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

Abstract

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.

Fisetin Prevents Acetaminophen-Induced Liver Injury by Promoting Autophagy

“…A previous study has shown that FST promoted autophagic degradation of phosphorylated tau via the activation of TFEB and Nrf2 transcription factors (Kim et al, 2016), suggesting that FST exerts pro-autophagy effects. However, another study has also shown that FST exerts autophagy repressing effects (Yen et al, 2017) at doses < 20 µM in tunicamycin-mediated cells. This observation appears contrary to our conclusions, yet consistent with data from Figures 3 and 4, where we show that FST (5 mM) decreased L-02 cell viability, inhibited autophagy and upregulated inflammasome associated proteins.…”

Endoplasmic Reticulum Stress Inducer Tunicamycin Alters Hepatic Energy Homeostasis in Mice

“…Tunicamycin, a pharmacological ER stress inducer, can stimulate tumor cell apoptosis [ 17 , 18 ]. Therefore, it has been used to develop an anti-cancer drug [ 19 , 20 ].…”

Antidepressant Flavonoids and Their Relationship with Oxidative Stress

“…It has also been reported to decrease the levels of isoflurane-induced cytosolic calcium and reactive oxygen species [ 121 ]. Likewise, cellular ROS production induced by several oxidative damaging agents was attenuated by pretreatment with chrysin (2) [ 122 ], naringenin (3) [ 123 ], astilbin (4) [ 53 ], icariin (5) [ 124 ], 7,8-dihydroxyflavone (6) [ 125 ], hyperoside (7) [ 126 ], baicalein (8) [ 127 ], rutin (11) [ 128 ], luteolin (12) [ 129 ], fisetin (15) [ 130 ], nobiletin (16) [ 131 ], kaempferitrin (10) [ 132 ], and quercetin (17) [ 133 ].…”