Quercetin 3-O-methyl ether protects FL83B cells from copper induced oxidative stress through the PI3K/Akt and MAPK/Erk pathway
Hsiao-Ling Tseng1, Chia-Jung Li2, Lin-Huang Huang3, Chun-Hao Tsai1, Chun-Yao Chen1, Chun-Nan
Lin4#, Hsue-Yin Hsu1#
These authors contributed equally to this work.
Authors’ Affiliations: 1Department of Life Sciences, 2Institute of Medical Sciences, Tzu Chi University,
Hualien, Taiwan. 3School of Medicine, Institute of Traditional Medicine, National Yang-Ming University,
Taipei, Taiwan. 4Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung,
Taiwan.
Hsiao-Ling Tseng [email protected] Chia-Jung Li [email protected] Lin-Huang Huang [email protected] Chun-Hao Tsai [email protected] Chun-Yao Chen [email protected]
# Corresponding authors: Hsue-Yin Hsu
Department of Life Sciences, Tzu Chi University, Hualien City, Taiwan.
Mailing address: No. 701, Sec. 3, Zhongyang Rd., Hualien City, 970, Taiwan (R.O.C.) Tel: +886-3-8565301#2610 Fax: +886-3-8572526 E-mail: [email protected]
Chun-Nan Lin
Faculty of Fragrance and Cosmetics, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Department of Biological Science and Technology, School of Medine, China Medical University, Taichung, Taiwan.
Tel: +886 7 3121101 Fax: +886 7 5562365 E-mail: [email protected] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 17 18 19 20 21 22 23 24 25 26 27 28 1
Abstract
Quercetin is a bioflavonoid that exhibits several biological functions in vitro and in vivo. Quercetin 3-O-methyl ether (Q3) is a natural product reported to have pharmaceutical activities, including antioxidative and anticancer activities. However, little is known about the mechanism by which it protects cells from oxidative stress. This study was designed to investigate the mechanisms by which Q3 protects against Cu2+-induced cytotoxicity. Exposure to Cu2+ resulted in the death of mouse liver FL83B cells,
characterized by apparent apoptotic features, including DNA fragmentation and increased nuclear condensation. Q3 markedly suppressed Cu2+-induced apoptosis and mitochondrial dysfunction,
characterized by reduced mitochondrial membrane potential, caspase-3 activation, and PARP cleavage, in Cu2+-exposed cells. The involvement of PI3K, Akt, Erk, FOXO3A, and Mn-superoxide dismutase (SOD)
were shown to be critical to the survival of Q3-treated FL83B cells. The liver of both larval and adult zebrafish showed severe damage after exposure to Cu2+ at a concentration of 5 M. Hepatic damage
induced by Cu2+ was reduced by cotreatment with Q3. Survival of Cu2+-exposed larval zebrafish was
significantly increased by cotreatment with 15 M Q3. Our results indicated that Cu2+-induced apoptosis
in FL83B cells occurred via the generation of ROS, upregulation and phosphorylation of Erk, inactivation of Akt, and the downregulation of FOXO3A and MnSOD. Hence, these results also demonstrated that Q3 plays a protective role against oxidative damage in zebrafish liver and remarked the potential of Q3 to be used as an antioxidant for hepatocytes.
Keywords: Quercetin-3-O-methyl ether; ROS; Cu2+, Erk
29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 2
1. Introduction
Copper, an essential element for cellular physiological processes in most eukaryotic organisms, is toxic in excessive amounts as it can initiate oxidative stress and cause direct damage to proteins, lipids, and DNA through the formation of free radicals . The oxidative stress is the result of the production of reactive oxygen species (ROS) that causes particular damage in the liver or brain . Oxidative damage in the liver is typically manifested by the development of liver cirrhosis with episodes of hemolysis, leading to hepatic necrosis, vascular collapse, and death . Therefore, mechanisms to protect the liver from oxidative damage induced by elevated copper concentrations are of interest; dietary antioxidants are of particular interest .
Mitochondria, important targets of copper toxicity, produce a considerable quantity of ROS and, therefore, play an important role in copper-induced apoptosis . Increased mitochondrial permeability, a step in copper-related apoptotic and necrotic cell death, involves the formation of pores in the mitochondrial outer and inner membranes that are reported to be regulated by ROS . Copper-induced apoptosis was suggested to be dependent on induction of the mitochondrial dysfunction, whereas the prominent contribution of mitochondria to ROS generation also suggested an important role of mitochondria in copper-induced cell death . The mitochondrial dysfunction may lead to the activation of a series of caspases . The activation of caspase-3 leads to a series of apoptotic events, including the cleavage of PARP, a downstream substrate in the caspase-dependent apoptotic pathway, which protect DNA from oxidative damage .
The cell on account of susceptibility to oxidative damage is naturally provided with efficient antioxidant system glutathione (GSH), the ROS scavenger which helps to maintain a reduced state within the aerobic cells against oxidative stress . GSH is known to be important in the mobilization of copper in hepatocytes . In addition, superoxide dismutases (SODs) are metalloenzymes that act in the conversion of superoxide anion (•O2−) free radicals into hydrogen peroxide (H2O2) and thus form a crucial part of the cellular antioxidant defense mechanism against mitochondrial superoxide induced by copper . Once these enzymes are overwhelmed by excessive ROS production, irreversible damage and cell death can occur.
Recently, the Akt signaling pathway and the extracellular signal-regulated kinase (Erk) pathway have been viewed as critical regulators of apoptosis to various stimuli, including oxidative stress . 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 3
FOXO3A is a member of the FOXO (Forkhead box, class O) transcription factors that regulates a wide range of biological functions, including stress resistance and apoptosis . Specifically, FOXO3A is a tumor suppressor that is regulated by various external stimuli, such as oxidative stress . In response to survival signals transmitted by Akt/PKB and the related kinases, FOXO3A is phosphorylated to create a binding site for 14-3-3, which results in its nuclear exclusion and consequent inability to bind DNA . Additionally, the FOXO3A protein is critical for activation of downstream target genes such as Mn-superoxide dismutase (SOD) during periods of oxidative stress . Since FOXO3A transcription factor is regulated by various stimuli, deregulation of FOXO3A may be due to defects in signal transduction .
Quercetin, a naturally occurring flavonoid protection in cells against oxidative stress .
Quercetin-3-O-methyl-ether (Q3), shown in Figure 1, is one of the quercetin derivatives frequently found in medicinal
plants . In spite of the limited research concerning with cellular bioactivities, the mechanism of Q3 on antioxidation remains unclear . To explore the effect of Q3, cultured mouse hepatocytes were used in this study. Since the protective effect of Q3 in tissue cannot be simulated only by such in vitro studies and zebrafish are thought to be excellent models for toxicological evaluations due to their ability to tolerate soft water for testing effects of metals in vivo . Zebrafish were further employed to assess the effect of Q3 in liver, a main site of oxidative stress in vivo, under copper-induced oxidative stress in this study.
2. Materials and Methods 2.1. Reagents and antibodies
Trizol reagent, 5,5′,6,6′-tetrachloro-1,1′,3.3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and 5-chloromethylfluorescein diacetate (CMFDA) were purchased from Invitrogen (Carlsbad, CA, USA). CuCl2, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide
(DMSO), 4′,6-diamino-2 phenylindole (DAPI), diacetyldichlorofluorescein (DCFDA), Tween-20, F-12K medium, trypsin-EDTA, penicillin/streptomycin mixture, and all other analytical chemicals and protein extraction chemicals were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Life Technologies (Grand Island, NY, USA). Anti-β actin (101313), anti-caspase-3 (22302), anti-PARP (75098), anti-Erk1/2 (113094), anti-phospho-Erk1/2 (Thr202/Tyr204) (59582), anti-p38 MAPK (103009), anti-phospho-p38 MAPK (Thr180/Tyr182)(48614), anti-JNK 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 4
(101165), phospho-JNK (Thr183/Tyr185) (79029), PI3K (111173), Akt (13990), anti-phospho-Akt (Ser473) (28932), anti-FOXO3A (100277), anti-MnSOD (30728), and anti-14-3-3 (100801) were purchased from GeneTex Inc. (ICON-Genetex, Taipei, Taiwan).
2.2. Cell culture and viability
A mouse liver cell line, FL83B, was maintained in F-12K medium with 10% (v/v) heat-inactivated FBS, penicillin G (100 U/mL), streptomycin (100 mg/mL), and L-glutamine (2 mM) at 37°C with 5% CO2. Q3 dissolved in DMSO or CuCl2 was added to the cells and maintained for an additional 24 or 36 h.
The final concentration of DMSO in the various treatments never exceeded 1%. After treatment, 0.5 mg/mL MTT was added to the cell culture at 37 C for 4 h. Cells were then mixed with DMSO at room temperature to solubilize the formazan that formed intracellularly. The optical density (OD) of each culture well was measured using a microplate reader (Bio-tek Instruments, VT, USA) at 570 nm to quantitate the amount of formazan formed within the cells. The OD of formazan formed in the control cells at 0 h was considered as 100% viability.
2.3. DNA fragmentation and electrophoresis
DNA fragments from FL83B cells treated with Cu2+ and/or Q3 were analyzed by agarose gel
electrophoresis. Apoptotic DNA was isolated using DNA lysis buffer through the processes described previously . Isolated DNA, mainly derived from the apoptotic bodies occurred in cells, was subjected to 2.0% agarose electrophoresis at 50V for 3 h. DNA fragments, consisting of multimers of 160–200 base pairs, were visualized under ultraviolet light after staining with ethidium bromide.
2.4. Nuclear condensation and DAPI staining assay
To observe the effects of Q3 on Cu2+-induced cell damage, FL83B cells were seeded into 6-well
dishes. DAPI (0.4 μg/mL) was added to the cells for an additional 15 min after treatment to allow visualization of the DNA condensation, a characteristic feature of apoptotic cells. Nuclear structures were analyzed at 200× magnification using an Olympus IX71 fluorescence microscope (Melville, NY) to determine the presence of DNA condensation.
2.5. Western blotting analysis
Cells were harvested after treatments and the expression of proteins was performed as described in our previous research . 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 5
2.6. Evaluation of mitochondrial membrane potential
Changes in FL83B cell mitochondrial membrane potentials were determined by JC-1 . After treatment, cells were harvested and incubated in 1 mL PBS containing 15 μM JC-1 for 30 min at 37°C in the dark. Cells were then washed and resuspended in PBS and examined immediately by fluorescence-associated cell sorting analysis (BD Biosciences, San Jose, CA, USA) and the data were analyzed using BD Cell Quest software. At least 1000 cells from each treatment were analyzed.
2.7. Measurement of intracellular ROS level
DCFDA can be deacetylated inside cells and then reacted quantitatively with intracellular radicals resulting in its conversion to DCF, a fluorescent byproduct that is retained within the cells . FL83B cells were incubated at 37°C with 100 μM of Cu2+ for 24 or 36 h with or without 60 μM of Q3. After treatment,
cells were rinsed with PBS and 10 mM DCFDA was added. After a 30 min incubation at 37°C, DCFDA fluorescence images were observed and captured under a fluorescent microscope, as described above. 2.8. Fluorescence measurements of cellular glutathione
FL83B cells were cultured in 10 cm dishes for 24 h. After treating with Cu2+ and/or Q3 for 24 and 36
h, cells were gently washed twice with PBS. Cellular nonprotein thiols, induced by oxidative stress, were measured by incubating with 2 μM of CMFDA for 30 min at 37°C. The fluorescence of 2 fields for each indicator was measured from the cells with an excitation and emission wavelengths of 488 nm and 530 nm, respectively.
2.9. Animals and husbandry
Fertilized eggs from zebrafish (Danio rerio) were obtained from the Taiwan Zebrafish Core Facility at Academia Sinica (TZCAS, Taipei, Taiwan). All experiments were conducted in compliance with the institutional guidelines for animal care as published by the National Laboratory Animal Center, National Science Council, Taiwan. Fish maintenance was performed as previously described . The zebrafish liver fatty acid binding protein (LFABP) is a 14 kDa cytoplasmic protein that binds to long-chain fatty acids and promotes the intracellular diffusion of fatty acid . A LFABP:GFP transgenic zebrafish line was created for studying the regulatory networks responsible for LFABP gene expression in livers with green fluorescent protein (GFP). 2.10. Exposure studies 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 6
The exposure experiment involved aqueous exposure of larval or adult zebrafish for 10 h. At least 60 zebrafish were placed in each experimental tank containing different treatments. Throughout the exposure, all tanks were inspected daily for dead animals, which were removed. After treatment, the zebrafish were anesthetized with a saturated solution of 4-ethylaminobenzoate (bezocaine; Sigma). Whole body fluorescence was measured immediately after anesthetization, serving as the basis for the evaluation of liver damage induced by Cu2+.
2.11. Histological evaluation
The abdominal digestive organs of adult zebrafish were collected and fixed in a formaldehyde fixative and dehydrated in a graded series of ethanol using a TP 1020 Tissue Processor (Leica Microsystems, Wetzlar, Germany). After embedding in paraffin, 7-μm-thick tissue sections of the liver were cut and serial sections were stained by hematoxylin and eosin (H&E) prior to examination for hemorrhage and cellular hypertrophy.
2.12. Reverse transcriptase-polymerized chain reaction (RT-PCR)
To detect GFP or GAPDH transcription, individual zebrafish were sacrificed by cold shock, and the total RNA was extracted from the abdominal organs with Trizol, according to manufacturer instructions. cDNA was synthesized using an RT-PCR system (SuperScript III Reverse Transcriptase, Invitrogen), as described previously .
2.13. Statistical analysis
Each experiment was performed at least 3 times, and all data are represented as means standard deviation (SD) of quadruplicate measurements. Student’s t-test was used to analyze the statistical significance of the results, with P 0.05 being considered to be statistically significant.
3. RESULTS
3.1. Protective effect of Q3 on Cu2+-induced cytotoxicity in FL83B cells
This part of the study was designed to evaluate whether Q3 protects FL83B cells from oxidative stress induced by Cu2+. To examine the effect of Cu2+-induced cytotoxicity in hepatocytes, an MTT assay
was carried out to measure FL83B cell viability after treatment with Cu2+ and Q3. As shown in Figure 2A,
the viability of FL83B cells exposed to Q3 for 24 h were decreased by exposure to concentrations ranging 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 7
from 10 to 60 M; in the presence of 40 and 60 M of Q3, there were 82% 4% and 67% 2% of the cells which remained viable, respectively. Cu2+
, at concentrations higher than 100 M, significantly
decreased the viability of FL83B cells in a dose-dependent manner. Following exposure to 100 M of Cu2+for 24 h, there were 58% 3% viable cells as compared to controls (Fig. 2B). The viability of cells
treated with 100 M of Cu2+ for 36 h was significantly decreased to 41% 3% (P 0.01) of the control
value. Therefore, 100 M of Cu2+ was used to induce FL83B cells injury in the subsequent Q3
cotreatment experiments. When cells, were exposed to Cu2+ and cotreated with Q3 at 60 M, , viability
was significantly increased to 70% 2% (P 0.05) and 83% 4% (P 0.01) at 24 and 36 h, respectively, as compared to the Cu2+-treated alone.
3.2. Protective effects of Q3 on Cu2+-induced apoptosis
To gain insight into the protective effects of Q3 on nuclear alterations, cells were stained with DAPI. As shown in Figure 3A, the cells underwent remarkable nuclear changes upon exposure to Cu2+. The
nuclei were intact, round, and uniformly stained in control cells, as well as in Q3-treated cells. However, after exposed to Cu2+, the cells manifested nuclear condensation. Nuclear changes exhibited in FL83B
cells at 100 M of Cu2+ were reduced by cotreating the cells with Q3. Nuclear fragmentation, as shown in
Figure 3B, showed the identical phenomenon. FL83B cells treated with 100 M of Cu2+ resulted in
marked DNA fragmentation, showing distinctive ladder patterns of approximately 160–200 base pair fragments, which was abolished by cotreating cells with Q3.
The damage to the mitochondria in Cu2+-treated FL83B cells was detected by the loss of
mitochondrial membrane potential (Fig. 3C). The majority of the untreated cells, with a polarized mitochondrial membrane potential, were identified in the upper right quadrant. The cells showed progressive loss of mitochondrial membrane potential after exposure to Cu2+. As indicated by JC-1, the
loss of mitochondrial membrane potential occurred in a time-dependent manner. According to the time course analysis, Cu2+-induced mitochondrial changes in FL83B cells were reduced by Q3 cotreatment.
The cleavage of caspase-3 and its substrate, PARP, which occurs after the loss of mitochondrial membrane potential, induces apoptosis through proteolytic cleavage. This process was significantly increased in Cu2+-treated FL83B cells at both 24 and 36 h. The presence of cleaved PARP was decreased
188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 8
significantly in Cu2+-treated FL83B cells after they had been cotreated with Q3.
3.3. Protective effects of Q3 on Cu2+-induced ROS production and cellular responses
After exposure to 100 M of Cu2+ for 24 and 36 h, fluorescence from most cells stained with
DCFDA indicated that intracellular ROS had accumulated significantly in FL83B cells, but the accumulation was significantly inhibited by cotreatment with Q3 (Fig. 4A). The presence of ROS in Q3-treated cells was slightly increased, as compared to the control cells. In addition, treatment with 100 M of Cu2+, 60 M of Q3, or cotreatment with Cu2+ and Q3 for 24 h dramatically increased glutathione
(GSH) production, whereas GSH content declined significantly in FL83B cells treated with Cu2+ for 36 h
(Fig. 4B). Treatment with 100 M of Cu2+ significantly increased the expression and phosphorylation of
Erk at 36 h. However, the increased expression and phosphorylation of Erk was significantly inhibited by cotreatment of Q3 (Fig. 4C, 4E). Whilst the expression of p38 was increased in cells treated with Cu2+ or
Q3 alone, however, no increase in phosphorylation of p38 was found in cells treated with Q3 or Cu2+ at
36 h (Fig. 4C, 4E). Furthermore, expression of JNK was increased in cells treated with Cu2+ and Q3,
individually or following cotreatment, at 36 h, whereas the decreased phosphorylation of JNK was observed following cotreatment (Fig. 4C, 4E). As shown in Figure 4D and 4E, expression of PI3K and phosphorylation of Akt were blocked by Cu2+, but enhanced by Q3 or Q3 cotreatment followed by Cu2+.
A higher level of FOXO3A was also observed in cells treated with Q3, alone or combined with Cu2+, for
36 h, but decreased in cells exposed only to Cu2+. Decreased expression of MnSOD and increased
expression of 14-3-3 were found in Cu2+-treated cells, whereas expression of 14-3-3 was decreased
significantly in cells treated with Q3 alone or cotreated with Cu2+ and Q3 for 36 h.
3.4. Protective effects of Q3 on Cu2+-induced damages in zebrafish
Copper toxicity was further evaluated using a zebrafish animal model (Fig. 5A). The survival times of zebrafish exposed to different concentrations of Cu2+, indicated that Cu2+ at a concentration higher than
10 M was lethal to larval zebrafish within 4 days, but concentrations of 2 M were not lethal over a 10-day observation period. Therefore, 5 M of Cu2+, which showed 50% larval survival at day 10, was used
to evaluate the protective effect of Q3 on Cu2+-induced damages in vivo. Survival curves, shown in Figure
5B, indicate that 15 M Q3 significantly prevented zebrafish lethality due to Cu2+-induced toxicity. To
estimate the protective effects of Q3 on liver injuries induced by Cu2+, LFABP:GFP expression in
215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 9
zebrafish livers was evaluated at 10 h after treatment (Fig. 5C). A significant decrease in GFP was found after exposure to 5 M Cu2+ for 10 h, whereas the expression of GFP was reversely increased in zebrafish
cotreated with Q3 and Cu2+ (Fig. 5C, D).
As shown in Figure 5E, the intensity of GFP expressed in the livers of adult zebrafish was the same as that in the larval zebrafish. Histological changes which cellular hypertrophy and hemorrhage were included, were found in livers of adult zebrafish, stained by H&E. These observations suggested that the susceptibility of Cu2+-induced liver damage in adult zebrafish was reduced by cotreatment with 15 M
Q3.
4. Discussion
Our experiments showed a close relationship between copper-induced oxidative stress and apoptosis. ROS, including superoxide and hydroxyl radicals, can be induced by copper and lead to cell death by causing cellular damage through lipid peroxidation, protein deficits, decreased levels of GSH and SOD, and mitochondrial dysfunction . ROS production in copper-treated cells has been reported to be correlated with the degree of unsaturation of cardiolipin acyl chains . The oxidation of the cardiolipin acyl chains is closely associated to the cytosolic release of cytochrome c from the mitochondria, which triggers the catalytic activation of caspases and cell death . Poly-(ADP-ribose) polymerase (PARP), the downstream substrate of activated caspases, plays an important role in DNA stability is cleaved in response to caspase-dependent apoptosis . Recent reports drag in mitochondria as the major target for liver toxicity resulting from copper overloaded and dissipation of mitochondrial transmembrane potential Δm, followed by induction of mitochondrial permeability transition (MPT) eventually lead to the release of factors and activate cellular apoptosis through different cascades . The opening of MPT pore is an important event in the apoptotic death of liver cells . The greater generation of ROS with lower Δm in Cu2+-treated FL83B cells provide a more favorable condition for MPT induction that eventually leads to mitochondrial swelling and disruption of the outer membrane. In accordance with the cell viability, cleavage of PARP with DNA fragmentation as well as nuclear condensation in Cu2+-treated cells emphasize that mitochondria-mediated apoptosis is involved in copper-induced cell death. In this study, Cu2+ was found
to stimulate increased production of ROS, which resulted in toxic effects leading to loss of cell viability. 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 10
Cu2+-induced cytotoxicity in hepatocytes was suggested to occur as a result of mitochondrial ROS formation . Our results clearly demonstrated that Cu2+ at concentrations higher than 100 M induced
apoptotic cell death in a mitochondria-dependent manner. The mechanism of copper toxicity is mainly induced by the generation of ROS, thus the initial blockage of ROS generation might be useful for the protection of cells from oxidative stress. To determine whether Q3 prevents oxidative stress by inhibiting ROS generation, intracellular ROS concentrations were measured in FL83B cells after Cu2+ exposure. The
addition of Q3 significantly reduced Cu2+-induced ROS formation, protecting the cells against Cu2+
-induced cell death, and suggesting that Q3 inhibited ROS accumulation in Cu2+-treated FL83B cells.
Glutathione (GSH) is a ubiquitous intracellular peptide with diverse functions that participate in antioxidant defense, maintenance of thiol status and modulation of cell proliferation . GSH is present at high concentrations in the cells and scavenges various ROS for maintaining cells against oxidant threat . Whilst the Cu2+-exposed FL83B cells demonstrated a marked rise in oxidative stress, characterized by
excessive ROS production, reduced GSH synthesis and the considerable oxidative DNA damage. Here, we had presented an evidence of the protection propensity of GSH by employing the experimental approach in which the DNA damage was assessed by measuring nuclear condensation and DNA fragmentation. DNA damage in the presence of Cu2+ have revealed that the oxidation of nucleobases is caused by •O2− as well as OH• radicals generated in the catalytic cascade by metal ions . The action of ROS induced by Cu2+ is offset by the increase of GSH in cells cotreated with Q3, suggesting that Q3 provides antioxidant protective benefits to the cells.
ROS are regulators of mitogen-activated protein kinases (MAPKs) which mediate intracellular signal transduction in response to different physiological stimuli and stressing conditions . At least 3 subfamilies of MAPKs, JNK, p38, and Erk have been described to control many cellular events, including differentiation, proliferation, and apoptosis . An increase in intracellular ROS, as well as the activation of MAPKs, were reported to be in part responsible for the induction of growth arrest and apoptosis . ROS-mediated oxidative stress was suggested to play a pivotal role in hepatotoxicity and be critical for the regulation of various protein interactions in MAPKs . Moreover, activation of the PI3K/Akt pathway also plays a key role in regulating cell proliferation, growth, and survival. Phosphorylation of the MAPKs and 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 11
PI3K/Akt is known to be involved in protecting cells against oxidative stress . Here we found that Cu2+ at a concentration of apoptosis enhanced the phosphorylation of Erk1/2, but not JNK, p38 and,Akt in FL83B cells. These results demonstrated that Cu2+-induced oxidative stress in FL83B cells was mediated by
overexpression and phosphorylation of Erk, strongly suggesting that Q3 protected against Cu2+-induced
oxidative stress via blocking the expression and phosphorylaton of Erk. In some studies, Erk had been implicated in apoptotic events through mitochondria and the downstreamed caspase activation, or the suppression of Akt . Low levels of phosphorylated Akt with elevated activation of Erk coincided with the apoptosis in Cu2+-treated FL83B cells. The dephosphorylated Akt in Cu2+-treated cells was abolished by Q3, suggesting that Q3 protection occurs through the PI3K/Akt signaling pathway. Furthermore, the decreased expression and increased phosphorylation of Akt were observed in Q3 treated cells, indicating that the activation of the PI3K/Akt and MAPK/Erk pathways were involved in the Q3-mediated protection.
FOXO3A, an important regulator of cellular function, activates downstream target genes, such as MnSOD, and creates binding sites for the chaperone proteins such as 14-3-3 when it is phosphorylated by Akt . FOXO3A is phosphorylated on multiple sites besides that for Akt, the key regulator of FOXO3A. The phosphorylated FOXO3A cluster at S295/345/426 is targeted by Erk and mediates MDM2-dependent ubiquitination and protein degradation . The dephosphorylation of Akt and elevated expression of 14-3-3 indicated that PI3K/Akt and 14-3-3 were not involved in the phosphorylation and consequent degradation of FOXO3A in Cu2+-treated FL83B cells. Instead, the increased expression of 14-3-3 with the reduction of FOXO3A and MnSOD in Cu2+-treated cells indicated the ubiquitination of FOXO3A was mediated by the upregulation and phosphorylation of Erk. On the contrary, expression of FOXO3A, MnSOD and 14-3-3 in Q3-treated cells overwhelms that induced by Cu2+. Thus, negatively regulated Akt, in cells treated with Q3, increased FOXO3A accumulation in the nucleus, causing an upregulation of the downstream target, MnSOD. Although the decrease of FOXO3A in Cu2+-treated cells may arise from an increase in protein degradation, owing to Cu2+-mediated over-production of ROS, the notable nuclear damage caused by Cu2+ makes it possible that the drop of MnSOD reflects changes in gene expression at transcriptional or translational levels. 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 12
The present results show that Q3 markedly suppressed Cu2+-induced oxidative stress in FL83B cells
by enhancing the biosynthesis of GSH and SOD upon ROS generation, as well as by activating specific PI3K/Akt and MAPK/Erk. This study also provided evidence that activation of the mitochondrial apoptosis pathway, in response to Cu2+, leads to the reduction of mitochondrial membrane potential and to
the cleavage of PARP. The cleavage of PARP in Cu2+-treated FL83B cells was associated with nuclear
condensation and DNA fragmentation. Thus, this study demonstrated the possible molecular mechanism by which Q3 exerts its antioxidant effect in Cu2+-treated normal liver cells is via the PI3K/Akt/ and
MAPK/Erk pathway (Fig. 6).
Zebrafish are small vertebrates that can be easily manipulated in large numbers. Organogenesis in zebrafish takes only a few days to produce functional organs and allows for the visual analysis of the development of internal structures in the semi-transparent, living animals . Many studies have suggested that zebrafish provide a powerful model for the study of oxidative stress and metal toxicology . Whist the oxidative damage in liver was shown to be one of the effects of copper toxicity in zebrafish, yet studies using zebrafish to examine the effect of antioxidants on Cu2+-induced oxidative stress remain scarce . To simulate the effect of Q3 in organisms, zebrafish were therefore used as a model in vivo to elucidate the effect of Q3 on Cu2+-induced oxidative cytotoxicity in this study. Our results are consistent with previously reported findings regarding the ability of zebrafish to tolerate in a low micromolar range of water-dissolved copper, whereas the lethality increases rapidly with increasing concentrations . Because Cu2+ is deposited in the liver, ROS are produced mainly in the liver of zebrafish . Cellular stress induced by Cu2+ becomes rapidly evident in the liver after treatment, indicating our proper utility of liver models
for studying Cu2+ toxicity in zebrafish. Histopathological studies showed that Cu2+ induced a marked
degeneration in hepatocytes and caused focal damage to the liver tissue; they also demonstrated that these lesions were markedly diminished in zebrafish cotreated with Q3. Furthermore, 15 M Q3, alone, did not cause significant cellular damage in vitro nor induce any change in liver histology in zebrafish. These results in cotreatment of Q3 are expected given the further development against the copper-induced damage in organisms. Together, these observations showed that cotreatment with Q3 resulted in decreased hepatic damages, indicating that Q3 provides a protective effect against Cu2+-induced oxidative
325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 13
stress in liver. Based on our results, it is hypothesized that the liver protection efficacy of Q3 may be related to its ability to enhance the antioxidant machinery in cells through upregulation of GSH and MnSOD.
5. Conclusions
These results suggest that Q3 might be a promising candidate for the prevention or treatment of Cu2+-induced hepatic injuries. Accordingly, Q3 is proposed to be an antioxidant that functions through the
stabilization of mitochondria, scavenging ROS, and activating protein kinases. Q3 suppresses the activation of Erk and enhances the expression of Mn-SOD and GSH in response to oxidative stress, leading to the attenuation of cellular and histological damages induced by copper. However, the bioavailability and mechanism of Q3 on copper-induced hepatic damages are essential to be explored in mammals before using as a hepatoprotector.
Acknowledgements
This work was supported by grant TCIRP96005-01 (Tzu-Chi University, Hualien, Taiwan). 352 353 354 355 356 357 358 359 360 361 362 363 364 365 14
Reference
Chung, Y. W., Kim, H. K., Kim, I. Y., Yim, M. B., and Chock, P. B. (2011). Dual function of protein kinase C (PKC) in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced manganese superoxide dismutase (MnSOD) expression: activation of CREB and FOXO3a by PKC-alpha phosphorylation and by PKC-mediated inactivation of Akt, respectively. J Biol Chem 286, 29681-29690.
Cossarizza, A., and Salvioli, S. (2001). Flow cytometric analysis of mitochondrial membrane potential using JC-1. Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] Chapter 9, Unit 9 14.
Craig, P. M., Wood, C. M., and McClelland, G. B. (2007). Oxidative stress response and gene expression with acute copper exposure in zebrafish (Danio rerio). Am J Physiol Regul Integr Comp Physiol 293, R1882-1892.
Dias, N., and Bailly, C. (2005). Drugs targeting mitochondrial functions to control tumor cell growth. Biochemical pharmacology 70, 1-12.
Dickinson, D. A., and Forman, H. J. (2002). Cellular glutathione and thiols metabolism. Biochemical pharmacology 64, 1019-1026.
Enomoto, S., Okada, Y., Guvenc, A., Erdurak, C. S., Coskun, M., and Okuyama, T. (2004). Inhibitory effect of traditional Turkish folk medicines on aldose reductase (AR) and hematological activity, and on AR inhibitory activity of quercetin-3-O-methyl ether isolated from Cistus laurifolius L. Biol Pharm Bull 27, 1140-1143.
Eruslanov, E., and Kusmartsev, S. (2010). Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol 594, 57-72.
Foo, N. P., Lin, S. H., Lee, Y. H., Wu, M. J., and Wang, Y. J. (2011). alpha-Lipoic acid inhibits liver fibrosis through the attenuation of ROS-triggered signaling in hepatic stellate cells activated by PDGF and TGF-beta. Toxicology 282, 39-46.
Galindo, M. F., Jordan, J., Gonzalez-Garcia, C., and Cena, V. (2003). Reactive oxygen species induce swelling and cytochrome c release but not transmembrane depolarization in isolated rat brain mitochondria. Br J Pharmacol 139, 797-804.
Ghosh, N., Ghosh, R., Mandal, V., and Mandal, S. C. (2011). Recent advances in herbal medicine for treatment of liver diseases. Pharm Biol 49, 970-988.
Gross, D. N., van den Heuvel, A. P., and Birnbaum, M. J. (2008). The role of FoxO in the regulation of metabolism. Oncogene 27, 2320-2336.
Gu, T. L., Tothova, Z., Scheijen, B., Griffin, J. D., Gilliland, D. G., and Sternberg, D. W. (2004). NPM-ALK fusion kinase of anaplastic large-cell lymphoma regulates survival and proliferative signaling through modulation of FOXO3a. Blood 103, 4622-4629.
Harris, E. D. (2003). Basic and clinical aspects of copper. Critical reviews in clinical laboratory sciences 40, 547-586.
Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature 407, 770-776.
Her, G. M., Chiang, C. C., Chen, W. Y., and Wu, J. L. (2003). In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio). FEBS 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 15
Lett 538, 125-133.
Hernandez, P. P., and Allende, M. L. (2008). Zebrafish (Danio rerio) as a model for studying the genetic basis of copper toxicity, deficiency, and metabolism. Am J Clin Nutr 88, 835S-839S.
Herrmann, M., Lorenz, H. M., Voll, R., Grunke, M., Woith, W., and Kalden, J. R. (1994). A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic acids research 22, 5506-5507.
Hetman, M., Kanning, K., Cavanaugh, J. E., and Xia, Z. (1999). Neuroprotection by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. J Biol Chem 274, 22569-22580.
Higuchi, H., Adachi, M., Miura, S., Gores, G. J., and Ishii, H. (2001). The mitochondrial permeability transition contributes to acute ethanol-induced apoptosis in rat hepatocytes. Hepatology 34, 320-328.
Hong, M. Y., Chapkin, R. S., Barhoumi, R., Burghardt, R. C., Turner, N. D., Henderson, C. E., Sanders, L. M., Fan, Y. Y., Davidson, L. A., Murphy, M. E., Spinka, C. M., Carroll, R. J., and Lupton, J. R. (2002). Fish oil increases mitochondrial phospholipid unsaturation, upregulating reactive oxygen species and apoptosis in rat colonocytes. Carcinogenesis 23, 1919-1925.
Huang, W. T., Li, C. J., Wu, P. J., Chang, Y. S., Lee, T. L., and Weng, C. F. (2009). Expression and in vitro regulation of pituitary adenylate cyclase-activating polypeptide (pacap38) and its type I receptor (pac1-r) in the gonads of tilapia (Oreochromis mossambicus). Reproduction 137, 449-467.
Jomova, K., and Valko, M. (2011). Advances in metal-induced oxidative stress and human disease. Toxicology 283, 65-87.
Kadiiska, M. B., Hanna, P. M., and Mason, R. P. (1993). In vivo ESR spin trapping evidence for hydroxyl radical-mediated toxicity of paraquat and copper in rats. Toxicology and applied pharmacology 123, 187-192.
Krumschnabel, G., Manzl, C., Berger, C., and Hofer, B. (2005). Oxidative stress, mitochondrial permeability transition, and cell death in Cu-exposed trout hepatocytes. Toxicology and applied pharmacology 209, 62-73.
Li, C. J., Chu, C. Y., Huang, L. H., Wang, M. H., Sheu, L. F., Yeh, J. I., and Hsu, H. Y. (2012). Synergistic anticancer activity of triptolide combined with cisplatin enhances apoptosis in gastric cancer in vitro and in vivo. Cancer letters 319, 203-213.
Li, M., Chiu, J. F., Mossman, B. T., and Fukagawa, N. K. (2006). Down-regulation of manganese-superoxide dismutase through phosphorylation of FOXO3a by Akt in explanted vascular smooth muscle cells from old rats. J Biol Chem 281, 40429-40439.
Linbo, T. L., Stehr, C. M., Incardona, J. P., and Scholz, N. L. (2006). Dissolved copper triggers cell death in the peripheral mechanosensory system of larval fish. Environ Toxicol Chem 25, 597-603. Mao, Q. Q., Xian, Y. F., Ip, S. P., Tsai, S. H., and Che, C. T. (2011). Protective effects of peony
glycosides against corticosterone-induced cell death in PC12 cells through antioxidant action. J Ethnopharmacol 133, 1121-1125.
McBride, H. M., Neuspiel, M., and Wasiak, S. (2006). Mitochondria: more than just a powerhouse. Current biology : CB 16, R551-560. 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 16
Murota, K., and Terao, J. (2003). Antioxidative flavonoid quercetin: implication of its intestinal absorption and metabolism. Arch Biochem Biophys 417, 12-17.
Murphy, C. T. (2006). The search for DAF-16/FOXO transcriptional targets: approaches and discoveries. Exp Gerontol 41, 910-921.
Mut, M., Lule, S., Demir, O., Kurnaz, I. A., and Vural, I. (2012). Both mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 and phosphatidylinositide-3-OH kinase (PI3K)/Akt pathways regulate activation of E-twenty-six (ETS)-like transcription factor 1 (Elk-1) in U138 glioblastoma cells. The international journal of biochemistry & cell biology 44, 302-310. Nakano, H., Nakajima, A., Sakon-Komazawa, S., Piao, J. H., Xue, X., and Okumura, K. (2006). Reactive
oxygen species mediate crosstalk between NF-kappaB and JNK. Cell death and differentiation 13, 730-737.
Navarro, R., Busnadiego, I., Ruiz-Larrea, M. B., and Ruiz-Sanz, J. I. (2006). Superoxide anions are involved in doxorubicin-induced ERK activation in hepatocyte cultures. Annals of the New York Academy of Sciences 1090, 419-428.
Oikawa, S., and Kawanishi, S. (1998). Distinct mechanisms of site-specific DNA damage induced by endogenous reductants in the presence of iron(III) and copper(II). Biochimica et biophysica acta 1399, 19-30.
Ouariti, O., Boussama, N., Zarrouk, M., Cherif, A., and Ghorbal, M. H. (1997). Cadmium- and copper-induced changes in tomato membrane lipids. Phytochemistry 45, 1343-1350.
Park, K. R., Nam, D., Yun, H. M., Lee, S. G., Jang, H. J., Sethi, G., Cho, S. K., and Ahn, K. S. (2011). beta-Caryophyllene oxide inhibits growth and induces apoptosis through the suppression of PI3K/AKT/mTOR/S6K1 pathways and ROS-mediated MAPKs activation. Cancer letters 312, 178-188.
Partridge, L., and Bruning, J. C. (2008). Forkhead transcription factors and ageing. Oncogene 27, 2351-2363.
Parveen, R., Baboota, S., Ali, J., Ahuja, A., Vasudev, S. S., and Ahmad, S. (2011). Effects of silymarin nanoemulsion against carbon tetrachloride-induced hepatic damage. Arch Pharm Res 34, 767-774. Petrosillo, G., Ruggiero, F. M., and Paradies, G. (2003). Role of reactive oxygen species and cardiolipin
in the release of cytochrome c from mitochondria. FASEB J 17, 2202-2208.
Pourahmad, J., and O'Brien, P. J. (2000). Contrasting role of Na(+) ions in modulating Cu(+2) or Cd(+2) induced hepatocyte toxicity. Chemico-biological interactions 126, 159-169.
Rauen, U., Kerkweg, U., Weisheit, D., Petrat, F., Sustmann, R., and de Groot, H. (2003). Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron. Free Radic Biol Med 35, 1664-1678.
Roy, D. N., Mandal, S., Sen, G., and Biswas, T. (2009). Superoxide anion mediated mitochondrial dysfunction leads to hepatocyte apoptosis preferentially in the periportal region during copper toxicity in rats. Chemico-biological interactions 182, 136-147.
Rubio, S., Quintana, J., Eiroa, J. L., Triana, J., and Estevez, F. (2007). Acetyl derivative of quercetin 3-methyl ether-induced cell death in human leukemia cells is amplified by the inhibition of ERK. Carcinogenesis 28, 2105-2113.
Samuele, A., Mangiagalli, A., Armentero, M. T., Fancellu, R., Bazzini, E., Vairetti, M., Ferrigno, A., 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 17
Richelmi, P., Nappi, G., and Blandini, F. (2005). Oxidative stress and pro-apoptotic conditions in a rodent model of Wilson's disease. Biochim Biophys Acta 1741, 325-330.
Shukla, S., Shukla, M., Maclennan, G. T., Fu, P., and Gupta, S. (2009). Deregulation of FOXO3A during prostate cancer progression. Int J Oncol 34, 1613-1620.
Vincent, A. M., Russell, J. W., Low, P., and Feldman, E. L. (2004). Oxidative stress in the pathogenesis of diabetic neuropathy. Endocrine reviews 25, 612-628.
Weng, C. J., Chen, M. J., Yeh, C. T., and Yen, G. C. (2011). Hepatoprotection of quercetin against oxidative stress by induction of metallothionein expression through activating MAPK and PI3K pathways and enhancing Nrf2 DNA-binding activity. N Biotechnol 28, 767-777.
Winge, D. R., and Mehra, R. K. (1990). Host defenses against copper toxicity. Int Rev Exp Pathol 31, 47-83.
Xiao, X., Liu, J., Hu, J., Zhu, X., Yang, H., Wang, C., and Zhang, Y. (2008). Protective effects of protopine on hydrogen peroxide-induced oxidative injury of PC12 cells via Ca(2+) antagonism and antioxidant mechanisms. European journal of pharmacology 591, 21-27.
Xue, T., Luo, P., Zhu, H., Zhao, Y., Wu, H., Gai, R., Wu, Y., Yang, B., Yang, X., and He, Q. (2012). Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes. Toxicology and applied pharmacology 261, 280-291.
Yang, J. Y., Zong, C. S., Xia, W., Yamaguchi, H., Ding, Q., Xie, X., Lang, J. Y., Lai, C. C., Chang, C. J., Huang, W. C., Huang, H., Kuo, H. P., Lee, D. F., Li, L. Y., Lien, H. C., Cheng, X., Chang, K. J., Hsiao, C. D., Tsai, F. J., Tsai, C. H., Sahin, A. A., Muller, W. J., Mills, G. B., Yu, D., Hortobagyi, G. N., and Hung, M. C. (2008). ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation. Nature cell biology 10, 138-148.
You, B. R., and Park, W. H. (2012). Arsenic trioxide induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion. Oncology reports 28, 749-757.
Zaret, K. S. (2002). Regulatory phases of early liver development: paradigms of organogenesis. Nat Rev Genet 3, 499-512.
Zhuang, S., and Schnellmann, R. G. (2006). A death-promoting role for extracellular signal-regulated kinase. The Journal of pharmacology and experimental therapeutics 319, 991-997.
Zimmermann, S., and Moelling, K. (1999). Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286, 1741-1744. 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 18
Figure legends
Figure 1. Structure of quercertin-3-o-methyl ether.
Figure 2. Effect of Q3 on Cu2+-induced FL83B cell damages. (A) Dose-dependent cytotoxic effects of
Q3 and Cu2+ on FL83B cells after 24 h treatment. (B) Effect of Q3 on Cu2+-induced cytotoxicity in FL83B
cells. Cells were cotreated for 30 min with Q3 concentrations of 10, 20, 40, and 60 M, and then exposed to 100 M of Cu2+ for 24 and 36 h. Cell viability was determined by the MTT assay and expressed as
percentages of the corresponding values for the control group. Data are presented as the means S.D. (n 8). *p 0.05 and **p 0.01 compared with the control group. #p 0.05 and ##p 0.01 compared with Cu2+-treated group.
Figure 3. Protective effects of Q3 on nuclear and mitochondrial damages induced by Cu2+. (A)
Nuclear condensation in FL83B cells treated with Q3 and Cu2+ for 24 or 36 h. Nuclear morphology,
visualized by fluorescence microscopy (200), showed that Q3 cotreatment obviously attenuated the effect of Cu2+ exposure. (B) DNA fragmentation in FL83B cells cotreated with Q3 and Cu2+ for 36 h.
DNA fragmentation in Cu2+-exposed FL83B cells was eliminated by cotreatment with Q3. (C) Cleavage
of PARP as evaluated by western blot analysis. PARP, a protein associated with caspase-dependent cell death in FL83B cells, indicated that Q3 attenuated the damages in Cu2+-exposed cells. (D) Mitochondrial
membrane potential assayed by JC-1. The results indicate that a significant loss of mitochondrial membrane potential was induced by Cu2+-exposed FL83B cells and was regained after cotreatment with
Q3. *p 0.01 compared with the control group.
Figure 4. Protective effects of Q3 on Cu2+-induced oxidative stress in FL83B cells. (A) ROS
accumulation in FL83B cells treated with Q3 and/or Cu2+. ROS production was assessed using a ROS
assay dye, DCFDA. Representative images were taken by fluorescence microscopy (200). (B) GSH production in FL83B cells treated with Q3 and/or Cu2+. Changes in GSH levels in FL83B cells were
determined by flow cytometry with CMFDA fluorescence dye and expressed as percentages of the corresponding values for the control group. Cells were treated with Q3 at 60 μM or Cu2+ at 100 μM, or
cotreated with Q3 and Cu2+ for 24 or 36 h. Levels of MAPKs, PI3K/Akt, FOXO3A, MnSOD, and 14-3-3
in cells treated with Q3 and/or Cu2+ were evaluated by Western blot analysis for 36 h (C & D). (E)
520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 19
Quantification of the gels shown in (C & D). The protein expression levels were normalized to -actin and expressed as fold changes to the respective Cu2+ groups. Data are representative of 3 independent experiments. * P < 0.05 compared to Cu2+ groups.
Figure 5. Protective effects of Q3 on zebrafish exposed to 5 M Cu2+ in vivo. Survival times of
embryonic zebrafish exposed to (A) Cu2+, and (B) Cu2+ exposure with or without Q3. (C) Comparison of
the LFABP expression in embryonic zebrafish exposed to Cu2+ and/or Q3 for 10 h (10 hpe). (D)
Comparison of the liver-specific expression of the LFABP gene in embryonic zebrafish treated for 10 h (10 hpe). PCR products from transcripts of LFABP zebrafish genes were performed at 0, 2, 6, and 10 h after 5 M Cu2+ exposure. Ten hours was selected to evaluate the effect of Q3 on Cu2+-induced liver
damage, as a significant decrease in GFP expression was found. GAPDH was used as a control and was amplified in a similar PCR reaction. Densitometric results are presented as means S.D. (n 3). (E) Analysis of GFP expression and oxidative damage in the liver of adult transgenic zebrafish exposed to Q3 and/or Cu2+ for 10 h (hpe). GFP expression in the liver of adult zebrafish without treatment is strong,
whereas it is almost non-detectable in animals exposed to Cu2+. Hemorrhaging, shown in the livers of
Cu2+-exposed zebrafish, indicated that Q3 significantly reduced the Cu2+-induced oxidative damages in
liver.
Figure 6. A proposed mechanism of Q3 on Cu2+-induced oxidative stress in FL83B cells. A scheme
illustrating signaling pathways affected by Q3 to block the Cu2+-induced oxidative response. ROS levels
are elevated during stress caused by Cu2+ and drive expression and activation of MAPKs, the most
important of which is Erk, in FL83B cells. Overexpression and phosphorylation of Erk regulate apoptosis in Cu2+-treated cells through the mitochondria-dependent pathway. It also suppresses the activation of
PI3K/Akt and the consequent series of transcription and translation events contributing to the synthesis of GSH against ROS induced by Cu2+. Copper exposure induces ROS, which activate Erk to inhibit the
localization of FOXO3A inside the nucleus to suppress the transcription of FOXO-dependent antioxidant genes, MnSOD. Increased expression of MnSOD and GSH followed by the phosphorylation of Akt in cells cotreated with Q3 abolishes the ROS generated by Cu2+ stimuli. Because ROS is the second
messenger for initiating apoptosis, suppression of ROS levels by scavenging or reduction of oxidative 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 20
responses by GSH or MnSOD after cotreatment with Q3 shuts down major signaling pathways involved in the activation of transcription and translation events, contributing to the cell death. Deregulation of FoxO3A through the defected PI3K/Akt pathway in Cu2+-treated hepatocytes is one of the major themes
in Q3-mediated antioxidation. 574 575 576 577 21
578
579
580
581
582
583
