-independent Manner
Because it has been shown that ROS-mediated DNA damage triggers activation of PARP and subsequent cell death (18, 25), we studied the role of PARP in ajoene-induced apoptotic cell death. Treatment of adipocytes with 200 μM ajoene induced the proteolytic cleavage of PARP-1 (116 kDa), resulting in the accumulation of the 85-kDa cleavage product (Figure 5A, top). Western blotting and quantitative data revealed that PARP-1 cleavage was apparent 4 hours after ajoene treatment and gradually increased. Moreover, pretreatment with the antioxidant NAC blocked ajoene-mediated degradation of PARP-1 (Figure 5B). Surprisingly, ajoene did not activate caspase-3, either through proteolytic cleavage of the proenzyme (Figure 5A, middle) or through protease activity (Figure 5C). Taken together, these data indicate that ROS-mediated cell death involved PARP degradation but in a caspase-independent manner.
We next examined whether ajoene-induced cell death involved AIF, because AIF-induced cell death is also caspase independent (17). The adipocytes exposed to ajoene (200 μM) showed a translocation of AIF from the mitochondria to the nuclei in a time-dependent manner (Figure 6A). Quantitative analysis also showed that the amount of nuclear AIF gradually increased from 4 to 12 hours. Moreover, the pretreatment with the antioxidant NAC prevented translocation of AIF to nuclei (Figure 6B).
This study elucidates the biological effect of ajoene, a component of garlic, on 3T3-L1 adipocytes. Decreases in adipose tissue mass may involve the loss of mature fat cells through apoptosis (4), and, therefore, we are especially interested in identifying compounds that influence apoptosis and studying their mechanism of action. To our knowledge, our study reports the first insight into the mechanism of ajoene-induced apoptosis by ROS in 3T3-L1 adipocytes. ROS generation is first shown here to activate MAPK cascade and PARP-1. Our results show that increased ROS production is involved in AIF release in a caspase-independent manner.
To elucidate the mechanism of apoptosis by ajoene, we studied MAPK expression and ROS production. Our results show that treatment with ajoene led to phosphorylation of both ERK1/2 and JNK in 3T3-L1 adipocytes. In general, JNK activation is associated with apoptosis induction, whereas ERK activation is cytoprotective (14). Although it is generally accepted that activation of ERK1/2 leads to cell proliferation (26), there are conditions in which ERK1/2 activation results in cell death (27, 28). Furthermore, previous studies have shown that H2O2 (ROS stimulator) rapidly activates ERK in PC12 cells (29), and ROSs are involved in cell death through the ERK1/2 signaling pathway (30). Our results also showed that ROS levels peaked 20 minutes after ajoene treatment, whereas ERK1/2 was activated at 30 minutes and JNK was activated at 180 minutes, which suggests that within this context, ROS is likely to be upstream of MAPK activation. Our data indicate that ajoene differentially influences the phosphorylation status of members of the MAP kinase superfamily; the phosphorylation of ERK1/2 rapidly decreased, whereas the phosphorylation of JNK slowly and modestly decreased. In addition, our observation that pretreatment of cells with NAC inhibited both ROS generation and ERK1/2 and JNK protein expression suggests that ROS directly influences MAPK signaling. Catalase is a scavenger for hydrogen peroxide and did not block ajoene-induced ROS generation. NAC, however, is a thiol-reducing agent in addition to its action as a free radical scavenger (31). Ajoene, which contains thiol groups, may be reduced by NAC and thereby lose its ability to induce apoptosis through generation of ROS and subsequent MAPK activation.
Some studies have implicated the members of the MAPK family as regulators of mitochondrial-dependent apoptosis (32). A recent report showed that JNK up-regulation is followed by the activation of Bax and downstream mitochondrial depolarization, with the release of AIF and cytochrome c, in a caspase-independent manner (33). Therefore, we hypothesized that these signaling pathways may be involved in the ROS-induced mitochondrial changes by ajoene. Our results showed that ajoene did not affect the activity of caspase-3 either by proteolytic cleavage of the proenzyme or by protease activity. This suggests that the apoptotic cell death induced by ajoene treatment is caspase independent.
It has been reported that AIF mediates cell death through a caspase-independent pathway. Death stimuli cause translocation of AIF from the mitochondria to the nucleus where it initiates nuclear condensation (17, 34). Once the nucleus condenses, large-scale chromatin fragmentation ensues, followed by cell death. Consistent with these findings, we found translocation of AIF from mitochondria to the nucleus and DNA fragmentation. In addition, the treatment of cells with NAC inhibited AIF translocation to the nucleus, suggesting that the increased intracellular ROS level is critical for the AIF relocalization after ajoene treatment. These observations indicate that ajoene induces ROS generation followed by AIF translocation and cell death, which can be reversed by preventing generation of ROS.
PARP is a nuclear enzyme that facilitates DNA repair in response to DNA damage (35, 36). Enhanced activation of PARP enzyme is a major contributor to oxidative stress-induced cell dysfunction and tissue injury (37, 38). ROSs cause single-strand DNA breaks (39). Single-strand DNA breaks can activate nuclear PARP, which ADP-ribosylates different nuclear proteins at the expense of cleaving NAD+. If PARP activation exceeds a certain limit, it can lead to cellular NAD+ and ATP depletion, ultimately resulting in cell death (37, 38, 39, 40). Our data also showed that increased level of ROS by ajoene induced cleavage of 116-kDa PARP-1, resulting in the accumulation of an 85-kDa product. Further treatment with NAC attenuated cleavage of PARP-1.
In summary, we have shown that ajoene treatment induced apoptosis through the caspase-independent cell death pathway by enhancement of intracellular ROS level. The enhancement of intracellular ROS level promotes MAPK and PARP-1 activation and subsequent AIF release, ultimately resulting in apoptosis. Thus, ajoene may be a new therapeutic tool for the treatment of obesity by regulating fat cell number through the induction of adipocyte apoptosis.
This work was supported by the Georgia Research Alliance, AptoTec, and the Georgia Research Alliance Eminent Scholar endowment held by C.A.B.