The Neuroprotective Effects of Phytoestrogens on Amyloid beta  Protein-induced Toxicity Are Mediated by Abrogating the Activation of Caspase Cascade in Rat Cortical Neurons*

Chuen-Neu WangDagger , Chih-Wen Chi§, Yun-Lian LinDagger , Chieh-Fu ChenDagger , and Young-Ji ShiaoDagger

From the Dagger  National Research Institute of Chinese Medicine, Republic of China and the § Institute of Pharmacology, National Yang-Ming University, Taipei 112, Taiwan, Republic of China

Received for publication, July 19, 2000, and in revised form, November 2, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Amyloid beta  protein (Abeta ) elicits a toxic effect on neurons in vitro and in vivo. In present study we attempt to elucidate the mechanism by which Abeta confers its neurotoxicity. The neuroprotective effects of phytoestrogens on Abeta -mediated toxicity were also investigated. Cortical neurons treated with 5 µM Abeta -(25-35) for 40 h decreased the cell viability by 45.5 ± 4.6% concomitant with the appearance of apoptotic morphology. 50 µM kaempferol and apigenin decreased the Abeta -induced cell death by 81.5 ± 9.4% and 49.2 ± 9.9%, respectively. Abeta increased the activity of caspase 3 by 10.6-fold and to a lesser extent for caspase 2, 8, and 9. The Abeta -induced activation of caspase 3 and release of cytochrome c showed a biphasic pattern. Apigenin abrogated Abeta -induced cytochrome c release, and the activation of caspase cascade. Kaempferol showed a similar effect but to a less extent. Kaempferol was also capable of eliminating Abeta -induced accumulation of reactive oxygen species. These two events accounted for the remarkable effect of kaempferol on neuroprotection. Quercetin and probucol did not affect the Abeta -mediated neurotoxicity. However, they potentiated the protective effect of apigenin. Therefore, these results demonstrate that Abeta elicited activation of caspase cascades and reactive oxygen species accumulation, thereby causing neuronal death. The blockade of caspase activation conferred the major neuroprotective effect of phytoestrogens. The antioxidative activity of phytoestrogens also modulated their neuroprotective effects on Abeta -mediated toxicity.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Senile plaque and neurofilament tangles are the hallmarks of Alzheimer's disease (AD),1 which is one of the major neurodegenerative diseases. Amyloid beta  protein (Abeta ), the major protein component of senile plaque, has been suggested to play an important role in pathogenesis of AD (1, 2). Abeta , a 39- to 42-amino acid peptide, is derived proteolytically from amyloid precursor protein, which is expressed widely throughout the brain (3, 4). Multiple lines of evidence have demonstrated that fibril Abeta participates in the induction of neuronal death and neuritic changes (5-8). The Abeta -related fragments (Abeta -(1-40), Abeta -(1-42), and Abeta -(25-35)) exhibit toxicity to neurons either in vitro or in vivo (9-11). The mechanisms by which Abeta elicits deleterious effect on neurons remain to be established.

Extensive studies have shown that Abeta -induced neurotoxicity in multiple cell types may be mediated by several different mechanisms. The neurotoxic effect may be attributable to the disturbance in calcium homeostasis (12-14) and consequently inducing the accumulation of reactive oxygen species (ROS) (13, 15-18). ROS provokes membrane damage compromising membrane integrity and increasing the permeability of ions, including calcium. The increase of calcium influx leads to generating more ROS, thereby initiating the positive feedback loop. Cultured neurons treated with Abeta or transgenic mice expressing Abeta renders neurons vulnerable to apoptosis, indicating that caspase activation plays a role in Abeta -induced neurotoxicity. Several caspases involved in apoptosis have been described to be activated by Abeta (6, 19-22). However, the mechanisms by which Abeta activates caspase cascade remain unclear. Furthermore, the activation of cyclin-dependent kinase has also been indicated to be a mediator of neurotoxicity induced by Abeta (23).

Several agents have been shown to be neuroprotective in in vitro system by targeting to specific pathway responsible for Abeta -induced toxicity. These agents include antioxidants or free radical scavengers (12, 15, 17, 18, 24-26), calcium ion channel blockers (13, 26); growth factors (27), inhibitor of cyclin-dependent kinase (23), and caspase inhibitors (15, 19, 21). Recent evidence shows that estrogen deficiency in postmenopausal women is one of the most significant risk factors for onset of AD (28, 29). Thus, estrogens have become a research focus. It has been shown that estrogen protects neurons against a number of toxic insults, including Abeta (30-34). The neuroprotective effects of estrogen are suggested to be independent of their classic nuclear estrogen receptors (32, 34).

Flavonoids, the so-called phytoestrogens, occur ubiquitously in food plants and herbal medicines. They not only bind to estrogen receptor but also exert antioxidative and antiproliferative activity (35-37). Furthermore, evidence has demonstrated that phytoestrogens interfere with a number of intracellular processes, including enzyme activation and cAMP accumulation (38, 39). Flavonoids are typically classified into four groups. Those are flavone, flavonol, flavanone, and isoflavone. Thus, apigenin, luteolin, kaempferol, quercetin, naringenin, 2,3-dihydroluteolin, genistein, and prunetin were chosen for the study of neuroprotection (Fig. 1).



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Fig. 1.   The structures and chemical names of phytoestrogens.

In the present report, we demonstrate that apigenin blocked the release of cytochrome c and activation of caspase cascade induced by Abeta . Kaempferol only inhibited the activation of caspase cascade, and the effect was less potent than that of apigenin. However, kaempferol was more effective than apigenin in counteract the deleterious effect of Abeta on cortical neurons. Kaempferol exhibited antioxidative activity and decreased the ROS accumulation induced by Abeta , whereas apigenin lacked antioxidative activity and showed a marginal effect on ROS level. Furthermore, quercetin or probucol facilitated the neuroprotective effect of apigenin on Abeta -mediated toxicity. Therefore, these results indicated that blockade of activation of caspase cascade conferred the neuroprotective effects of phytoestrogens on Abeta -mediated neurotoxicity. The inhibition of caspase cascade in combination with antioxidative activity will further eliminate Abeta -mediated neurotoxicity.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

Media and supplements for cell culture were from Life Technologies (Gaithersburg, MD). Amyloid beta  peptide-(25-35) (Abeta -(25-35)), poly-D-lysine, cytosine-beta -D-arabinofuranoside, probucol, genistein, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), trypan blue, dimethyl sulfoxide (Me2SO), and fluorescein diacetate were purchased from Sigma Chemical Co. (St. Louis, MO). Enhanced chemiluminescence detection reagents and anti-mouse IgG antibody conjugated with horseradish peroxidase were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). 2,2'-Azinobis(3-ethylbenzthiazoline sulfonic acid) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Fluorescein diacetate (FDA) and 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) were purchased from Molecular Probes (Leiden, The Netherlands). Ac-DEVD-CHO, Ac-LEHD-CHO, AC-IETD-CHO, and a casepase-3 cellular activity assay kit were obtained from Calbiochem (Darmstadt, Germany). Colorimetric caspase substrates were from BIOSOURCE (Nivelles, Belgium). Monoclonal antibodies for cytochrome c were obtained from PharMingen (San Diago, CA). All other reagents were purchased from Sigma or Merck (Darmstadt, Germany).

Methods

Cell Culture-- Primary cultures of neonatal cortical neurons were prepared from the cerebral cortex of Harlan Sprague-Dawley rat pups at postnatal day 1 (40, 41). Briefly, each pup was decapitated and the cortex was digested in 0.5 mg/ml papain at 37 °C for 15 min. The tissue was dissociated in Hypernate A medium (containing B27 supplement) by aspirating trituration. Cells were plated (5 × 104 cells/cm2) onto poly-D-lysine-coated dishes and maintained in Neurobasal medium containing B27 supplement (40), 10 units/ml penicillin, 10 µg/ml streptomycin, and 0.5 µg/ml glutamine (5% CO2/9% O2) for 3 days. Cells were then exposed to cytosine-beta -D-arabinofuranoside (5 µM) for 1 day to inhibit proliferation of non-neuronal cells. The cells were used for the experiment on the fifth day.

Measurement of Cell Viability-- The reduction of MTT, cleavage of FDA, and trypan blue exclusion were used to evaluate the cell viability. Cells were incubated with minimum essential medium containing 0.5 mg/ml MTT for 1 h. The medium was aspirated, and the formazan particle was dissolved with lysis buffer (10% sodium dodecyl sulfate, 3.3 mM HCl, 50% dimethylformamide). A600 nm was measured by using enzyme-linked immunosorbent assay reader (42). Cells were loaded with 15 µM FDA for 5 min at 25 °C, and then 1 ml of 1% deoxycholate was added to lyse the cells. The fluorescent intensity of the lysate was determined by using a spectrofluorometer with excitation and emission wavelength of 490 nm and 514 nm, respectively (26, 43). Cell viability was also assessed by using trypan blue exclusion as described previously (42).

Measurement of ROS-- Intracellular reactive oxygen species were measured by CM-H2DCFDA assay (26). In brief, cells were loaded with 50 µM CM-H2DCFDA for 30 min, and then 1 ml of 1% deoxycholate was added to lyse the cells. The fluorescent intensity of the lysate was determined by using a spectrofluorometer with excitation and emission wavelength of 488 nm and 525 nm, respectively.

Measurement of Cytochrome c Release-- Treated cells were collected in harvest buffer (50 mM Hepes, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 10 µg/ml leupeptin) and suspended by passage through a 200-µl pipette tip. The cell suspensions were centrifuged at 10,000 × g for 10 min, and supernatants were diluted with the same volume of immunoprecipitation buffer (harvest buffer containing 2% Nonidet P-40). Immunoprecipitation of cytochrome c was performed by using anti-cytochrome c antibody. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis. Immunoreactive proteins were detected by enhanced chemiluminescence detection reagents.

Measurement of Cellular Activity of Caspases-- Treated cells were harvested in cell lysis buffer (50 mM Hepes, pH 7.4, 1 mM dithiothreitol, 0.1 mM EDTA, 0.1% Chaps, and 0.1% Triton X-100). The detailed experiments were performed according to the manufacturer's protocol.

Other Methods-- The antioxidant activity of phytoestrogens were determined by trolox equilibrium antioxidant capacity (TEAC) method as described by Pellegrini et al. (44). The activity of lactate dehydrogenase (LDH) was determined by a method described previously (45). Results are expressed as means ± S.D. and were analyzed by ANOVA with post hoc multiple comparison with a Bonferroni test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phytoestrogens Protected Neurons from Abeta -induced Neurotoxicity-- Treatment of rat cortical neurons with the toxic fragment of fibril Abeta (f-Abeta -(25-35)) for 40 h decreased cell viability in a concentration-dependent manner as determined by MTT reduction or trypan blue exclusion (Fig. 2A). 10 µM Abeta decreased the MTT reduction and trypan blue exclusion by 58.2 ± 3.9% and 63.3 ± 11.5%, respectively. However, treatment with Abeta elicited a marginal effect on the ability to cleave fluorescein diacetate and had no effect on the release of LDH. The effect of Abeta on cell viability of cortical neurons was time-dependent as measured by MTT reduction (Fig. 2B). The appearance of irregularly shaped cell bodies and discontinuous neurite was concomitant with the decrease of cell viability (Fig. 2C). The percentage of cells with injured morphology was elevated as the concentration of Abeta was increased.



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Fig. 2.   Effects of Abeta -(25-35) on the cell viability of cortical neurons. Cortical neurons were incubated with various concentrations of Abeta -(25-35) for 40 h (A), and neurotoxicity was measured by the FDA assay (closed circles), MTT assay (closed squares), trypan blue exclusion (opened squares), and LDH release (closed triangles). Neurons were exposed to 5 µM of Abeta -(25-35) for 0-40 h, after the incubation periods, and cell viability was determined by the MTT assay (B). Results are means ± S.D. (where large enough to be shown) from four independent experiments and expressed relative to the cells treated with vehicle alone. C, the representative photographs of neurons treated with 0 (a), 5 (b), 10 (c), and 20 µM (d) of Abeta -(25-35) for 24 h.

The effects of a series of phytoestrogens on Abeta -induced neurotoxicity were investigated. Cells were incubated with various concentrations of pytoestrogens for 2 h then exposed to 5 µM Abeta for 40 h. Cell viability was verified by MTT reduction analysis. Results showed that kaempferol and apigenin reduced the Abeta -induced neurotoxicity in a concentration-dependent manner (Fig. 3, A and B). Kaempferol at 50 µM decreased the percentage of cell death from 45.5 ± 4.6% to 7.9 ± 3.6%. Apigenin was less potent to attenuate Abeta -induced neurotoxicity (Fig. 3B). For the morphology, 50 µM kaempferol diminished the extent of cells with injured morphology induced by Abeta (Fig. 3C). The protective effect of kaempferol on Abeta -mediated neurotoxicity was further confirmed by the measurement of trypan blue exclusion (data not shown). Luteolin, quercetin, flavanones, isoflavones, and beta -estradiol did not show any significant effects on Abeta -induced neurotoxicity (Table I).



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Fig. 3.   Kaempferol and apigenin prevented cell death of cortical neurons induced by Abeta -(25-35). Cortical neurons were pretreated with various concentrations of kaempferol (A) or apigenin (B) for 2 h and then exposed to 5 µM of Abeta -(25-35) for 40 h. Cell death was measured by MTT assay. Results are means ± S.D. (where large enough to be shown) from four independent experiments and expressed relative to cells treated with vehicle alone. Significant differences between cells treated with Abeta -(25-35) and Abeta -(25-35) plus phytoestrogen are indicated by **, p < 0.01 and ***, p < 0.001. C, neurons were pretreated with vehicle (0.1% Me2SO) (a, b) or 50 µM kaempferol (c, d) for 2 h and then exposed to 5 µM of Abeta -(25-35) (b, d) for 40 h. The morphology was visualized by phase-contrast microscope. The results are representative photographs, and similar results were repeated three times.


                              
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Table I
Protection of cortical neurons against Abeta -induced cell death by phytoestrogens and estrogen
Cortical neurons were incubated with vehicle (0.1% Me2SO), various phytoestrogens, or beta -estradiol at highest nontoxic concentration for 2 h then exposed to 5 µM Abeta for 40 h. The cell viability was assessed by MTT reduction analysis. The data are means ± S.D. and expressed relative to cells treated with vehicle alone. Significant differences between cells treated with Abeta and Abeta plus phytoestrogen or estrogen are indicated in footnotes.

Abeta Induced the Activation of Caspase Cascade-- Inhibitors of caspases were employed to investigate whether apoptosis was involved in Abeta -mediated toxicity. Ac-DEVD-CHO and Ac-LEHD-CHO are the cell-permeable inhibitors for caspases 3 and 9, respectively. These inhibitors block the activity of caspases but do not interfere with its activation. Both inhibitors reduced Abeta -induced cell death in a concentration-dependent manner (Fig. 4, A and B). In contrary, Ac-IETD-CHO, the cell-permeable inhibitor of caspase 8, did not decrease Abeta -induced cell death (data not shown). Morphological study also showed that Ac-DEVD-CHO and Ac-LEHD-CHO eliminated the cells with injured morphology induced by Abeta (Fig. 4C).



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Fig. 4.   Ac-DEVD-CHO and Ac-LEHD-CHO prevented cell death of cortical neurons induced by Abeta -(25-35). Cortical neurons were incubated with various concentrations of Ac-DEVD-CHO (caspase 3 inhibitor) (A) and Ac-LEHD-CHO (caspase 9 inhibitor) (B) for 2 h then exposed to 5 µM of Abeta -(25-35) for 40 h. Cell death were measured by MTT assay. Results are means ± S.D. from four independent experiments and expressed relative to cells treated with vehicle alone. Significant differences between cells treated with Abeta -(25-35) and Abeta -(25-35) plus phytoestrogen are indicated by ***, p < 0.001. C, neurons were pretreated with vehicle (0.1% Me2SO) (a, b), 20 µM of Ac-DEVD-CHO (c, d), or 20 µM of Ac-LEHD-CHO (e, f) for 2 h and then exposed to 5 µM of Abeta -(25-35) (b, d, f) for 40 h. The morphology was visualized by phase-contrast microscope. The results are representative photographs, and similar results were repeated three times.

Analysis of the activity of caspases were performed to further determine whether the activation of caspase cascade was involved in the Abeta -mediated neurotoxicity. Treatment of neurons with Abeta induced activation of caspase 2, 3, 8, and 9 in a time-dependent manner as measured by substrate cleavage (Fig. 5). Exposure of neurons to Abeta for 24 h, the specific activity of caspase 2, 3, 8, and 9 were increased by 6.4-, 11.6-, 6.0-, and 4.7-fold of control, respectively. The activity of caspase 3 was enhanced to a larger extent by Abeta in comparison with caspase 2, 8, and 9. Abeta exhibited a biphasic effect on the activation of caspase 3. There was a transit activation of caspase 3 at 2 h and followed by a sustained activation from 8 to 24 h. The similar result for caspase 9 was also obtained. However, the first wave of activation of caspase 9 at 2 h was not statistically significant. Caspases 2 and 8 did not show early phase of activation. The significant activation of caspases 2 and 8 occurred from 12 to 24 h.



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Fig. 5.   Abeta -(25-35)-induced activation of caspases in cortical neurons. Cortical neurons were treated with 5 µM of Abeta -(25-35) for indicated time periods. Activity of caspase 9, 3, 2, and 8 was determined by the cleavage ability of Ac-LEHD-pNA (a), Ac-DEVD-pNA (b), Ac-VDVAD-pNA (c), and Ac-IETD-pNA (d) in cell extracts, respectively. Results are means ± S.D. (where large enough to be shown) from four independent experiments. Significant differences between control and Abeta -treated cells are indicated by *, p < 0.05 and ***, p < 0.001.

The Effects of Caspase Inhibitors and Phytoestrogens on Caspase Cascade-- The inhibitor of caspases 3, 9, and 8 decreased the activity of caspase 3 and 9 at 2 h (Table II). Both inhibitors of caspase 3 and 9 inhibited the activity of caspase 2, 3, and 9 at 24 h (Table III). The inhibitor of caspase 3 diminished the activity of caspase 2, 3, and 9 by 72.8 ± 8.7, 90.8 ± 4.4, and 60.9 ± 15.0%, respectively, and the inhibitor of caspase 9 decreased the activity of caspases to a lesser extent. However, both inhibitors did not show significant effect on the activity of caspase 8. The inhibitor of caspase 8 decreased the activity of caspase 8 and 9 by 17.0 and ± 4.0% and 43.4 ± 4.0%, respectively.


                              
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Table II
The inhibition of caspase activation at 2 h by phytoestrogens and caspase inhibitors
Cortical neurons were incubated with phytoestrogens (50 µM) or caspase inhibitor (10 µM) for 2 h and exposed to 5 µM Abeta for 2 h. Cell viability was assessed by MTT reduction analysis. The data are means ± S.D. of four independent experiments and expressed relative to cell treated with Abeta alone. Significant differences between cells treated with Abeta and Abeta plus phytoestrogen or caspase inhibitor are indicated in footnotes.


                              
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Table III
The inhibition of caspase activation at 24 h by either phytoestrogen or caspase inhibitors
Cortical neurons were incubated with phytoestrogens (50 µM) or caspase inhibitor (10 µM) for 2 h and exposed to 5 µM Abeta for 24 h. Cell viability was assessed by MTT reduction analysis. The data are means ± S.D. of four independent experiments and expressed relative to cell treated with Abeta alone. Significant differences between cells treated with Abeta and Abeta plus phytoestrogen or caspase inhibitor are indicated in footnotes.

Apigenin and kaempferol did not show significant effects on the activation of caspase cascade at 2 h (Table II). However, apigenin inhibited the activity of caspase 2, 3, 8, and 9 at 24 h by about 55 to 60% (Table III). Kaempferol also diminished the activity of these caspases but to a lesser extent. Quercetin only showed inhibitory effect on the activity of caspase 9 at 24 h (Table III).

Apigenin Inhibited Abeta -induced Release of Cytochrome c-- Kaempferol and apigenin attenuated Abeta -induced activation of caspases 9 and 3. The release of cytochrome c from mitochondria, the upstream signaling component of these two caspases, was therefore investigated (Fig. 6). The release of cytochrome c induced by Abeta exerted a biphasic pattern similar to the activation of caspase 3. The first phase occurred at 2 h (Fig. 6A) and followed by the second phase from 12 to 24 h (data not shown). Apigenin significantly inhibited Abeta -induced cytochrome c release at 2 and 12 h by 34.1 and 55.7%, respectively. However, kaempferol did not affect Abeta -induced cytochrome c release either at 2 or 12 h.



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Fig. 6.   Apigenin blocked Abeta -(25-35)-induced release of cytochrome c from mitochondria in cortical neurons. A, cortical neurons were treated with 5 µM of Abeta -(25-35) for different time period. B, cells were incubated with 50 µM kaempferol or 50 µM apigenin for 2 h then exposed to 5 µM Abeta -(25-35) for 2 h (opened column) or 12 h (closed column). After treatment, cytochrome c release was analyzed by immunoblotting. The top parts of A and B are the representative immunoblots of cytochrome c. The bottom parts are the level of cytochrome c relative to the cells treated with vehicle alone. Results are means ± S.D. from three independent experiments. Significant differences between control and Abeta -treated cells (A), or cells treated with Abeta -(25-35) and Abeta -(25-35) plus phytoestrogen (B) are indicated by *, p < 0.05 and ***, p < 0.001.

Antioxidative Activity Potentiated the Neuroprotection of Apigenin-- The effect of Abeta on intracellular level of ROS was examined by using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Abeta induced ROS accumulation significantly from 8 to 24 h (Fig. 7A). Treatment with 5 µM Abeta for 8 and 16 h elevated the level of ROS to 129 ± 5% and 158 ± 10% of control, respectively. Abeta increased the level of ROS in a concentration-dependent manner (Fig. 7B). Kaempferol, luteolin, and quercetin reduced the level of ROS by 30-50% in control cells and by 50-60% in Abeta -treated cells (Fig. 7C). Apigenin decreased the ROS level by 25% in Abeta -treated cells but did not affect that in control cells. The antioxidant capacity of phytoestrogens was also determined by TEAC method (Table IV). The results showed that apigenin had lower antioxidant capacity than quercetin and luteolin. Kaempferol also showed higher antioxidant capacity than apigenin. Thus, the sequence of antioxidant capacity for these phytoestrogens did not correlate well to their neuroprotective ability. The results also implied that high antioxidative activity of flavonoid per se was not able to protect neuron against Abeta -induced neurotoxicity.



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Fig. 7.   Effects of Abeta -(25-35) on the level of ROS in cortical neurons. Cells were incubated with 5 µM of Abeta -(25-35) for the time periods indicated (A) or treated with various concentrations of Abeta -(25-35) for 16 h (B). Cortical neurons were incubated with or without 50 µM of various phytoestrogens for 18 h (opened column). Cells were incubated with or without 50 µM of various phytoestrogens for 2 h and then exposed to 5 µM of Abeta -(25-35) (closed column) for 16 h (C). Thereafter, the level of ROS was measured by the CM-DCFDA. Results are means ± S.D. from four independent experiments and expressed as percentage relative to the cells treated with vehicle alone. Significant differences between vehicle- and phytoestrogen-treated cells (opened column) or cells treated with Abeta and Abeta plus phytoestrogen (closed column) are indicated by ***, p < 0.001.


                              
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Table IV
Antioxidant capacity of phytoestrogens and estrogen
The unit of antioxidant capacity (TEAC) is defined as the concentration of Trolox having the equivalent antioxidant activity to 1 mM of the phytoestrogens or estrogen. The data are means ± S.D. of five independent experiments.

Apigenin was much more able than kaempferol to block caspase activation (Table III). Apigenin, however, exhibited the neuroprotective effect to a lesser extent (Fig. 3). We speculated, therefore, that the antioxidative activity and ability for ROS reducing may modulate the neuroprotective effect of kaempferol. To address the hypothesis that antioxidative activity may enhance the neuroprotective effect of apigenin, the effect of antioxidants on neuroprotection of apigenin was evaluated (Fig. 8). Quercetin did not reduce Abeta -mediated cell death from 1 to 50 µM. Cotreatment with quercetin and apigenin enhanced the neuroprotective effect of apigenin (Fig. 8A). Probucol, an antioxidant, was more potent at potentiating the neuroprotection effect of apigenin (Fig. 8B). The results demonstrated that, although antioxidant activity of quercetin and probucol per se did not show neuroprotective effect, they did modulate the neuroprotective effect of apigenin.



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Fig. 8.   Quercetin and probucol facilitated the neuroprotective effect of apigenin. Cortical neurons were pretreated with various concentrations of quercetin (A) or probucol (B) in the presence (closed symbol) or absence (opened symbol) of 50 µM of apigenin for 2 h, and then exposed to 5 µM of Abeta -(25-35) for 40 h. Cell death was measured by the MTT assay. Results are means ± S.D. (where large enough to be shown) from four independent experiments and expressed relative to cells treated with vehicle alone. Significant differences between cells treated with Abeta -(25-35) plus apigenin and Abeta -(25-35) plus apigenin and antioxidant are indicated by ***, p < 0.001.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present report, we demonstrate that kaempferol and apigenin prevented death of cultured neurons exposed to fibril Abeta -(25-35). The action modes of these phytoestrogens were neither due to an activation of the nuclear estrogen receptor nor solely based on an antioxidative mechanism. The anti-apoptotic signaling activity conferred neuroprotective effect of kaempferol and apigenin. Antioxidative activity of flavonoids or other antioxidants did not exhibit direct effects on neuroprotection. However, antioxidative activity facilitated the neuroprotective effect of apigenin.

Kaempferol and apigenin protected neurons from Abeta -mediated toxicity, whereas quercetin and luteolin failed to protect neurons. This result suggests that the substitution of hydroxyl group at C-3' position severely impairs the neuroprotective ability of kaempferol. The deficiency of neuroprotective effects of narigenin and 2,3-dihydroluteolin also implicates that the double bond between the C-2 and C-3 positions is required for apigenin and kaempferol to exert their neuroprotective effects. 4-Hyroxytamoxifen, an estrogen receptor antagonist, did not abrogate the protective effects, suggesting that the mode of action is independent of nuclear estrogen receptor (data not shown). That 17beta -Estradiol did not protect cortical neurons against Abeta -induced toxicity further confirms the hypothesis that the effect of phytoestrogen segregates from the action of estrogen receptor. However, some studies have shown that estrogens counteract the toxic effect of Abeta on PC12 phenochromocytoma cells and human neuroblastoma cells (32, 33, 45, 46). A possible explanation of these discrepancies could be Abeta exerting toxic effect on cell line and primary culture of cortical neurons via distinct mechanisms. Phytoestrogens reducing the loss of MTT reduction by Abeta has also been described in PC12 phenochromocytoma cells. The report (45), however, concludes that phytoestrogens affect the plasma membrane sensitivity to formazan crystal rather than protect cells against Abeta -induced cell death (45). Furthermore, quercetin shows protective effect to PC12 cells but not to primary culture of cortical neurons. These results suggest that cell lines and the primary culture of cortical neurons are protected by pytoestrogens through distinct mechanism.

Both caspase 3 inhibitor and caspase 9 inhibitor prevented neuronal death induced by Abeta , suggesting that Abeta -exerted neurotoxicity is mediated by an apoptotic pathway. Evidence supporting this speculation includes the facts that Abeta elevated the activity of caspase 3, 9, 8, and 2 and promoted the release of cytochrome c from mitochondria. The results also showed that caspase 3 displayed a biphasic activation by Abeta treatment. This activation consisted of a transient activation at 2 h and a sustained activation after 8 h. For caspase 9, the first wave activation was not supported by the statistics. Nevertheless, the elevation of caspase 9 activity at 2 h may be biologically relevant. The release of cytochrome c also showed a similar pattern as the activation of caspase 3 and 9, although Abeta activated caspase 2 and 8 after 16 h and to a lesser extent. These data suggest that the apoptosis signaling induced by Abeta is mediated primarily by activation of caspase 3 and 9 and cytochrome c release. The activation of caspase 2 and 8 and the production of ROS may be the secondary responses. Caspase 8 inhibitor was found unable to prevent neuronal death induced by Abeta , which further confirms that caspase 8 may not play the major role in Abeta -induced neuronal death.

Although the Abeta -mediated neurotoxicity becomes the focus of intense interest. The underlining mechanisms are still controversial. The study of Giovanni et al. shows that cortical neurons from caspase 3 knockout mice are resistant to Abeta -mediated cell death, suggesting that caspase 3 is the major component mediating Abeta -induced apoptosis (22). However, the studies of Troy et al. (21) show down-regulation of caspase 3 does not block Abeta -(1-42)-induced cell death. They also show that sympathetic neurons from caspase 2 null mice are resistant to Abeta -(1-42)-mediated cell death, implicating an important role of caspase 2 in Abeta -(1-42)-induced apoptosis (21). Moreover, cell death is blocked by the down-regulation of caspase 2 in hippocampal neurons, sympathetic neurons, or neuronal PC12 cells with antisense oligonucleotides. Beside caspase 3 and 2, other caspases are also thought to be involved in Abeta -induced apoptosis. Nakagawa et al. (20) shows that caspase-12-deficient cortical neurons are not susceptible to the apoptosis induced by Abeta -(1-40) (20). Another study, however, shows that the apoptotic pathway activated by Abeta requires both caspase 8 and Fas-associated death domain (FADD) (47). Our results demonstrate that Abeta elicited the activation of caspase 2, 3, 8, and 9 in cortical neurons. The release of cytochrome c and activation of caspase 9 and caspase 3 may be the major pathway mediating the Abeta -induced apoptosis of neurons. Kaempferol and apigenin abrogated the activation of all four caspases (Table III). Apigenin was more potent than kaempferol in blocking the release of cytochrome c and activation of caspase cascade induced by Abeta . However, kaempferol was more effective than apigenin to protect neurons from Abeta -induced cell death. On the basis of these data, we speculate that there may be other factors involved in the protective effect of kaempferol. ROS scavenging activity of kaempferol may be the most possible candidate to promote its neuroprotective effect.

Many reports have demonstrated or proposed that ROS is responsible for Abeta -induced neurotoxicity (13, 15-18). Behl et al. (17) shows that Abeta increases the intracellular level of H2O2 and lipid peroxide. This result suggests that free radical damage is one factor accounting for Abeta cytotoxicity. The studies of Kruman et al. (48) further provide evidence that 4-hydroxynonenal, an aldehydic product of membrane lipid peroxidation, is a key mediator of neuronal apoptosis induced by Abeta . Therefore, it is possible that scavenging of ROS may also contribute to the neuroprotective activity of kaempferol and apigenin. The level of ROS in Abeta -treated neurons was elevated after 8-h incubation, implying that ROS production may be another mediator for Abeta -induced cell death. The level of ROS in Abeta -treated neurons were reduced by both neuroprotective and nonprotective flavonoids. Furthermore, nonprotective flavonoids, quercetin and luteolin, were more potent in reducing the level of ROS and had higher antioxidative activity. These results indicate that the antioxidative activity of flavonoids per se do not confer the neuroprotective effect on Abeta -mediated toxicity. Nevertheless, quercetin and probucol, which possess antioxidative activity, did show a significant facilitating effect on the neuroprotection of apigenin. These data provide the convincing explanation for the inconsistent results between inhibition of caspase cascade and the neuroprotective effect of kaempferol and apigenin.

Taken together, the results presented here provide a plausible mechanism by which Abeta provokes death of cortical neurons. In this model (see Fig. 9), Abeta -mediated apoptosis consists of the first and second waves of caspases activation. Abeta primarily induces the release of cytochrome c from mitochondria and subsequent activation of caspase 9 and 3 at 2 h (Figs. 5 and 6). The inhibitor of caspase 8 blocked the activation of caspase 9 and 3 at 2 h (Table II), indicating caspase 8 may be involved in the first wave of caspase activation. Thereafter, caspase 3 evokes a second wave of cytochrome c release and activation of caspase cascade from 12 to 24 h. In the second wave of caspase activation, there is a positive feedback between caspase 3 and cytochrome c release, thereby establishing a signaling cascade and amplifying the signal (49). The inhibitor of caspase 3 abrogated the activation of caspase 2 at 24 h (Table III), indicating that caspase 2 may be the downstream factor of caspase 3 and complicated in the second wave of caspase activation. The sustained activation of caspase 8 occurred after 12 h, indicating that caspase 8 may also participate in the second wave of caspase activation. Furthermore, The damaged mitochondria in the second wave of caspase activation may also cause the accumulation of ROS, or alternatively, the generation of ROS after 8 h may also be involved in the release of cytochrome c and the second wave activation of caspase cascade. Both activation of caspase cascade and elevation of ROS may account for the Abeta -mediated toxicity. Both apigenin and kaempferol, but not quercetin, abrogated the second wave of cytochrome c release or activation of caspase 2, 3, 8, and 9 (Table III, Fig. 6). Despite apigenin abrogating the initial wave of cytochrome c release, both flavonoids did not affect the initial wave activation of caspase 3 and 9. These results suggest that apigenin and kaempferol protect neurons against Abeta -induced toxicity by diminishing the second wave activation of the caspase cascade. Our results clearly demonstrate that kaempferol and apigenin exhibited differentially neuroprotective effects on Abeta -induced toxicity by abrogating the release of cytochrome c and activation of the caspase cascade. Antioxidative activity of flavonoids or other antioxidants facilitated the caspase-dependent neuroprotective effect of phytoestrogen, thereby conferring a significant neuroprotective ability against Abeta -mediated toxicity.



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Fig. 9.   Proposed model for role of caspase cascade, ROS accumulation, and the protective effect of flavonoids on cell death of cortical neurons evoked by Abeta -(25-35). Schematic illustration describes the pathways contributing to cell death by Abeta -(25-35). Opened arrowheads indicate the putative target sites of apigenin and kaempferol. The closed arrowhead indicates the target side for antioxidants. For a summary see "Discussion."

Our results demonstrate that inhibition of caspase and scavenging of ROS act cooperatively to save neurons from Abeta -mediated toxicity. Therefore, inhibition of caspase cascade and decrease in the level of ROS are proposed as neuroprotective strategies in AD. Base on these results, the development of neuroprotective agents such as a compound that combines potent antioxidant and caspase inhibitory properties may prevent the incidence of AD or retard the progression of AD. Thus, our findings point toward new approach to drug discovery for clinical therapies of AD.


    FOOTNOTES

* This study was supported by the National Science Council (Grants NSC88-2314-B-077-014 and NSC89-2314-B-077-007), Taiwan, Republic of China.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: National Research Institute of Chinese Medicine, No. 155-1 Sec. 2, LiNung St., Peitou, Taipei 112, Taiwan, Republic of China. Tel.: 886-2-2820-1999 (ext. 4212); Fax: 886-2-2826-4266; E-mail: yshiao@cma23.nricm.edu.tw.

Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M006406200


    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid beta  protein; Ac-DEVD-CHO, acetyl-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-aldehyde; Ac-LEDH-CHO, acetyl-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Leu-Glu-Asp-His-aldehyde; Ac-IETD-CHO, acetyl-Ala- Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Ile- Glu-Thr-Asp-aldehyde; CM-H2DCFDA, 5-(and-6)-chloromethyl-2',7'- dichlorodihydrofluorescein diacetate; LDH, lactate dehydrogenase; FDA, fluorescein diacetate; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; ROS, reactive oxygen species; TEAC, trolox equilibrium antioxidant capacity.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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