Fisetin Protects PC12 Cells from Tunicamycin-Mediated Cell Death via Reactive Oxygen Species Scavenging and Modulation of Nrf2-Driven Gene Expression, SIRT1 and MAPK Signaling in PC12 Cells

Background: Fisetin (3,7,3′,4′-tetrahydroxyflavone) is a dietary flavonol and exhibits antioxidant, anti-inflammatory, and neuroprotective activities. However, high concentration of fisetin is reported to produce reactive oxygen species (ROS), induce endoplasmic reticulum (ER) stress and cause cytotoxicity in cancer cells. The aim of this study is to investigate the cytoprotective effects of low concentration of fisetin against tunicamycin (Tm)-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Methods: Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptotic and autophagic markers were analyzed by Western blot. Gene expression of unfolded protein response (UPR) and Phase II enzymes was further investigated using RT-Q-PCR or Western blotting. Intracellular ROS level was measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) by a fluorometer. The effects of fisetin on mitogen activated protein kinases (MAPKs) and SIRT1 (Sirtuin 1) signaling pathways were examined using Western blotting and specific inhibitors. Results: Fisetin (<20 µM) restored cell viability and repressed apoptosis, autophagy and ROS production in Tm-treated cells. Fisetin attenuated Tm-mediated expression of ER stress genes, such as glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP also known as GADD153) and Tribbles homolog 3 (TRB3), but induced the expression of nuclear E2 related factor (Nrf)2-targeted heme oxygenase (HO)-1, glutamate cysteine ligase (GCL) and cystine/glutamate transporter (xCT/SLC7A11), in both the presence and absence of Tm. Moreover, fisetin enhanced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38 MAPK. Addition of JNK and p38 MAPK inhibitor significantly antagonized its cytoprotective activity and modulatory effects on UPR. Fisetin also restored Tm-inhibited SIRT1 expression and addition of sirtinol (SIRT1 activation inhibitor) significantly blocked fisetin-mediated cytoprotection. In conclusion, this result shows that fisetin activates Nrf2, MAPK and SIRT1, which may elicit adaptive cellular stress response pathways so as to protect cells from Tm-induced cytotoxicity.

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.

Fisetin Inhibits Tm-Mediated Apoptotic and Autophagic Marker Protein Expression
Poly (ADP-ribose) polymerase-1 (PARP-1) is specifically proteolysed by caspases to a 24 and a 89 kDa fragment during the execution of the apoptotic program [36]. Figure 2a shows that treatment of PC12 cells with fisetin (10-20 µM) in the absence of Tm for 16 h decreased PARP-1 processing in a dose-dependent manner, although it did not significantly change cell viability (Figure 1b). Tm (1 µg/mL) treatment increased PARP-1 cleavage as compared with no Tm control, and co-treatment with fisetin counteracted this reaction (Figure 2b). Band quantification shows that Tm induced a significant increase in the ratio of cleaved/full length PARP-1, and fisetin (10-20 µM) dose-dependently attenuated Tm-mediated cleavage. This result indicates that fisetin serves as an anti-apoptotic agent in PC12 cells.

Fisetin Inhibits Tm-Mediated Apoptotic and Autophagic Marker Protein Expression
Poly (ADP-ribose) polymerase-1 (PARP-1) is specifically proteolysed by caspases to a 24 and a 89 kDa fragment during the execution of the apoptotic program [36]. Figure 2a shows that treatment of PC12 cells with fisetin (10-20 µM) in the absence of Tm for 16 h decreased PARP-1 processing in a dose-dependent manner, although it did not significantly change cell viability (Figure 1b). Tm (1 µg/mL) treatment increased PARP-1 cleavage as compared with no Tm control, and co-treatment with fisetin counteracted this reaction (Figure 2b). Band quantification shows that Tm induced a significant increase in the ratio of cleaved/full length PARP-1, and fisetin (10-20 µM) dosedependently attenuated Tm-mediated cleavage. This result indicates that fisetin serves as an antiapoptotic agent in PC12 cells. (c) Effect of fisetin on Tm-mediated conversion of LC3B and formation of Atg12-Atg5 conjugate. Cells were treated with the indicated reagent for 16 h and cell lysates were prepared and subjected to immunoblotting. The blots are representative from one of three independent experiments. Data obtained from immunoblots were then analyzed using ImageJ software. Data represent the mean ± SD of three independent experiments. (a) ** p < 0.01 represents significant differences compared with no fisetin control; (b,c) ** p < 0.01 represents significant differences compared with vehicle control (without Tm). # p < 0.05; ## p < 0.01 represent significant differences compared with the Tm-treated vehicle group. (c) Effect of fisetin on Tm-mediated conversion of LC3B and formation of Atg12-Atg5 conjugate. Cells were treated with the indicated reagent for 16 h and cell lysates were prepared and subjected to immunoblotting. The blots are representative from one of three independent experiments. Data obtained from immunoblots were then analyzed using ImageJ software. Data represent the mean ± SD of three independent experiments. (a) ** p < 0.01 represents significant differences compared with no fisetin control; (b,c) ** p < 0.01 represents significant differences compared with vehicle control (without Tm). # p < 0.05; ## p < 0.01 represent significant differences compared with the Tm-treated vehicle group.
Autophagy and apoptosis are important and interconnected stress response mechanisms. Microtubule-associated protein 1 light chain 3 (LC3) is the mammalian ortholog of yeast Atg8, and is required for the formation of autophagosome membrane. The conversion of LC3β from LC3β-I (free form, 18 kDa) to LC3β-II (phosphatidylethanolamine-conjugated form, 16 kDa) is an initiating step in autophagy in mammals [37]. PC12 cells cultured in the absence of Tm did not cause the conversion of LC3β (Figure 2a). In comparison, Figure 2c shows that an increase in the LC3β-II in PC12 cells was observed in the presence of Tm (1 µg/mL), confirming that autophagy was induced by Tm. Co-treatment of cells with 10 and 20 µM fisetin dose-dependently reduced Tm-mediated increase in the ratio of LC3β-II/LC3β-I. The formation of Atg12-Atg5 conjugate is a key factor in regulating the formation of autophagosome [38]. In parallel to the results found for LC3β conversion, Tm treatment for 16 h also enhanced Atg12-Atg5 conjugate formation and co-treatment of fisetin (10 and 20 µM) blocked its formation. This result suggests that fisetin represses Tm-mediated autophagy in PC12 cells.

Fisetin Inhibits Tm-Mediated Endoplasmic Reticulum (ER) Stress Gene Expression
We further investigated the effect of fisetin on Tm-mediated ER stress response. The first target was X-box-binding protein 1 (XBP1) mRNA splicing, a critical signal transducer in the unfolded protein response. The levels of the unspliced XBP1 (XBP1u) and active spliced XBP1 (XBP1s) mRNA were measured by RT-PCR and subsequent polyacrylamide electrophoresis. It was found that Tm (1 µg/mL) treatment significantly increased XBP1 splicing, but co-treatment with fisetin (5-20 µM) did not change the relative level of XBP1s to that of XBP1u ( Figure 3a). A similar phenomenon was also observed for eIF2α phosphorylation, another ER stress signal transducer upstream of ATF4. Tm (1 µg/mL) treatment markedly induced eIF2α phosphorylation, while fisetin (10-20 µM) did not change its level (Figure 3b). These results indicate that fisetin did not affect Tm-activated IRE1-XBP1 or PERK-eIF2 pathway. Autophagy and apoptosis are important and interconnected stress response mechanisms. Microtubule-associated protein 1 light chain 3 (LC3) is the mammalian ortholog of yeast Atg8, and is required for the formation of autophagosome membrane. The conversion of LC3β from LC3β-I (free form, 18 kDa) to LC3β-II (phosphatidylethanolamine-conjugated form, 16 kDa) is an initiating step in autophagy in mammals [37]. PC12 cells cultured in the absence of Tm did not cause the conversion of LC3β (Figure 2a). In comparison, Figure 2c shows that an increase in the LC3β-II in PC12 cells was observed in the presence of Tm (1 µg/mL), confirming that autophagy was induced by Tm. Cotreatment of cells with 10 and 20 µM fisetin dose-dependently reduced Tm-mediated increase in the ratio of LC3β-II/LC3β-I. The formation of Atg12-Atg5 conjugate is a key factor in regulating the formation of autophagosome [38]. In parallel to the results found for LC3β conversion, Tm treatment for 16 h also enhanced Atg12-Atg5 conjugate formation and co-treatment of fisetin (10 and 20 µM) blocked its formation. This result suggests that fisetin represses Tm-mediated autophagy in PC12 cells.

Fisetin Inhibits Tm-Mediated Endoplasmic Reticulum (ER) Stress Gene Expression
We further investigated the effect of fisetin on Tm-mediated ER stress response. The first target was X-box-binding protein 1 (XBP1) mRNA splicing, a critical signal transducer in the unfolded protein response. The levels of the unspliced XBP1 (XBP1u) and active spliced XBP1 (XBP1s) mRNA were measured by RT-PCR and subsequent polyacrylamide electrophoresis. It was found that Tm (1 µg/mL) treatment significantly increased XBP1 splicing, but co-treatment with fisetin (5-20 µM) did not change the relative level of XBP1s to that of XBP1u ( Figure 3a). A similar phenomenon was also observed for eIF2α phosphorylation, another ER stress signal transducer upstream of ATF4. Tm (1 µg/mL) treatment markedly induced eIF2α phosphorylation, while fisetin (10-20 µM) did not change its level (Figure 3b). These results indicate that fisetin did not affect Tm-activated IRE1-XBP1 or PERK-eIF2 pathway.  (f) Cells were treated with the indicated reagent for 8 h and GRP78 and CHOP in cell lysates were analyzed using immunoblotting. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with vehicle control (without Tm). ## p < 0.01 represents significant differences compared with the Tm-treated vehicle group.
We then investigated whether fisetin affected Tm-mediated ER-target gene expression. GRP78 and CHOP are canonically upregulated during apoptosis induced by ER stress. It was found that Tm treatment caused a strong increase in GRP78 mRNA expression, up by ~20-fold, while co-treatment with fisetin (10-20 µM) dose-dependently reduced its expression and 90% inhibition was noted at 20 µM ( Figure 3c). Figure 3d shows that Tm induced CHOP mRNA by ~22-fold and co-treatment with 15 and 20 µM fisetin could dose-dependently attenuate the upregulation by 77% and 88%, respectively. It has been reported that tribbles-related protein 3 (TRB3), a target gene of CHOP, was responsible for Tmmediated PC12 apoptosis [39]. In parallel to the results found for CHOP mRNA expression, Figure  3e shows that treatment of PC12 cells with 1 µg/mL Tm increased TRB3 mRNA expression by 24.7fold, and 15 and 20 µM fisetin could dose-dependently attenuate its upregulation. In conclusion, fisetin (10-20 µM) inhibited Tm-upregulated UPR mRNA expression in a dose-dependent manner.
Western blotting reveals that treatment of PC12 cells with Tm modestly increased GRP78 and CHOP protein expression. Fisetin dose-dependently attenuated GRP78 expression; while its effect on Tm-mediated CHOP protein expression was less prominent (Figure 3f). The less obvious inhibition on CHOP protein expression could be due to the poor detection sensitivity and specificity of antibodies against rat CHOP.

Fisetin Scavenges Reactive Oxygen Species (ROS) Production
It has been reported that Tm induces apoptosis in leukemia U937 cells through ROS generation [40]. Figure 4a shows that treatment of PC12 cells with fisetin (5-20 µM) alone for 16 h could decrease ROS level by about 15-20%. Treatment of PC12 cells with Tm (1 µg/mL) for 16 h modestly increased intracellular ROS production by about 11% (Figure 4b). Fisetin (10-20 µM) and N-acetyl-cysteine (NAC) (1 mM), a well-known antioxidant, completely abolished Tm-mediated ROS overproduction to a level lower than that of vehicle control. Current data support the notion that fisetin serves as a ROS scavenger in PC12 cells [28]. (f) Cells were treated with the indicated reagent for 8 h and GRP78 and CHOP in cell lysates were analyzed using immunoblotting. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with vehicle control (without Tm). ## p < 0.01 represents significant differences compared with the Tm-treated vehicle group.
We then investigated whether fisetin affected Tm-mediated ER-target gene expression. GRP78 and CHOP are canonically upregulated during apoptosis induced by ER stress. It was found that Tm treatment caused a strong increase in GRP78 mRNA expression, up by~20-fold, while co-treatment with fisetin (10-20 µM) dose-dependently reduced its expression and 90% inhibition was noted at 20 µM (Figure 3c). Figure 3d shows that Tm induced CHOP mRNA by~22-fold and co-treatment with 15 and 20 µM fisetin could dose-dependently attenuate the upregulation by 77% and 88%, respectively. It has been reported that tribbles-related protein 3 (TRB3), a target gene of CHOP, was responsible for Tm-mediated PC12 apoptosis [39]. In parallel to the results found for CHOP mRNA expression, Figure 3e shows that treatment of PC12 cells with 1 µg/mL Tm increased TRB3 mRNA expression by 24.7-fold, and 15 and 20 µM fisetin could dose-dependently attenuate its upregulation. In conclusion, fisetin (10-20 µM) inhibited Tm-upregulated UPR mRNA expression in a dose-dependent manner.
Western blotting reveals that treatment of PC12 cells with Tm modestly increased GRP78 and CHOP protein expression. Fisetin dose-dependently attenuated GRP78 expression; while its effect on Tm-mediated CHOP protein expression was less prominent (Figure 3f). The less obvious inhibition on CHOP protein expression could be due to the poor detection sensitivity and specificity of antibodies against rat CHOP.

Fisetin Scavenges Reactive Oxygen Species (ROS) Production
It has been reported that Tm induces apoptosis in leukemia U937 cells through ROS generation [40]. Figure 4a shows that treatment of PC12 cells with fisetin (5-20 µM) alone for 16 h could decrease ROS level by about 15-20%. Treatment of PC12 cells with Tm (1 µg/mL) for 16 h modestly increased intracellular ROS production by about 11% (Figure 4b). Fisetin (10-20 µM) and N-acetyl-cysteine (NAC) (1 mM), a well-known antioxidant, completely abolished Tm-mediated ROS overproduction to a level lower than that of vehicle control. Current data support the notion that fisetin serves as a ROS scavenger in PC12 cells [28].  Intracellular ROS production was measured by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). Data represent the mean ± SD of three independent experiments. (a) ** p < 0.01 represents significant differences compared with no fisetin control; (b) ** p < 0.01 represents significant differences compared with control. ## p < 0.01 represents significant differences compared with the Tm-treated vehicle group.

Fisetin Induces Nrf2-Driven Oxidative Stress Response Gene Expression
Nrf2 (nuclear E2 related factor 2)-ARE (antioxidant response element) is a primary sensor and oxidative stress regulator [23]. Fisetin (10 µM) was reported to cause activation of Nrf2-ARE and upregulation of heme oxygenase-1 (HO-1), cystine/glutamate transporter (xCT/SLC7A11) and glutamate cysteine ligase (GCL) in HT22 cells [25,41]. Figure 5a shows that fisetin (5-20 µM) treatment for 6 h significantly induced HO-1 mRNA expression in an inverted U curve, and the greatest increase (22-fold) was found for 10 µM. Consequently, HO-1 protein expression was significantly stimulated after fisetin treatment ( Figure 5b). Band quantification shows that a hormetic dose-response curve was noted after 8 h treatment and ~4.5-fold induction was caused by 10 and 15 µM fisetin (Figure 5c). Treatment with fisetin for 16 h induced less HO-1 protein expression than treatment for 8 h, and plateau induction (~3.7-fold) was reached by doses equal to or higher than 15 µM. Data represent the mean ± SD of three independent experiments. (a) ** p < 0.01 represents significant differences compared with no fisetin control; (b) ** p < 0.01 represents significant differences compared with control. ## p < 0.01 represents significant differences compared with the Tm-treated vehicle group.

Fisetin Induces Nrf2-Driven Oxidative Stress Response Gene Expression
Nrf2 (nuclear E2 related factor 2)-ARE (antioxidant response element) is a primary sensor and oxidative stress regulator [23]. Fisetin (10 µM) was reported to cause activation of Nrf2-ARE and upregulation of heme oxygenase-1 (HO-1), cystine/glutamate transporter (xCT/SLC7A11) and glutamate cysteine ligase (GCL) in HT22 cells [25,41]. Figure 5a shows that fisetin (5-20 µM) treatment for 6 h significantly induced HO-1 mRNA expression in an inverted U curve, and the greatest increase (22-fold) was found for 10 µM. Consequently, HO-1 protein expression was significantly stimulated after fisetin treatment (Figure 5b). Band quantification shows that a hormetic dose-response curve was noted after 8 h treatment and ~4.5-fold induction was caused by 10 and 15 µM fisetin (Figure 5c). Treatment with fisetin for 16 h induced less HO-1 protein expression than treatment for 8 h, and plateau induction (~3.7-fold) was reached by doses equal to or higher than 15 µM. h; subsequently, the cell lysates were subjected to immunoblotting for HO-1 and α-tubulin. Immunoblots were then analyzed using ImageJ software. Data represent the mean ± SD of three independent experiments. * p < 0.05, and ** p < 0.01 represents significant differences compared with vehicle control.
Parallel induction effects were also observed for Nrf2-driven mRNA expression of glutathione metabolism related genes, such as GCLC (catalytic subunit of GCL), GCLM (modifier subunit of GCL) and xCT (Figure 5d-f). These results suggest that fisetin induced a hormetic effect on Nrf2-driven mRNA expression of Phase II antioxidant enzymes.
We further investigated how fisetin affected Nrf2-driven gene expression in the presence of Tm. Figure 6a shows that Tm (1 µg/mL) only slightly stimulated HO-1 mRNA expression by 1.84-fold, and co-treatment with fisetin (10-20 µM) enhanced HO-1 upregulation dose-dependently. Figure 6b shows that in parallel to the results observed in mRNA expression, treatment of PC12 cells with Tm (1 µg/mL) for 16 h slightly upregulated HO-1 protein expression and fisetin enhanced HO-1 protein expression dose-dependently.
To further investigate whether the increased survival rate seen in fisetin-treated cells was dependent on HO-1 activity, PC12 cells were treated with ZnPP (zinc protoporphyrin IX), a potent competitive inhibitor of HO enzyme activity, for 30 min, followed by various concentrations of fisetin for 30 min before exposure to Tm (1 µg/mL) for 16 h. Figure 6c shows that the addition of ZnPP (0.25 and 1 µM) caused cytotoxicity on PC12 cells in all groups, and attenuated the fisetin-mediated cytoprotective effect dose-dependently. These results indicate that HO-1 activity may provide a survival advantage in PC12 cells.
We then turned to investigate how Tm affects GSH-related gene expression. The expressions of GCLC and GCLM were not affected by Tm (1 µg/mL) (Figure 6d,e). Fisetin (5-20 µM) induced GCLM expression dose-dependently (Figure 6e), while GCLC expression was impacted following a hormetic dose-response curve (Figure 6d). and α-tubulin. Immunoblots were then analyzed using ImageJ software. Data represent the mean ± SD of three independent experiments. * p < 0.05, and ** p < 0.01 represents significant differences compared with vehicle control.
Parallel induction effects were also observed for Nrf2-driven mRNA expression of glutathione metabolism related genes, such as GCLC (catalytic subunit of GCL), GCLM (modifier subunit of GCL) and xCT (Figure 5d-f). These results suggest that fisetin induced a hormetic effect on Nrf2-driven mRNA expression of Phase II antioxidant enzymes.
We further investigated how fisetin affected Nrf2-driven gene expression in the presence of Tm. Figure 6a shows that Tm (1 µg/mL) only slightly stimulated HO-1 mRNA expression by 1.84-fold, and co-treatment with fisetin (10-20 µM) enhanced HO-1 upregulation dose-dependently. Figure 6b shows that in parallel to the results observed in mRNA expression, treatment of PC12 cells with Tm (1 µg/mL) for 16 h slightly upregulated HO-1 protein expression and fisetin enhanced HO-1 protein expression dose-dependently.
To further investigate whether the increased survival rate seen in fisetin-treated cells was dependent on HO-1 activity, PC12 cells were treated with ZnPP (zinc protoporphyrin IX), a potent competitive inhibitor of HO enzyme activity, for 30 min, followed by various concentrations of fisetin for 30 min before exposure to Tm (1 µg/mL) for 16 h. Figure 6c shows that the addition of ZnPP (0.25 and 1 µM) caused cytotoxicity on PC12 cells in all groups, and attenuated the fisetin-mediated cytoprotective effect dose-dependently. These results indicate that HO-1 activity may provide a survival advantage in PC12 cells. and α-tubulin were employed and the bands were quantitated using ImageJ. The data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with vehicle control (without Tm); # p < 0.01, and ## p < 0.01 represent significant differences compared with the Tm-treated vehicle group; (c) Effects of inhibition of HO-1 activity on cell viability. PC12 cells were pretreated for 30 min with Znpp, and fisetin (5-20 µM) was then added 30 min prior to Tm exposure for 16 h. MTT was used to analyze the cell viability. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with the Tm-treated respective vehicle group. ## p < 0.01 represents significant differences compared with the respective no inhibitor group.
It was reported that xCT expression was upregulated by both Nrf2 and ATF4 [42]. We found that, different from other Nrf2-driven genes, Tm strongly stimulated xCT expression by 10-fold. In parallel to the results observed in the absence of Tm, fisetin (5-20 µM) enhanced xCT overexpression in an inverted U response curve in the presence of Tm (Figure 6f).

Fisetin Activates Mitogen-Activated Protein Kinase (MAPK) Signaling Pathways
Induction of Nrf2 by flavonoids has been associated with the activation of various members of the MAPK family [43]. To investigate how fisetin affects MAPK activation, PC12 cells were treated with 10, 15 and 20 µM fisetin for 30 min prior to Tm (1 µg/mL) exposure for 2 and 4 h, and cell lysates were immunoblotted with specific antibodies. Figure 7a,b show that treatment of 1 µg/mL Tm for 2 and 4 h induced ERK and JNK activation, but not p38 MAPK activation. Co-treatment with fisetin (10-20 µM) significantly augmented ERK, JNK and p38 MAPK activation with different patterns: enhancement of ERK and JNK activation was noted during the test period, while p38 MAPK phosphorylation was only found 2 h after Tm insult. It was reported that xCT expression was upregulated by both Nrf2 and ATF4 [42]. We found that, different from other Nrf2-driven genes, Tm strongly stimulated xCT expression by 10-fold. In parallel to the results observed in the absence of Tm, fisetin (5-20 µM) enhanced xCT overexpression in an inverted U response curve in the presence of Tm (Figure 6f).

Fisetin Activates Mitogen-Activated Protein Kinase (MAPK) Signaling Pathways
Induction of Nrf2 by flavonoids has been associated with the activation of various members of the MAPK family [43]. To investigate how fisetin affects MAPK activation, PC12 cells were treated with 10, 15 and 20 µM fisetin for 30 min prior to Tm (1 µg/mL) exposure for 2 and 4 h, and cell lysates were immunoblotted with specific antibodies. Figure 7a,b show that treatment of 1 µg/mL Tm for 2 and 4 h induced ERK and JNK activation, but not p38 MAPK activation. Co-treatment with fisetin (10-20 µM) significantly augmented ERK, JNK and p38 MAPK activation with different patterns: enhancement of ERK and JNK activation was noted during the test period, while p38 MAPK phosphorylation was only found 2 h after Tm insult. To study the cytoprotective role of the MAPK pathways, PC12 cells were pre-incubated for 30 min with 5 µM inhibitor for each pathway, U0126 (ERK), SP600125 (JNK) and SB203580 (p38 MAPK), and 5-20 µM fisetin was then added 30 min prior to Tm (1 µM) exposure for 16 h. Figure 7c,d show that addition of JNK inhibitor SP600125 and p38 MAPK specific inhibitor SB203580 significantly MTT was used to analyze the cell viability. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with the Tm-treated respective vehicle group. # p < 0.05 and ## p < 0.01 represent significant differences compared with the respective no inhibitor group.
To study the cytoprotective role of the MAPK pathways, PC12 cells were pre-incubated for 30 min with 5 µM inhibitor for each pathway, U0126 (ERK), SP600125 (JNK) and SB203580 (p38 MAPK), and 5-20 µM fisetin was then added 30 min prior to Tm (1 µM) exposure for 16 h. Figure 7c,d show that addition of JNK inhibitor SP600125 and p38 MAPK specific inhibitor SB203580 significantly exacerbated Tm-mediated cytotoxicity and attenuated the cytoprotective effects of fisetin. On the other hand, MEK inhibitor U0126 did not exert any effect on cell viability in the presence of Tm ( Figure S2). These results indicate that JNK and p38 MAPK signaling pathways, but not ERK, may be associated with the cytoprotective effect of fisetin.

Contribution of JNK and p38 MAPK Pathways to ER Stress and Nrf2-Driven Gene Expression
We further investigated whether JNK and p38 MAPK are involved in fisetin-mediated oxidative stress response gene expression. Figure 8a,b show that SB203580 significantly attenuated fisetin-enhanced HO-1 and xCT expression. This implies that fisetin-activated p38 MAPK signaling may be involved in upregulation of Nrf2-driven gene expression. exacerbated Tm-mediated cytotoxicity and attenuated the cytoprotective effects of fisetin. On the other hand, MEK inhibitor U0126 did not exert any effect on cell viability in the presence of Tm ( Figure S2). These results indicate that JNK and p38 MAPK signaling pathways, but not ERK, may be associated with the cytoprotective effect of fisetin.

Contribution of JNK and p38 MAPK Pathways to ER Stress and Nrf2-Driven Gene Expression
We further investigated whether JNK and p38 MAPK are involved in fisetin-mediated oxidative stress response gene expression. Figure 8a,b show that SB203580 significantly attenuated fisetinenhanced HO-1 and xCT expression. This implies that fisetin-activated p38 MAPK signaling may be involved in upregulation of Nrf2-driven gene expression.
Furthermore, we investigated the role of JNK and p38 MAPK on ER stress responsive gene expression. Figure 8c,d show that Tm-mediated CHOP expression was increased by both SP600125 and SB203580, but GRP78 was only enhanced by SP600125. Furthermore, the 20 µM fisetin-inhibited expressions of CHOP and GRP78 were reversed by both SP600125 and SB203580. This indicates that both fisetin-mediated JNK and p38 MAPK activation may participate in down-regulation of ER stress. This result is in agreement with a previous finding that Tm-induced p38 MAPK activation may serve as an upstream negative regulator of ER stress, and confer adaptive cytoprotection against Tmmediated cell injury [35]. The mRNA levels of CHOP and GRP78 were analyzed from RNA isolated after 6 h treatment of Tm. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with the Tm-treated respective vehicle group. ## p < 0.01 represents significant differences compared with the respective no inhibitor group.  . (a,b) The mRNA levels of HO-1 and xCT were analyzed from RNA isolated after 12 h treatment of Tm; (c,d) The mRNA levels of CHOP and GRP78 were analyzed from RNA isolated after 6 h treatment of Tm. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with the Tm-treated respective vehicle group. ## p < 0.01 represents significant differences compared with the respective no inhibitor group. Furthermore, we investigated the role of JNK and p38 MAPK on ER stress responsive gene expression. Figure 8c,d show that Tm-mediated CHOP expression was increased by both SP600125 and SB203580, but GRP78 was only enhanced by SP600125. Furthermore, the 20 µM fisetin-inhibited expressions of CHOP and GRP78 were reversed by both SP600125 and SB203580. This indicates that both fisetin-mediated JNK and p38 MAPK activation may participate in down-regulation of ER stress. This result is in agreement with a previous finding that Tm-induced p38 MAPK activation may serve as an upstream negative regulator of ER stress, and confer adaptive cytoprotection against Tm-mediated cell injury [35].
To further examine the cytoprotective role of SIRT1, we examined whether the increased survival rate seen in fisetin-treated cells was dependent on SIRT1 activity. We pre-treated cells with sirtinol (SIRT1 activation inhibitor) for 30 min, followed by fisetin for 30 min before exposure to Tm (1 µg/mL) for 16 h. Figure 9c shows that the addition of sirtinol (15 µM) did not significantly cause cytotoxicity in PC12 cells in the absence of Tm. However, sirtinol completely blocked the fisetinmediated cytoprotective effect against Tm. These results indicate that SIRT1 activity is involved in fisetin-induced cytoprotection. Cell lysates were prepared and the levels of SIRT1 and α-tubulin were analyzed by immunoblotting. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with control. ## p < 0.01 represents significant differences compared with the Tm-treated vehicle group; (c) Effects of inhibition of SIRT1 activity on cell viability. PC12 cells were pretreated for 30 min with 15 µM sirtinol and 10 or 20 µM fisetin was then added 30 min prior to Tm (1 µg/mL) exposure for 16 h. MTT was used to analyze the cell viability. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with the Tm-treated respective vehicle group. ## p < 0.01 represents significant differences compared with the respective no inhibitor group.
Tm (1 g/mL) (a,b) Fisetin reverses Tm-mediated SIRT1 downregulation. PC12 cells were treated with fisetin (5-20 µM) 30 min prior to Tm (1 µg/mL) treatment for 16 h at 37 • C. Cell lysates were prepared and the levels of SIRT1 and α-tubulin were analyzed by immunoblotting. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with control. ## p < 0.01 represents significant differences compared with the Tm-treated vehicle group; (c) Effects of inhibition of SIRT1 activity on cell viability. PC12 cells were pretreated for 30 min with 15 µM sirtinol and 10 or 20 µM fisetin was then added 30 min prior to Tm (1 µg/mL) exposure for 16 h. MTT was used to analyze the cell viability. Data represent the mean ± SD of three independent experiments. ** p < 0.01 represents significant differences compared with the Tm-treated respective vehicle group. ## p < 0.01 represents significant differences compared with the respective no inhibitor group.
To further examine the cytoprotective role of SIRT1, we examined whether the increased survival rate seen in fisetin-treated cells was dependent on SIRT1 activity. We pre-treated cells with sirtinol (SIRT1 activation inhibitor) for 30 min, followed by fisetin for 30 min before exposure to Tm (1 µg/mL) for 16 h. Figure 9c shows that the addition of sirtinol (15 µM) did not significantly cause cytotoxicity in PC12 cells in the absence of Tm. However, sirtinol completely blocked the fisetin-mediated cytoprotective effect against Tm. These results indicate that SIRT1 activity is involved in fisetin-induced cytoprotection.

Discussion
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][26][27][28]. 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 [30,31], 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 [30,32]. We therefore investigate whether low concentration of fisetin alone could upregulate unfolded protein response (UPR) gene expression in order to conquer ER stress. Figure S5a 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 S5b-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 [40,47]. 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 H 2 DCFH to analyze H 2 O 2 levels [49]. In addition, studies using antioxidants to demonstrate involvement of ROS [50,51] 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 [52,53]. 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,[54][55][56]. 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 [44,45]. 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.

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 × 10 5 /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 × 10 5 /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 OD 550 of cell-free blank from OD 550 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.

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).  (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 ∆∆C t method. XBP1s and XBP1u mRNA levels were measured using regular PCR as described in our previous publication [60].

Intracellular ROS Analysis
Cellular reactive oxygen species were analyzed using the fluorescence probe 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA) (Invitrogen), which passively diffuses into the cell and is cleaved and oxidized to 2 ,7 -dichlorofluorescein (DCF). PC12 cells were stained with 20 µM H 2 DCFDA 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 × 10 5 /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.

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.

Conclusions
Fisetin (5-20 µM) protected PC12 cells from Tm-induced cell death. Fisetin mitigated Tm-mediated apoptosis, autophagy and reactive oxygen species (ROS) production. Its cytoprotective effects were likely associated with modulation of UPR and resulted in down-regulation of ER stress target genes, GRP78, CHOP, and TRB3. Furthermore, treatment of cells with fisetin induced expression of Nrf2 target genes, HO-1, GCLC, GCLM and xCT. Fisetin restored SIRT1 expression and enhanced MAPK activation, which may confer an adaptive response to modulate Tm-mediated stress responses and cytotoxicity.