Endoplasmic reticulum (ER) is a dynamic organelle responsible for the synthesis, correct folding, post-translational modification and transport of secretory proteins. Disturbance of ER function leads to the accumulation of unfolded or misfolded proteins in the ER lumen, and is referred to as ER stress [1
]. The correlations between ER stress and neuronal degenerative diseases, including Alzheimer’s (AD), Parkinson’s (PD) and amyotrophic lateral sclerosis (ALS), are well documented [2
]. ER stress can also be chemically induced by tunicamycin (Tm), an antibiotic produced by Streptomyces lysosuperificus.
Tm induces ER stress by inhibiting microsomal enzyme N
-acetylglucosamine-1-phosphate transferase, which is responsible for the glycosylation of newly synthesized proteins [3
The unfolded protein response (UPR) is a series of events that cells activate in order to cope with ER stress and to re-establish ER homeostasis [4
]. UPR contains three parallel signalling branches: IRE1 (inositol-requiringprotein1)-XBP1 (X-box-binding protein 1), ATF6 (activating transcription factor 6) and PERK (protein kinase RNA-like ER kinase)-eIF2α (eukaryotic translation initiation factor 2α)-ATF4 (activating transcription factor 4). The three transcription factors activated, XBP1, ATF6, and ATF4, individually and collectively upregulate the transcription of ER chaperones, such as glucose-regulated proteins 78 (GRP78), ER biosynthetic machinery, and ER associated degradation (ERAD) components in order to resolve ER stress [6
]. In the case of chronic or unmitigated ER stress, cell death pathways are induced [7
]. ER stress-induced apoptosis is mediated mainly by C/EBP homologous protein (CHOP/GADD153) [8
Emerging data also suggest that, in addition to UPR, autophagy is part of the global ER stress response [10
]. Autophagy is an evolutionarily conserved mechanism that sequesters undesired cellular material into autophagosomes for delivery to lysosomes for degradation [12
]. Autophagosome formation is controlled by a well-orchestrated action of a distinctive set of autophagy-related (Atg) proteins. A key reaction involves the lipidation of Atg8 in autophagic membranes, which is facilitated by the Atg12–Atg5 conjugate [13
Flavonoids are a group of bioactive compounds that are commonly found in fruits, vegetables, nuts and beans. Flavonoids and their metabolites can transport across the blood–brain barrier (BBB), making them ideal targets in the therapeutic utility for neurodegenerative disorders [14
]. Numerous studies indicate that the anti-neurodegeneration effects of flavonoids are consistent with their roles as activators of mitogen activated protein kinases (MAPKs), SIRT1 (Sirtuin 1) or Nrf2 (nuclear E2 related factor 2)-ARE (antioxidant response element) [15
]. The MAPK family, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2
-terminal protein kinase), and p38, plays an essential role in transduction extracellular signals to cellular response via a cascade of phosphorylation events [19
]. SIRT1 is an NAD+
-dependent class III histone deacetylase, which regulates cellular metabolism, stress resistance, cellular survival, cellular senescence/aging, and inflammation-immune function [20
]. It has been shown that SIRT1 overexpression in neurons promotes neurite outgrowth and cell survival [21
]. Recently, it has been reported that the combination of tyrosol, a natural antioxidant phenol, and S
-adenosylmethionine increased SIRT1 protein and its nuclear relocalization in ethanol-treated HepG2 cells and protected cells from oxidative stress [22
]. Nrf2-ARE is a primary sensor and oxidative stress regulator [23
]. Nrf2 activation upregulates the expression of a group of functionally diverse cytoprotective proteins, such as NAD(P)H, NAD(P)H:quinone oxidoreductase 1 (NQO1), superoxide dismutase (SOD), glutathione S
-transferase (GST), glutathione peroxidase (GPx), heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL), catalase, and thioredoxin [16
Fisetin (3,7,3′,4′-tetrahydroxyflavone, Figure 1
a) is a flavonol with a wide range of bioactivites, including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. It has been reported that fisetin protects rat pheochromocytoma PC12 cells from MPP+
-induced toxicity by upregulation of the expression of Nrf2-induced antioxidant enzymes [24
]. Fisetin has been shown to promote neuronal differentiation and long-term potentiation, maintain cognitive function and enhance memory in an animal model [25
]. Fisetin also protects PC12 cells from hypoxia-induced PC12 cell death via activating the HIF1α, MAPK and PI3K/Akt signaling pathways [28
]. Taken together, these studies show that fisetin is a potential compound with neuroprotective activity. However, the cytoprotective effect of fisetin on neuronal cells exposed to ER stress inducer is still unknown. The aim of this study is to dissect the molecular and biochemical pathways involved in fisetin-mediated cytoprotection in Tm-treated PC12 cells.
Phytochemicals are being studied for the therapeutic usage in the prevention and treatment of neurodegenerative disorders. Fisetin, a flavonoid existing in many different plant-based food products, has been reported to possess antioxidant, neurotrophic and neuroprotective activities by directly scavenging ROS or affecting signaling pathways in the maintenance of neuronal function and cell survival [25
]. However, little is known regarding how fisetin affects signaling molecules in response to ER stress in neuronal cells.
We found here that Tm caused PC12 cell death. Addition of fisetin (5–20 µM) significantly reversed the cytotoxicity. We also tested whether fisetin could protect PC12 cells from another ER stress inducer, sesquiterpene lactone thapsigargin (Tg). It was found that Tg (0.3 µg/mL) caused 45% PC12 cell death and co-treatment with fisetin (5–20 µM) exerted a dose-dependent cytoprotective effect. The above results indicate that fisetin can prevent ER stress-induced cell death in PC12 cells (Figure S3
). Furthermore, we also found that Tm (1 µg/mL) caused 33% adherent PC12 cell death, and only those cells supplemented with 15 and 20 µM fisetin could slightly attenuate the cytotoxicity, indicating that the cytoprotective effect of fisetin is valid to PC12 cells grown in both suspension and as monolayers on poly-l
-lysine-coated plates (Figure S4
Autophagy and apoptosis are cell death mechanisms with complex interactions between each other [46
]. We found that fisetin inhibited Tm-mediated apoptosis, as indicated by decreased levels of activated PARP-1. Fisetin also decreased Tm-induced LC3-II accumulation and Atg12–Atg5 conjugate, indicating that Tm-mediated autophagy was also attenuated. Furthermore, our data show that in the absence of Tm, fisetin alone could protect PC12 cells from apoptotic damage. In contrast to the results observed for higher concentrations of fisetin, which induced apoptosis [30
], this study is the first to document that low concentration of fisetin has anti-apoptotic and anti-autophagic effects in PC12 cells under ER stress.
To dissect the mechanism underlying the protective effect of fisetin, we first investigated whether fisetin inhibited Tm-induced ER stress gene expression. Our results show that fisetin did not inhibit XBP1 splicing or eIF2α phosphorylation. However, it significantly attenuated Tm-mediated mRNA expressions of GRP78, CHOP and TRB3. Significant inhibition against GRP78 protein overexpression was also noted. These results suggest that fisetin down-regulated ER stress-target gene expression without directly affecting Tm-activated IRE or PERK signaling.
It has been reported that high concentration of fisetin induces ER stress gene expression in cancer cells [30
]. We therefore investigate whether low concentration of fisetin alone could upregulate unfolded protein response (UPR) gene expression in order to conquer ER stress. Figure S5
a shows that no XBP1s RNA could be induced by fisetin (5–20 µM) after 6 h treatment. It has been reported that fisetin (<10 µM) dose- and time-dependently induces ATF4 expression in immortalized mouse hippocampal HT22 cells [41
]. In concert with this, we found that treatment of PC12 cells with fisetin (5–20 µM) alone for 6 h caused modest stimulation with regard to mRNA expression of ATF4, GRP78 and CHOP following a hormetic dose–response curve, and the highest induction was found at 10–15 µM by 1.5- to 2.5-fold (Figure S5
b–d). This suggests that the modest induction of UPR by fisetin alone may play a part in protecting cells from Tm-mediated ER stress.
It was reported that Tm induced ROS production in vitro and in vivo [40
]. In the current study, we found that both fisetin and NAC, a synthetic precursor of GSH, can block Tm-mediated ROS production, as measured by the reduced DCF fluorescence. However, ROS are difficult to measure and prone to artifacts that can generate false-positive signals [48
]. Many inherent problems, such as being oxidized by other reactive molecules, exist in using fluorescence probe H2
DCFH to analyze H2
]. In addition, studies using antioxidants to demonstrate involvement of ROS [50
] are not always conclusive. However, until newer ROS-detection techniques are evolved, a clear picture of Tm-induced ROS in PC12 cells will be lacking.
Nrf2 and ATF4 are critical transcription factors involved in GSH metabolism [52
]. Therefore, compounds that upregulate both Nrf2 and ATF4 are potentially useful in neuroprotection through their effects on GSH metabolism. It has been reported that fisetin concurrently regulates Nrf2- and ATF4-driven gene expression, and increases GSH levels at both normal and under oxidative stress conditions in HT22 cells [41
]. In accordance with this, we found that in addition to the abovementioned ATF4 induction, fisetin alone also stimulates Nrf-2-driven mRNA expression of Phase II antioxidant enzymes, HO-1, GCL and xCT in a hormetic effect.
Tm alone only weakly or insignificantly induced HO-1, GCLC and GCLM expression. On the other hand, xCT was stimulated by Tm alone more than 10-fold, supporting the notion that it was upregulated by both ATF4 and Nrf2. Fisetin treatment further enhanced HO-1 expression dose-dependently, and the cytoprotective role of HO-1 was confirmed by the addition of competitive inhibitor, Znpp. Fisetin also upregulated the genes involved in GSH metabolism in the presence of Tm. In conclusion, fisetin may exert a cytoprotective effect by increasing HO-1 expression and maintaining the GSH level via upregulating GCL and xCT expression [25
We also found that fisetin activated ERK, JNK and p38 MAPK, and attenuation of JNK and p38 MAPK phosphorylation reduced its cytoprotective effects. Many studies reveal that the upregulation of HO-1 requires p38 MAPK activation [35
]. In agreement with this, we found that SB203580 (p38 inhibitor), but not SP600125 (JNK inhibitor), attenuated fisetin-mediated HO-1 overexpression. It was also reported that p38 MAPK mediates cell survival in response to oxidative stress via induction of other Phase II antioxidant enzymes [57
]. Inhibition of fisetin-activated p38 MAPK also strongly enhanced expression of ER stress-induced apoptotic CHOP, and a less prominent effect was found for those cells treated with JNK inhibitor. The above results suggest that fisetin-induced p38 MAPK phosphorylation, and to a lesser extent, JNK phosphorylation, may both confer adaptive responses to resist Tm-mediated ER stress and cytotoxicity.
The development of sirtuin-activating compounds (STACs) as nutraceuticals in the management of chronic diseases has attracted considerable research interest in recent years, with fisetin found to be a SIRT1 activator and inducer [44
]. We found that fisetin reversed Tm-inhibited SIRT1 expression in PC12 cells. In the presence of SIRT1 inhibitor sirtinol, no fisetin-mediated cytoprotective effect could be observed. These results indicate that fisetin suppresses neuronal toxicity, possibly via modulating SIRT1 activation and expression.
4. Materials and Methods
Fisetin (≥98%), tunicamycin (Tm), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), RPMI-1640 medium, and other chemicals were from Sigma-Aldrich Co. (St. Louis, MO, USA), unless otherwise indicated. 1,4-Diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene (U0126), a selective and potent inhibitor of MEK activity and activation of ERK1/2, and 4-(4′-fluorophenyl)-2-(4′-methylsulfinylphenyl)-5-(4′-pyridyl)-imidazole (SB 203580), a p38 MAP kinase inhibitor, were purchased from Promega (Madison, WI, USA). SP600125 (1,9-pyrazoloanthron), an inhibitor of JNK, was from Calbiochem (San Diego, CA, USA). Sirtinol, a specific inhibitor of SIRT1 and SIRT2, was from Santa Cruz (Dallas, TX, USA).
4.2. PC12 Cell Culture
The rat adrenal pheochromocytoma cell line PC12 was obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and maintained in RPMI-1640 medium, which contains 2 mM glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/mL penicillin and streptomycin, supplemented with 10% heat-inactivated horse serum (Hyclone, Logan, UT, USA) and 5% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) in 5% CO2 incubator at 37 °C.
4.3. Drug Treatments and Cell Viability Assay
For experiments testing the ability of tunicamycin (Tm) to induce cytotoxicity, cells were incubated in serum-free RPMI medium in the presence of Tm [34
]. PC12 cells (5 × 105
/mL) were seeded in 24-well plates and pretreated with the indicated concentration of fisetin or an equivalent volume of DMSO vehicle control (final concentration of 0.1%) for 30 min, followed by Tm treatment for 16 h.
To measure the cell viability of adherent PC12 cells that had undergone Tm-induced damage, PC12 cells (5 × 105/mL) were seeded on poly-l-lysine-coated 6-well plates in low serum (0.5% fetal bovine serum and 1% horse serum) medium for 16 h. Fisetin or an equivalent volume of DMSO vehicle (final concentration of 0.1%) was then added and incubated for 30 min prior to Tm treatment for an additional 16 h.
A cell-free blank with medium and tested reagent was employed in parallel. Cell viability was assessed by the mitochondrial-dependent reduction of MTT to purple formazan [58
]. The cell viability was calculated by subtracting the OD550
of cell-free blank from OD550
of each sample and was expressed as percentage of the control (100%).
An esterase-dependent cell viability analysis Calcein AM (Invitrogen) was used for reconfirmation of MTT data [59
]. Briefly, cells were incubated with 5 µM Calcein AM for 30 min at 37 °C, and the fluorescent signal was monitored using 485 nm excitation and 530 nm emission wavelengths.
4.4. Protein Extraction and Immunoblotting
RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL, USA) was used for preparation of whole cell extracts and the protein concentration was measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of cell lysates were separated on SDS-PAGE and then transferred onto Hybond-P PVDF (GE Healthcare, Buckinghamshire, UK) using a CAPS transfer buffer at 30 mA overnight at 4 °C. The membranes were blocked in a freshly made blocking buffer (5% skim milk in PBS with 0.05% Tween 20, pH 7.4, PBS-T) for 6 h at room temperature. After washing with PBS-T, the membranes were incubated with an appropriate dilution (1:1000‒1:5000) of primary antibody (Table 1
) overnight at 4 °C on a rocking platform. The membranes were then washed and incubated with suitable horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA, at a dilution of 1:10,000‒1:25,000) for 1 h at room temperature. The blots of were incubated by ECL Prime (GE Healthcare), and the chemiluminescent signals were then visualized with X-ray film. Densitometry of the bands was analyzed by ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA).
4.5. RNA Extraction, Real-Time RT-PCR, and Semi-Quantitative RT-PCR
Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare) was used for preparation of total RNA. High-Capacity cDNA Archive Kit (Applied Biosystems, Waltham, MA, USA) was used to prepare cDNA from 1 µg RNA. Real-time PCR (StepOne Real-Time PCR System, Applied Biosystems) was performed with 2 µL of the cDNA, 200 nM primers (Table 2
) and Fast SYBR Green Master Mix (Applied Biosystems) in 25 µL reaction mixture. The amplification conditions were as follows: 95 °C for 2 min, 40 cycles at 94 °C for 15 s, and 60 °C for 60 s. Target gene expression was measured and normalized to the respective β-actin expression level. The identity and purity of the amplified product was checked through analysis of the melting curve carried out at the end of amplification. Relative expression was evaluated with the ΔΔCt
XBP1s and XBP1u mRNA levels were measured using regular PCR as described in our previous publication [60
4.6. Intracellular ROS Analysis
Cellular reactive oxygen species were analyzed using the fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Invitrogen), which passively diffuses into the cell and is cleaved and oxidized to 2′,7′-dichlorofluorescein (DCF). PC12 cells were stained with 20 µM H2DCFDA at 37 °C for 30 min in the dark and then washed once in PBS. Fluorescence dye loaded cells were seeded in black 96-well plates (2 × 105/well) and treated with fisetin or in combination with Tm for 16 h. The signals were then read at EX485 nm/Em535 nm using a fluorometer.
4.7. Statistical Analysis
All experiments were repeated at least three times. The results were analyzed using Kruskal–Wallis H Test by SPSS version 18. If the Kruskal–Wallis H Test shows a significant difference between the groups, then pairwise comparisons were employed by Mann–Whitney U Tests, and a p-value of <0.05 was taken to be significant.