Am J Physiol Cell Physiol 292: C353–C361, 2007.
First published September 13, 2006; doi:10.1152/ajpcell.00388.2006.
Methoxyflavones protect cells against endoplasmic reticulum stress
and neurotoxin
Katsura Takano,1,3* Yoshiyuki Tabata,1,2* Yasuko Kitao,1 Rika Murakami,4 Hiroto Suzuki,4
Masashi Yamada,4 Munekazu Iinuma,5 Yukio Yoneda,3 Satoshi Ogawa,1 and Osamu Hori1
Departments of 1Neuroanatomy and 2Molecular Pharmacology, Kanazawa University Graduate
School of Medical Science, Kanazawa City, Ishikawa; 3Laboratory of Molecular Pharmacology,
Kanazawa University Graduate School of Natural Science and Technology, Kanazawa City, Ishikawa;
4
Meiji Dairies Corporation, Tokyo; and 5Gifu Pharmaceutical University, Gifu City, Gifu, Japan
Submitted 14 July 2006; accepted in final form 8 September 2006
neuronal degeneration; flavonoids
(ER) is an organelle where secretory proteins undergo posttranslational modification. Exposure
of cells to conditions such as glucose starvation, inhibition of
protein glycosylation, disturbance of Ca2⫹ homeostasis, and
oxygen deprivation accumulates unfolded proteins in the ER
(ER stress) and activates a set of pathways known as the
unfolded protein response (UPR; see Ref. 31). The UPR is
transmitted through activation of ER resident proteins, such as
inositol requiring kinase 1 ␣/␤, protein kinase-like ER kinase
(PERK), and activating transcription factor (ATF) 6, and UPRtarget genes include molecular chaperones in the ER, ERTHE ENDOPLASMIC RETICULUM
* K. Takano and Y. Tabata contributed equally to this work.
Address for reprint requests and other correspondence: O. Hori, Dept. of
Neuroanatomy, Kanazawa Univ. Graduate school of Medical Science, 13–1
Takara-Machi, Kanazawa City, Ishikawa, 920-8640, Japan (e-mail: osamuh
@nanat.m.kanazawa-u.ac.jp).
http://www.ajpcell.org
associated protein degradation (ERAD) molecules, and antioxidant genes (31). If the protein load in the ER exceeds its
folding capacity, or dome defects in the ER stress response
exist, cells tend to die, typically, with apoptotic features (ER
stress-induced cell death; see Ref. 31). Important roles for ER
stress and ER stress-induced cell death have been demonstrated
in a broad spectrum of pathological situations, including ischemia, diabetes, atherosclerosis, endocrine defects, and neurodegenerative disorders (7, 10, 12, 24, 28, 34, 38). In Parkinson’s
disease (PD) research, it was recently reported that neurotoxins, such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-pyridinium, and rotenone, induced not only oxidative stress
but also ER stress, and disturbance of the UPR, which was
observed in PERK null mice, rendered the sympathetic neurons
more vulnerable to 6-OHDA (28), suggesting the cross talk of
oxidative stress and ER stress in the experimental PD models.
Homocycteine-induced ER protein (Herp) is a membranebound, ubiquitin-like protein that is located in the ER (9, 14).
Although Herp is strongly induced by ER stress or deep
hypoxia, it decays rapidly consequent to proteasome-mediated
degradation, suggesting the possible contribution of Herp in
the ERAD. We recently reported that targeting disruption of
the Herp gene rendered F9 embryonic carcinoma cells vulnerable to ER stress (9). The ER stress-induced death in F9 Herp
null cells was associated with the aberrant ER stress signaling,
structural changes in the ER, and caspase activation. Using this
cell line, and other ER stress-sensitive cells such as mouse
insulinoma MIN6 cells and rat pheocromocytoma PC-12 cells,
we evaluated the protective effect of 103 plant-derived compounds against ER stress-induced cell death. We report here
that, unlike hydroxyflavones, methoxyflavones have strong
protective effects against ER stress in vitro and in vivo. They
mildly activated the UPR branches such as the eukaryotic
initiation factor (eIF) 2␣ and nuclear factor erythroid 2-related
factor (Nrf2) pathways without causing ER stress, at least in
part, through the activation of the protein kinase A (PKA)
pathway. Furthermore, neuroprotective property of tangeretin,
a methoxyflavone, was associated with enhanced expression of
molecular chaperons in the ER in a mouse model of PD. Our
results suggest that the regulation of ER stress by methoxyflavones could be a therapeutic target for preventing the ER
stress-related diseases.
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
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0363-6143/07 $8.00 Copyright © 2007 the American Physiological Society
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Takano K, Tabata Y, Kitao Y, Murakami R, Suzuki H,
Yamada M, Iinuma M, Yoneda Y, Ogawa S, Hori O. Methoxyflavones protect cells against endoplasmic reticulum stress and neurotoxin. Am J Physiol Cell Physiol 292: C353–C361, 2007. First
published September 13, 2006; doi:10.1152/ajpcell.00388.2006.—
Enhanced endoplasmic reticulum (ER) stress leads to cell death in
various pathophysiological situations. During a search for compounds
that regulate ER stress, we identified methoxyflavones, a group of
flavonoids, as strong protective agents against ER stress. Analysis in
mouse insulinoma MIN6 cells revealed that methoxyflavones mildly
activated the eukaryotic initiation factor 2␣ and nuclear factor erythroid 2-related factor pathways, but not the XBP1 pathway, and
induced downstream genes, including glucose-regulated protein
(GRP) 78, a molecular chaperone in the ER. The protective effect of
methoxyflavones was enhanced by agents that increase intracellular
cAMP levels such as forskolin, dibutyryl-cAMP and IBMX, but
suppressed by the protein kinase A (PKA) inhibitor H-89, suggesting
involvement of the PKA pathway in the regulation of ER stress by
methoxyflavones. Consistent with the results in cultured cells, pretreatment of mice with tangeretin, a methoxyflavone, enhanced expression of GRP78 and HO-1 without causing ER stress in renal
tubular epithelium and prevented tunicamycin-induced cell death.
Furthermore, preadministration of tangeretin in mice enhanced expression of GRP78 in the substantia nigra pars compacta and protected dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin that induces both oxidative and ER stress.
These results suggest that methoxyflavones play an important role in
the regulation of ER stress and could be a therapeutic target for the ER
stress-related diseases.
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EXPERIMENTAL PROCEDURES
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RESULTS
Prevention of ER stress-induced cell death by methoxyflavones. Preliminary studies with well-known compounds revealed that dantrolene, an antagonist of the ryanodine receptor,
and some anti-oxidants such as ␣-tocopherol partly prevented
tunicamycin-induced cell death in F9 Herp null cells (Fig. 1B).
Using these agents as positive controls, 103 plant-derived
compounds (IN1-IN103) were screened in F9 Herp null cells
treated with tunicamycin, and several methoxyflavones, including IN19 (tangeretin: 5,6,7,8,4⬘-pentamethoxyflavone), IN69
(nobiletin: 5,6,7,8,3⬘,4⬘-hexamethoxyflavone), IN72 (5,6,7,4⬘tetramethoxyflavone), and IN88 (sinensetin: 5,6,7,3⬘,4⬘-pentamethoxyflavone), were identified as candidate protective
agents against ER stress (Fig. 1, A–C). Methoxyflavones prevented tunicamycin-induced cell death more effectively than
dantrolene (Fig. 1, B and C), while hydroxyflavones, including
IN17 (5,7,2⬘,4⬘-tetrahydroxyflavone; Fig. 1B) and luteolin
(5,7,3⬘,4⬘-tetrahydroxyflavone, data not shown), had no effect
under the same conditions. Dantrolene-equivalent protective
activity of methoxyflavones was obtained at 10 –15 ␮M in F9
Herp null cells. Methoxyflavones, but not hydroxyflavones,
also inhibited tunicamycin-induced cell death in MIN6 cells
(Fig. 1DI) and, to a lesser extent, in PC-12 cells (data not
shown). In contrast, when MIN6 cells were treated with H2O2,
an agent that causes oxidative stress, methoxyflavones were
less effective compared with hydroxyflavones (Fig. 1DII). The
protective effect of methoxyflavones was also observed when
MIN6 cells were treated with other types of ER stressors such
as brefeldin A and A-23187 (data not shown).
Effect of methoxyflavones on ER stress signaling. To dissect
the mechanism underlying the protective effect of methoxyflavones, expression levels of UPR target genes, such as GRP78,
CHOP, and HO-1 (8, 17, 31), were analyzed in MIN6 cells.
IN19, IN88 (Fig. 2A), and other methoxyflavones (data not
shown) increased expression of all three genes, whereas the
effects were somewhat lower compared with hydroxyflavones
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Cell cultures and stress conditions. F9 Herp null cells and rat
astrocytes were maintained in DMEM with 20% FBS and minimal
essential medium with 10% FBS, respectively. MIN6 cells and PC-12
cells were provided by Dr. Jun-ichi Miyazaki, Osaka University (20)
and Dr. Haruhiro Higashida, Kanazawa University, and were maintained in DMEM with 15% FBS and with 5% FBS/5% horse serum,
respectively. ER stress was induced by treating cells with tunicamycin
(0.75–2 ␮g/ml; Sigma, St Louis, MO), brefeldin A (0.5 ␮g/ml;
Sigma), or A-23187 (2.5 ␮M; Sigma) for indicated times (6 – 48 h).
Each compound was added to the cells together with ER stressors.
Oxidative stress was induced by exposing cells to 66 ␮M H2O2
(Nacalai Tesque, Kyoto, Japan). Cells were pretreated with each
compound for 24 h, followed by exposure to H2O2 in the presence of
each compound for another 24 h.
Compounds and cell viability assays. One hundred and three
plant-derived compounds (IN1-IN103), including stilbenoids, xanthonoids, and flavonoids, were analyzed for their protecting effects
against ER stress in F9 Herp null cells. Hydroxyflavones such as
IN17, or methoxyflavones such as IN19, IN69, IN72, and IN88 were
isolated as described previously (11, 35). Luteolin, forskolin, dibutyryl-cAMP (DbcAMP), IBMX, and H-89 were purchased from
Wako (Osaka, Japan), and dantrolene, N-acetylcysteine, and ␣-tocopherol were obtained from Sigma. Cell viability under ER stress or
oxidative stress conditions was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,4-diphenyl-2H tetrazolium bromide assay (Nacalai Tesque)
or LIVE/DEAD cell toxicity assay (Molecular Probes, Eugene, OR),
as described previously (9).
Northern blotting and RT-PCR. MIN6 cells were treated with each
compound for indicated times (24 or 40 h), and, in some cases,
tunicamycin (2 ␮g/ml) was added to cells 6 h before harvesting. As a
positive control for RT-PCR, astrocytes were treated with tunicamycin (2 ␮g/ml) for 8 h. Total RNA (10 ␮g) isolated from MIN6 cells
or mouse kidney was subjected to Northern blotting using cDNA
fragments specific for glucose-regulated protein (GRP) 78, CCAAT/
enhancer-binding protein homologous protein (CHOP), heme oxygenase (HO)-1, or ␤-actin, as described previously (9). First-strand cDNA
was also synthesized with the first-strand cDNA synthesis kit (Takara,
Tokyo, Japan) and amplified with primers specific for XBP1, as
described previously (32).
Cell lysis and Western blotting. For analyzing the status of phosphorylation of eIF2␣, MIN6 cells were solubilized in buffer containing 1% Nonidet P-40 (NP40), 0.1% SDS, 0.2% deoxycholate and
subjected to Western blotting with anti-eIF2␣ antibody and antiphospho-eIF2␣ antibody (Cell Signaling Technology, Beverly, MA).
For estimating the status of Nrf2, nuclear proteins were extracted from
MIN6 cells as described previously (30) and subjected to Western
blotting with anti-Nrf2 antibody (Santa Cruz Biotechnology, Santa
Cruz, CA). Sites of primary antibody binding were visualized using
alkaline phosphatase-conjugated secondary antibodies.
Metabolic labeling. MIN6 cells were treated with each compound
for 24 h and metabolically labeled with 35S-Met/Cys (200 ␮Ci;
Amersham Pharmacia Biotech, Piscataway, NJ) for the last 3 h before
harvesting in Met-free medium. Cells were lysed in buffer containing
1% NP40, 0.1% SDS, 0.2% deoxycholate and subjected to SDSPAGE, followed by autoradiography.
Animal experiments. Animal experimental protocols were approved
by the Committee on Animal Experimentation of Kanazawa University (Takara-machi Campus). All animal experiments were performed
using C57BL/6 mice (male, 8 –12 wk of age). A mouse model of ER
stress was created by a single intraperitoneal injection of tunicamycin
(1 mg/kg), as described previously (39). For IN19 or vehicle administration, mice were intraperitoneally injected with IN19 (10
mg 䡠 kg⫺1 䡠 day⫺1) in saline, including 10% Cremophore EL (Sigma)
and 10% DMSO, or with the dissolving solution (vehicle) daily for 2
or 4 days. A mouse model of PD was created by intraperitoneal
injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP,
20 mg/kg) two times at a 2-h interval (15). Histological analysis was
performed at 4 and 7 days after MPTP injection as below. When mice
were pretreated with IN19 or vehicle for 4 days, MPTP was injected
in mice 2 h after IN19 or vehicle administration on the 4th day.
Histological analysis. At the indicated time points, kidney and
brain were removed from mice after perfusion with 4% of paraformaldehyde. The kidney was embedded in paraffin, and 5-␮m sections were
cut. Sections were subjected to hematoxylin and eosin (HE) staining,
immunostaining with anti-KDEL or anti-GRP78 antibody (StressGen;
Victoria, British Columbia, Canada), and TdT-UDP nick end labeling
(TUNEL) staining (Chemicon International, Temecula, CA). Coronal
brain sections (10 ␮m) were cut on a cryostat. Sections were processed
for immunostaining with anti-KDEL antibody or with anti-GRP78 antibody, together with anti-tyrosine hydroxylase (TH) antibody (Chemicon
International). We employed both anti-KDEL antibody and anti-GRP78
antibody to detect GRP78 expression, since anti-KDEL antibody displayed lower background compared with anti-GRP78 antibody in some
brain sections. FITC or Cy3-conjugated secondary antibodies were used
for visualization of immunolabeling.
Quantitative data analysis. Laser densitometry analysis was performed to semiquantitate results of Western blotting as described
previously (9). Tyrosine hydroxylase positive (TH⫹) neuronal cells in
the substantia nigra pars compacta (SNpc) were counted in two
representative sections (bregma ⫺3.16 and ⫺3.64 mm) as described
previously (15). Statistical analysis was performed by Student’s t-test.
FLAVONOIDS AND THE REGULATION OF ER STRESS
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Fig. 1. Effects of methoxyflavones and hydroxyflavones on endoplasmic reticulum (ER) stress-induced cell death. A: chemical structures of compounds. B and
C: protection of F9 homocystein-induced ER protein (Herp) null cells against ER stress-induced cell death. F9 Herp null cells (5 ⫻ 105 cells/condition) were
treated with 0.8 ␮g/ml tunicamycin (Tm) in the absence or presence of indicated compounds for 48 (B) or 36 (C) h, and cell viability was evaluated by
3-(4,5-dimethyl-2-thiazolyl)-2,4-diphenyl-2H tetrazolium bromide (MTT) assay (B) or by LIVE/DEAD cell toxicity assay (C). In B, the MTT value in the
absence of tunicamycin was determined as 100. Values shown are means ⫾ SD of 3 experiments. D: protection of MIN6 cells against ER stress (I) and oxidative
stress (II)-induced cell death. I: MIN6 cells (5 ⫻ 105 cells/condition) were treated with 1.5 ␮g/ml tunicamycin in the absence or presence of compounds for 48 h.
II: MIN6 cells (5 ⫻ 105 cells/condition) were pretreated with medium alone or with indicated compounds for 24 h, and incubated with 66 ␮M H2O2 for another
24 h in the absence or presence of compounds. Cell viability was evaluated by MTT assay as above. Values shown are means ⫾ SD of 4 experiments.
such as IN17 (Fig. 2A) or luteolin (data not shown). It was
previously reported that different types of flavonoids activate
eIF2␣ kinases, including PERK, and suppress protein synthesis
through phosphorylation of eIF2␣ (13). Consistent with these
observations, all flavone derivatives tested in the current study
enhanced phosphorylation of eIF2␣ in MIN6 cells after 24 h of
treatment, determined by Western blotting with anti-phosphoAJP-Cell Physiol • VOL
rylated eIF2␣ antibody. However, the effects were lower in
IN19 (Fig. 2BI) and other methoxyflavones (data not shown)
when compared with hydroxyflavones such as IN17 (Fig. 2BI)
or luteolin (data not shown). Accordingly, metabolic labeling
with 35S-methionine revealed that general protein synthesis in
MIN6 cells was suppressed by IN17, and, to a lesser extent, by
IN19 (Fig. 2BII).
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Fig. 2. Effects of methoxyflavones and hydroxyflavones on ER stress signaling. A: expression of unfolded protein response (UPR) target genes. MIN6 cells (107
cells/condition) were treated with IN17, IN19, or IN88 for 24 h, and total RNA (10 ␮g) was subjected to Northern blotting. Similar results were obtained in four
separate sets of experiments. B: activation of eukaryotic initiation factor (eIF) 2␣ (I) and suppression of general protein synthesis (II). I: MIN6 cells were treated
with IN17 or IN19 for 24 h, and whole cell extracts were subjected to Western blotting with anti-phospho (P)-eIF2␣ antibody or with anti-eIF2␣ antibody.
Quantification of relative intensities for P-eIF2␣ is shown (bottom). II: MIN6 cells were treated with IN17 or IN19 for 24 h and metabolically labeled with
35
S-Met/Cys for the last 3 h before harvesting. Cell lysates were subjected to SDS-PAGE, followed by autoradiography. C: nuclear accumulation of nuclear factor
erythroid 2-related factor (Nrf2). MIN6 cells were treated with IN17 or IN19 for 16 h, and nuclear extracts were subjected to Western blotting with anti-Nrf2 antibody.
NS indicates a nonspecific band in the same membrane. Quantification of relative intensities for Nrf2 is shown (bottom). D: activation of XBP1 signaling. MIN6 cells
were treated with IN19 for 24 h in the absence or presence of 2 ␮g/ml tunicamycin for the last 6 h before harvesting. As a control, cultured astrocytes were treated with
2 ␮g/ml tunicamycin for 8 h. Total RNA was subjected to RT-PCR with specific primers for XBP1. Similar results were obtained in 3 separate sets of experiments.
Because expression of HO-1 is regulated by both the eIF2␣ATF4 pathway (8) and Nrf2 pathway (31) and because Nrf2
was found to be a direct substrate of PERK (4), nuclear Nrf2
protein was assessed by Western blotting using nuclear extract
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from MIN6 cells. The levels of Nrf2 protein in the nucleus
were enhanced by treatment of cells with IN17 and, to a lesser
extent, with IN19 (Fig. 2C). In contrast to the activation of the
eIF2␣ and Nrf2 pathways, IN19 did not activate the XBP1
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pathway in MIN6 cells, another key pathway in the UPR (Fig.
2D), indicating that methoxyflavones did not cause ER stress
but activated the UPR branches such as the eIF2␣ and Nrf2
pathways.
Involvement of the protein kinase A pathway in the regulation of ER stress by methoxyflavones. It was recently reported
that methoxyflavones induced neurite outgrowth through the
activation of PKA signaling (22, 23). We hypothesized that this
pathway may also play a role in the regulation of ER stress.
Analysis with MIN6 cells revealed that forskolin, an adenylate
cyclase activator, or IBMX, a nonselective inhibitor of phosphodiesterase (PDE), protected cells against tunicamycin-induced cell death, whereas H-89, an inhibitor of PKA, accelerated cell death in the same condition (Fig. 3AI). Furthermore,
the protective effect of IN19 (see Fig. 5AII) or other methoxyflavones (data not shown) against ER stress was enhanced by
forskolin, DbcAMP, a cAMP analog, or IBMX but reversed by
H-89 (see Fig. 5AII), suggesting involvement of the PKA
pathway in the regulation of ER stress by methoxyflavones.
Consistently, forskolin or combination of forskolin with IBMX
upregulated expression of GRP78, CHOP, and HO-1 in MIN6
cells (Fig. 3B). Similar cell protection by forskolin or IBMX
against ER stress was observed in F9 Herp null cells (data not
shown).
Effect of methoxyflavones on ER stress-induced cell death in
vivo. To analyze the impact of methoxyflavones on ER stress in
vivo, a tunicamycin injection model in mice was employed
(39). First, IN19 (10 mg䡠kg⫺1 䡠day⫺1) was intraperitoneally
injected in mice for 2 or 4 consecutive days, and expression of
UPR target genes in the kidney was compared with vehicleinjected mice. Consistent with the results in cultured cells,
expression of GRP78 and HO-1 was enhanced in IN19-injected
kidneys (Fig. 4AI), whereas XBP1 was not activated (Fig.
4AII). Enhanced expression of GRP78 protein, which was
determined by immunostaining with anti-KDEL antibody (Fig.
4B) and with anti-GRP78 antibody (supplemental Fig. S1A; the
online version of this article contains supplemental data), was
observed mainly in tubular epithelium, although the expression
levels were lower than those in tunicamycin-injected kidneys
(2 days after a single injection; Fig. 4B). Next, tunicamycin
was intraperitoneally injected in the mice, and, 4 days after
injection, morphological changes and cell death were assessed
in the kidneys. HE and TUNEL staining revealed that tunicamycin treatment (1 mg/kg) partly destroyed the tubular structures (Fig. 4Cb) and caused cell death (Fig. 4Ce) in kidneys, as
described previously (39). However, when mice were pretreated with IN19 for four consecutive days, these histological
changes almost entirely disappeared (Fig. 4Cc, f), suggesting a
critical role of methoxyflavones in the regulation of ER stress
in vivo.
ER stress regulation in a mouse model of PD. Because
tangeretin (IN19) was shown to cross the blood-brain barrier
and was neuroprotective in a rat 6-OHDA lesion model of PD
(5), a possible association of neuroprotection and ER stress
regulation by IN19 was analyzed in a mouse PD model by
intraperitoneal injection of MPTP (39). Consistent with a
previous report in cultured cells (28), injection of MPTP
decreased the number of TH⫹ cells in the SNpc (⬃50% of
control mice) within 4 days but enhanced immunoreactivity
with anti-KDEL antibody in TH⫹ neuronal cells (Fig. 5A),
suggesting induction of ER stress in this in vivo model.
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Fig. 3. Effects of protein kinase A (PKA) activation on the regulation of ER
stress. A: regulation of ER stress-induced cell death by PKA-related compounds. MIN6 cells (5 ⫻ 105 cells/condition) were treated with 1.5 ␮g/ml
tunicamycin in the absence or presence of PKA-related compounds (I),
together with IN19 (II), for 48 h. Cell viability was evaluated by MTT assay.
Values shown are means ⫾ SD of 4 experiments. B: expression of UPR target
genes. MIN6 cells (107 cells/condition) were treated with indicated PKArelated compounds for 40 h, and total RNA (10 ␮g) was subjected to Northern
Blotting. Similar results were obtained in 4 separate sets of experiments.
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Intraperitoneal injection of IN19 (10 mg䡠kg⫺1 䡠day⫺1) for four
consecutive days also enhanced immunoreactivity of GRP78 in
TH⫹ neuronal cells in the SNpc before MPTP injection (Fig.
5B and supplemental Fig. S1B), and suppressed reduction of
TH⫹ neuronal cells in the SNpc 7 days after MPTP injection
(Fig. 5, C and D). These results suggest that the neuroprotective properties of IN19 in this mouse PD model are, at least in
part, associated with the regulation of ER stress.
DISCUSSION
Flavonoids are plant-derived polyphenolic compounds
that exhibit a broad spectrum of bioactivities, including
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anti-cancer, anti-inflammatory, and anti-atherogenic effects
(19). To date, beneficial bioactivities of flavonoids have
been attributed mainly to their anti-oxidant properties, such
as radical scavenging, chelating of metal ions, and induction
of anti-oxidant genes (19, 21, 33). Among flavonoids, however, methoxyflavones, which are abundant in citrus peels,
have another aspect. They also possess quite beneficial
properties, such as a hypolipidemic effect (16), suppression
of tumor growth/metastasis (3, 27), anti-inflammation (18),
and neuroprotective/neurotrophic function (5, 22, 23), although they have much less anti-oxidant activity compared
with hydroxyflavones or other flavonoids (1, 6). In fact, in
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Fig. 4. Protective effects of IN19 in tunicamycin-induced renal tubular damage. A and B: expression of UPR target genes after IN19 or tunicamycin injection.
A: mice were injected with IN19 (10 mg/kg) or vehicle daily for 2 or 4 days or injected with tunicamycin (1 mg/kg; 2 days after a single injection), and total
RNA, isolated from kidney, was subjected to Northern Blotting (I) or RT-PCR (II). Similar results were obtained in 3 separate sets of experiments. B: kidney
sections from IN19- or vehicle-injected mice (daily for 4 days) or tunicamycin-injected mice (2 days after a single injection) were subjected to immunostaining
with anti-KDEL antibody. C: inhibition of tunicamycin-induced tissue damage and cell death by IN19. Kidney sections were prepared from mice injected with
vehicle, tunicamycin (4 days after a single injection), or tunicamycin (4 days after a single injection) after IN19 preadministration (daily for 4 days) and subjected
to hematoxylin and eosin (HE) staining or TUNEL staining. Similar results were obtained in 3 separate sets of experiments.
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Fig. 5. Neuroprotection and ER stress regulation in a mouse model of Parkinson’s disease (PD). A: reduced numbers of tyrosine hydroxylase positive (TH⫹)
cells and enhanced immunoreactivity with anti-KDEL antibody after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection. Brain sections from mice
4 days after vehicle (a– c) or MPTP (d–f) injection were subjected to immunostaining with anti-tyrosine hydroxylase (TH) antibody and anti-KDEL antibody. SNpc,
substantia nigra pars compacta; MT, medial terminal nucleus of the accessory optic tract; VTA, ventral tegmental area. B: increased immunoreactivity with anti-KDEL
antibody after IN19 injection. Mice were injected with IN19 (10 mg/kg) daily for 4 days as described in Fig. 4, and brain sections were subjected to immunostaining
with anti-TH antibody and anti-KDEL antibody. Similar results were obtained at least in 3 separate sets of experiments. C: neuroprotection by IN19 after MPTP injection.
I: mice were pretreated with vehicle or IN19 for 4 consecutive days, and then injected with MPTP. After MPTP injection (7 days), brain sections, including the SNpc,
were subjected to immunostaining with anti-TH antibody. II: no. of TH⫹ cells in the SNpc. Values shown are means ⫾ SE of 4 experiments. *P ⬍ 0.01.
our experimental system, methoxyflavones were less effective against H2O2-induced cell death in MIN6 cells compared with IN17 (Fig. 1) or luteolin (data not shown). It was
reported that activation of the PKA pathway through the
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inhibition of PDE played an important role in the various
bioactivities of methoxyflavones (22, 23, 25), suggesting the
mechanisms beyond anti-oxidant property underlying the
beneficial effects of methoxyflavones.
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ACKNOWLEDGMENTS
We are grateful to Dr. Jun-ichi Miyazaki (Osaka University), and Dr.
Haruhiro Higashida (Kanazawa University) for providing MIN6 cells and
PC-12 cells, respectively. We thank Dr. David Ron (New York University) for
valuable suggestions. We also thank Takashi Tamatani, Michiyo Tamatani,
and Harumi Nishihama for technical assistance.
GRANTS
This work was partly supported by a Grant-in Aid for Scientific Research
(15032315) from the Ministry of Education, Science, Technology, Sports, and
Culture of Japan.
AJP-Cell Physiol • VOL
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In the current study, we demonstrated that methoxyflavones
strongly protected cells against ER stress in vitro and (Fig. 1)
in vivo (Fig. 4). Consistent with a previous report (13), methoxyflavones activated some branches of the UPR such as the
eIF2␣ and Nrf2 pathway and induced downstream genes such
as GRP78, HO-1, and CHOP without causing ER stress,
although the levels were somewhat less than hydroxyflavones
(Fig. 2). Activation of the PKA pathway was also involved in
the cell protection by methoxyflavones, at least in some types
of cells (Fig. 3A). Although activation of the PKA pathway
gives diverse biological effects on cells (29, 37), to our knowledge, this is the first report for the protective effect against ER
stress. It could be an upstream event in the signaling pathway
triggered by methoxyflavones, when compared with activation
of the UPR branches, since forskolin or combination of forskolin and IBMX enhanced expression of GRP78, CHOP, and
HO-1 in MIN6 cells (Fig. 3B). However, further studies are
required to dissect the detailed mechanism regarding the effect
of the PKA pathway in the regulation of ER stress. At this
moment, it is also not clear why hydroxyflavones failed to
protect cells against ER stress-induced cell death (Fig. 1DI). It
may reflect the fact that downstream genes of the eIF2␣
pathway include both cell survival- and cell death-related
genes (31), and persistent activation of the eIF2␣ pathway
could be linked to cell death (26). Another possibility is that
the effect of hydroxyflavones to the PKA pathway was less
than methoxyflavones, as previously described (25).
We also presented evidence that the neuroprotective property of methoxyflavones in a MPTP-injected mouse was associated with the enhanced expression of molecular chaperons in
the ER before MPTP injection (Fig. 5). Consistent with previous reports in vitro (28), intraperitoneal injection of MPTP in
mice enhanced immunoreactivity of GRP78/GRP94 in TH⫹
neuronal cells in the SNpc (Fig. 5A), suggesting induction of
ER stress in this experimental model in vivo. Our preliminary
results revealed that similar tendency was observed when
6-OHDA was injected on the medial forebrain bundle in the
mouse brain, suggesting the cross talk of oxidative stress and
ER stress in mouse PD models in vivo.
Recently, it was reported that a selective inhibitor of eIF2␣
dephosphorylation protected cells against ER stress and viral
infection (2). We speculate that there are natural products with
similar activities, and, in this context, methoxyflavones could
be good candidates. Methoxyflavones were reported to be
relatively safe to laboratory mice (36), and methoxyflavonecontaining citrus peels have been used for a long time in the
field of Chinese medicine or Japanese remedy. Therefore, one
of our further studies may aim to reanalyze the beneficial
effects of methoxyflavones and other flavonoids in terms of ER
stress regulation.
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