Equol Pretreatment Protection of SH-SY5Y Cells against Aβ (25–35)-Induced Cytotoxicity and Cell-Cycle Reentry via Sustaining Estrogen Receptor Alpha Expression

β-amyloid formation in the brain is one of the characteristics of Alzheimer’s disease. Exposure to this peptide may result in reentry into the cell cycle leading to cell death. The phytoestrogen equol has similar biological effects as estrogen without the side effects. This study investigated the possible mechanism of the neuron cell-protecting effect of equol during treatment with Aβ. SH-SY5Y neuroblastoma cells were treated with either 1 μM S-equol or 10 nM 17β-estradiol for 24 h prior to 1 μM Aβ (25–35) exposure. After 24 h exposure to Aβ (25–35), a significant reduction in cell survival and a reentry into the cell cycle process accompanied by increased levels of cyclin D1 were observed. The expressions of estrogen receptor alpha (ERα) and its coactivator, steroid receptor coactivator-1 (SRC-1), were also significantly downregulated by Aβ (25–35) in parallel with activated extracellular signal-regulated kinase (ERK)1/2. However, pretreatment of cells with S-equol or 17β-estradiol reversed these effects. Treatment with the ER antagonist, ICI-182,780 (1 μM), completely blocked the effects of S-equol and 17β-estradiol on cell viability, ERα, and ERK1/2 after Aβ (25–35) exposure. These data suggest that S-equol possesses a neuroprotective potential as it effectively antagonizes Aβ (25–35)-induced cell cytotoxicity and prevents cell cycle reentry in SH-SY5Y cells. The mechanism underlying S-equol neuroprotection might involve ERα-mediated pathways.


Introduction
Neuronal cell death is an important feature of human neurodegenerative diseases such as Alzheimer's disease (AD). This cell death is considered to occur as a consequence of aberrant activation of the cell cycle in neurodegeneration [1]. Under normal conduction, the cell cycle is tightly controlled by specific regulatory proteins. For instance, cyclins and cyclin-dependent kinases (CDKs) are two key classes of regulatory molecules that determine a cell's progress in the cell cycle [2]. As a key regulator of the G1-S transition, cyclin D1 interacts with CDK4 to form the cyclin D1-CDK4 complex and moves to the nuclei, thereby promoting cell cycle progression. Normal adult neuron cells never reenter the cell cycle (but stay in the G 0 stage) and are thus recognized as permanently postmitotic cells [3]. Conversely, neurons reenter the cycle, undergo DNA replication, and die after they are exposed to DNA-damaging agents, oxidative stress, or certain neurotoxins such as beta-amyloid (Aβ) aggregates [3]. The Aβ peptide is the major component of senile plaque derived from the Aβ precursor protein (APP); this peptide is a neuropathological hallmark of AD [4]. There are numerous different Aβ species including Aβ , Aβ , and Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). The Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) fragment is universally used in research as it has been found to elicit profound toxic manifestations in elderly people and to physiologically play a role in AD [5]. It has been previously shown that cell cycle activation accompanied by the upregulation of cyclin D1 in primary cultured rat cortical neurons was observed in response to exposure to Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and that such activation was followed by apoptotic neuronal death [6]. To elucidate the possible intracellular signaling pathway involved in the activation of the cell cycle by Aβ, extracellular signal-regulated kinase (ERK) 1/2-related pathways are the major focus of the present study because there is evidence of the involvement of ERK1/2 activation in Aβ-induced neuronal cell death [7]. It has been documented that activation of ERK 1/2 appears to be critical for G1 to S phase progression in cell cycle regulation [8]. A previous study showed that the overexpression of ERK 1/2 in cells exposed to Aβ was followed by an elevation in cyclin D1 expression, which resulted in changes in the cell-cycle distribution, particularly in the G1-S phase [9]. ERK1/2 is also the target of the regulatory action of estrogen and its regulation requires interaction with the known estrogen receptors (ERs), ERα and ERβ [10]. In addition to the reproductive system, both ERα and ERβ are broadly expressed in nonreproductive systems including the central nervous system [11]. Particularly, brain regions such as the hypothalamus, amygdala, and hippocampus appear to have distinct expression patterns of both ER subtypes [12]. Although it is recognized that ERβ is the predominant receptor in the hippocampus, where its absence has an impact on memory and cognitive function [13], ERα co-exists, and its coregulation may be important for ERβ to fulfill its cellular roles [14]. In other words, ERβ collaborating with ERα in its molecular actions is crucial for estrogen-mediated beneficial effects on hippocampus-dependent memory and cognition. The ERα subtype is of particular interest in the present study as it exhibits stronger transcription activity than ERβ and thus appears to be functionally superior to ERβ in the modulation of age-related memory decline [13][14][15]. It is noteworthy that ERα diminishing in the hippocampus with age leads to a decrease in the relative expression of ERα and ERβ, and nuclear ERα-mediated effects, all of which are putative molecular mechanisms for age-related memory decline in the presence of low estrogen levels [13]. In this regard, the molecular actions of both ER subtypes have been reported to be involved in the neuroprotection of estrogen against the pathogenic processes of AD [16]. Evidence suggests that estrogen is capable of protecting against Aβ-induced toxicity through ERα-mediated signaling pathways [17]. Moreover, the other major neuropathological hallmark of AD is intracellular aggregates of hyperphosphorylated Tau protein, which has recently been found to interact with ERα potentiating the reduction of ERα's transcriptional activity [18]. SRC-1 is one of the nuclear receptor coactivators which enhance the transcriptional activity of ERs to manipulate the relevant molecular events [19]. Studies performed in a human astrocytoma cell line demonstrated that estradiol treatment increased the cell number through the mediation of ERα, whereas the coactivator silencing by RNA interference of SRC-1 was able to block this effect [19].
Equol is a metabolite of daidzein, one of the major isoflavones in soybean food products, and is known as an ERs agonist [20]. Equol is capable of inducing transcriptional responses, especially through the binding of ERα [21]. The oral bioavailability of equol in humans seems to be high, resulting in a plasma concentration of 0.4~2 µM after taking a single bolus of 2 mg of equol [22]. Consumption of phytoestrogens has been found to avoid many side effects from estrogens [23]. Intriguingly, equol has been shown to be a promising neuroprotectant in in vitro models, and its neuroprotective effects are exerted through anti-neuroinflammatory mechanisms with the regulation of relevant signaling pathways at molecular levels [24]. However, whether the cell cycle regulatory event and ER-dependent signaling pathways involve the neuroprotective properties of equol remains an enigma. Thus, in this study, we investigated the effects of equol on protecting SH-SY5Y cells against Aβ-induced perturbations and the cellular mechanisms underlying equol's neuroprotective action in cell cycle events and ER pathways.

Treatments
Aβ (25-35) (Sigma Aldrich, St. Louis, MO, USA) was dissolved in sterile distilled water at a concentration of 1 mM, then incubated in a capped vial at 37 • C for 5 days to allow formation of the aggregated form. It was then stored frozen at −20 • C until use. 17β-Estradiol, S-equol, and ICI-182,780 (all from Cayman Chemical, Ann Arbor, MI, USA) were dissolved in 99.5% ethanol to make stock solutions, which were used for experiments at a final concentration of 10 nM for estradiol and 1 µM for equol and ICI-182,780 in culture medium. It should be noted that no cytotoxic effect of the vehicle (99.5% ethanol) per se on cells was observed via the analysis of cell viability in our preliminary experiments that were conducted to determine the appropriate concentrations of the aforementioned treatments for the present study.
To induce cell death, cells were incubated with (Aβ) or without (C) 1 µM Aβ (25-35) for 24 h. To study the effects of estradiol (E2) and equol (Eq), cells were preincubated with estradiol (E2 + Aβ) or equol (Eq + Aβ) for 24 h prior to Aβ (25-35) exposure. Estradiol was used as a positive control and ICI-182,780 was used as an ER antagonist. It was added 1 h before the estradiol or equol treatment.

Cell Viability Analysis
Cell viability was assessed using a modified 3-[4,5-dimethylthiazol-2]-2,5 diphenyltetrazolium bromide (MTT) assay (Sigma, St. Louis, MO, USA). Cells were seeded in 24-well dishes at a seeding density of 2 × 10 5 cells/well. After treatment, 300 µL of the MTT solution (5 mg/mL) was added to each well and incubated at 37 • C for 3 h. After removing the culture medium, 250 µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan, and then 200 µL of the solution was moved to a 96-well dish. The optical density was measured at 570 nm using a microplate reader. The absorbance of the control group was considered to have 100% cell viability.

Protein Extraction and Quantification
After treatment, cells were harvested, washed three times with PBS, and lysed using a cold RIPA lysis buffer supplemented with a protease inhibitor and an EDTA solution (Thermo, Hudson, NH, USA) at a ratio of 100:1:1, then centrifuged at 13,000 rpm and 4 • C for 30 min. The supernatant was collected, and the protein concentration was estimated with a BCA Protein Assay Kit (Sigma, St. Louis, MO, USA) using BSA as the standard.

Cell-Cycle Analysis
Cells (8 × 10 5 ) were seeded in 6-well dishes. After treatment, cells were trypsinized, washed in PBS, and centrifuged at 2000× g at 25 • C for 5 min, and then they were washed with PBS at least twice. Cells were fixed in 70% ethanol overnight. Before removing the ethanol, samples were centrifuged at 11 • C and 2200× g for 10 min. The pellet was then resuspended in 200 µL of DNA extraction buffer (containing 192 mL 0.2 M Na 2 HPO 4 and 8 mL 0.1 M citric acid at pH 7.8) and incubated for 30 min at 37 • C. PI dye (200 µL, containing 0.1% Triton-X100, 100 µg/mL RNase-A, and 80 µg/mL PI in PBS) was added, gently mixed, and incubated for 30 min at room temperature in the dark. After removing the PI dye, samples were resuspended with 1 mL of cold PBS prior to analysis by flow cytometry.

Statistical Analysis
Data are shown as the mean and standard deviation (SD). Statistical comparisons were performed using SAS 9.3 (Cary, NC, USA). One-way analysis of variance (ANOVA) and least squared difference (LSD) post-hoc analysis of multiple comparisons were used. The statistical significance was accepted at p < 0.05.
17β-estradiol binding to ERα is able to trigger transcriptional regulation of target genes, such as cyclin D1 [33]. In this regard, a recent study has reported that 17β-estradiol bound ERα has a role in controlling cell cycles [34]. In the present data, we presume that downregulated ERα expression in the presence of Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) might partially contribute to aberrant cell cycles. In normal conditions, neuron cells are postmitotic and stay in the G 0 phase, as indicated by the downregulation of proteins related to the cell cycle [35]. For instance, cyclin D1, a protein marker of the G 0 /G 1 phase, is expressed at the beginning of the G1 phase and continually accumulates in the nucleus during the G1 phase in the presence of the cell cycle reactivation [36]. When the cells progress into the S phase, cyclin D1 can secrete into the cytoplasm and its overexpression can reduce cell sizes and shorten the G1 phase resulting in the accelerated entry into the S phase [37]. Likewise, our results showed that Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) caused cells to leave the postmitotic phase and reenter the cell cycle in parallel with the increasing level of cyclin D1. This finding is in line with previous studies which found that Aβ (25-35) toxicity induces cell-cycle reentry [9,38]. However, only a tendency toward a decrease in cell number of the G1 phase in the Aβ-treated group was observed in this study. Such observation might be ascribed to a more rapid cell cycle progression in response to a higher level of cyclin D1 followed by Aβ treatment as mentioned above. Alternatively, it is plausible that there is a high degree of variability in the G1-phase progression due to the differences in nature between cells, which indicates that the cell itself may enter into G1 or exit from G1 at different time points from its neighboring cells [39]. In presenilin (PS)-1 familial AD brains, the presence of cyclin D1 accumulation was observed to be linked to cell-cycle activation and subsequently led to cell death [40]. Our results are in accordance with previous findings showing that when exposed to 25 µM Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), SH-SY5Y cells accumulated in the S phase, indicating that they did not progress beyond the S phase accompanied by apoptosis [9,41]. Taken together, we speculate that changes in ERα and cyclin D1 expressions concomitantly occurring with aberrant cell cycle reentry appear likely to underlie the cytotoxic mechanisms of Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Thus, apoptotic neuronal death is presumably the consequence of Aβ (25-35)-induced cytotoxicity [42]. However, it is noteworthy that more recent evidence indicates neuronal cell death triggered by a cell cycle reentry event could be independent of an apoptotic mechanism in AD [43]. More in-depth investigation is warranted to resolve this discrepancy. Nevertheless, S-equol prevented Aβ (25-35)-induced changes in the cell-cycle behavior, ERα, and cyclin D1 expressions, indicative of the neuroprotective potential of S-equol.
A common target for estrogen signaling and Aβ neurotoxicity is ERK 1/2 [9,10,44]. It was shown that ERK 1 and 2 are expressed in the pooled cerebrospinal fluid (CSF) of patients with AD, and elevated levels of ERK 1/2 in CSF are accompanied by increased levels of tau protein and the Aβ42 peptide [45]. Rapid activation of ERK 1/2 was reported in SH-SY5Y neuroblastoma cells exposed to Aβ (25-35) [9] and in mature hippocampal neurons [46]. Aberrant activation of ERK 1/2 was correlated with an elevated level of cyclin D1 that has been shown to be responsible for cell cycle reentry in neurons under Aβ-induced toxicity conditions, thereby potentiating the neuronal apoptosis responses [38]. The present data showed that Aβ (25-35) triggered ERK 1/2 activation, and pretreatments of S-equol and 17β-estradiol were able to prevent this response. In contrast, treatment with ICI-182,780 appeared to diminish the protective effects of S-equol and 17β-estradiol. These observations led us to propose that the neuroprotective mechanisms of the actions of S-equol and 17β-estradiol against Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) cytotoxicity might be mediated by the ERK1/2 pathways via ERα. Previous studies have shown that estrogen prevents cytotoxic effects of Aβ by activating MAPK which regulates ERK 1/2 expression and cyclin D1 to control cell cycle reentry [9,29]. Herein, we have shown that S-equol exhibited neuroprotective effects that mimicked the action of 17β-estradiol on Aβ (25-35)-treated SH-SY5Y cells through preventing cell cycle reentry downregulating cyclin D1 and ERα-mediated ERK 1/2 expressions, all of which might have involved suppression of Aβ (25-35)-induced cell cycle reentry by S-equol or 17β-estradiol pretreatments in the current study.

Conclusions
This study concludes that Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) caused diminished ERα levels, which mediated estrogen actions to disrupt normal cell cycle regulation and thus potentiates cell death. S-equol might act as a putative neuroprotective agent against Aβ (25-35) cytotoxicity, and its neuroprotective role might be, at least in part, attributed to its estrogenic potency. The observed putative neuroprotective effects of equol were associated with sustaining ERα levels and cell survival in our cell models. Furthermore, the molecular mechanism underlying this putative neuroprotection of S-equol is shown to involve the suppression of cell cycle reentry which might be synergized with ERα-involved activation of ERK 1/2 along with the prevented activation of cyclin D1.