molecules

Article
Effects of Carbazole Derivatives on Neurite
Outgrowth and Hydrogen Peroxide-Induced
Cytotoxicity in Neuro2a Cells
Yoshiko Furukawa 1, *,† , Atsushi Sawamoto 1,† , Mizuki Yamaoka 1 , Makiko Nakaya 1 ,
Yuhzo Hieda 2 , Tominari Choshi 2 , Noriyuki Hatae 3 , Satoshi Okuyama 1 , Mitsunari Nakajima 1
and Satoshi Hibino 2
1 Department of Pharmaceutical Pharmacology, College of Pharmaceutical Sciences, Matsuyama University,
4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan; asawamot@g.matsuyama-u.ac.jp (A.S.);
16131209@matsuyama-u.ac.jp (M.Y.); mu.yakuri.015@gmail.com (M.N.);
sokuyama@g.matsuyama-u.ac.jp (S.O.); mnakajim@g.matsuyama-u.ac.jp (M.N.)
2 Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Sanzo, Gakuen-cho, Fukuyama,
Hiroshima 729-0292, Japan; hieda@fukuyama-u.ac.jp (Y.H.); choshi@fukuyama-u.ac.jp (T.C.);
satoshihibino@ma.ccnw.ne.jp (S.H.)
3 School of Pharmaceutical Sciences, Health Science University of Hokkaido, Ishikari, Tobetsu,
Hokkaido 061-0293, Japan; nhatae@hoku-iryo-u.ac.jp
* Correspondence: furukawa@g.matsuyama-u.ac.jp; Tel.: +89-925-7111; Fax: +89-925-7162
† These authors contributed equally to this paper.




Received: 1 March 2019; Accepted: 5 April 2019; Published: 7 April 2019


Abstract: Many studies have demonstrated that oxidative stress plays an important role in
several ailments including neurodegenerative diseases and cerebral ischemic injury. Previously
we synthesized some carbazole compounds that have anti-oxidant ability in vitro. In this present
study, we found that one of these 22 carbazole compounds, compound 13 (3-ethoxy-1-hydroxy-8-
methoxy-2-methylcarbazole-5-carbaldehyde), had the ability to protect neuro2a cells from hydrogen
peroxide-induced cell death. It is well known that neurite loss is one of the cardinal features of
neuronal injury. Our present study revealed that compound 13 had the ability to induce neurite
outgrowth through the PI3K/Akt signaling pathway in neuro2a cells. These findings suggest that
compound 13 might exert a neurotrophic effect and thus be a useful therapy for the treatment of
brain injury.

Keywords: carbazole; neuro2a cells; neurite outgrowth; neuronal death; anti-oxidant

1. Introduction
Carbazole alkaloids, which are produced by plants such as those of the genus Murraya or
Clausena [1], have recently attracted much attention because 1) they have various abilities such
as anti-microbial, anti-tumor, anti-pileptic, anti-histaminic, anti-oxidant and anti-inflammatory
actions [2]; and 2) they are being used as lead compounds for drug development [3–5]. Recently,
we synthesized some carbazole derivatives having anti-oxidant ability, which was evaluated in
terms of their radical-scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and/or
2,20 -azinobis-(3-ethylbenzhiazolic-6-sulfonate) cations (ABTS) [5,6].
As the results of several recent studies have indicated that neurological disorders are linked to
elevated levels of oxidative stress [7,8] and that anti-oxidant(s) might have therapeutic potential [9],
we investigated herein, using neuroblastoma neuro2a cells, whether our carbazole compounds could
exert an anti-oxidant effect on neuronal cells. Neuro2a cells are a mouse neural crest-derived cell line

Molecules 2019, 24, 1366; doi:10.3390/molecules24071366 www.mdpi.com/journal/molecules

Molecules 2019, 24, 1366 2 of 10

and are frequently used to study neuroprotective abilities of various factors [10,11]. As a generator of
reactive oxygen species (ROS), we used hydrogen peroxide (H2 O2 ), which is known to easily penetrate
into cells and to generate high levels of ROS [12,13].
Neuro2a cells are also extensively used to study neuronal differentiation and neurite growth [14].
Molecules 2019, 24, x FOR PEER REVIEW 2 of 10
Neurite outgrowth is known to be crucial for neuronal plasticity and neuronal regeneration [15],
and thesecell actions
line and are considered
are frequently usedtotobestudy
important for developing
neuroprotective abilities of therapies to promote
various factors [10,11]. Asneuronal
a
generator
regeneration of reactive
in the case of oxygen species and
nerve injury (ROS), we used hydrogen
neurological peroxide
disorders [16].(H
We2O2), which is known to
thus evaluated the ability
easily penetrate into cells and to generate high levels of ROS [12,13].
of our carbazole compounds to promote neurite outgrowth from neuro2a cells.
Neuro2a cells are also extensively used to study neuronal differentiation and neurite growth [14].
As Neurite
a result,outgrowth
we successfully
is known found that one
to be crucial of our carbazole
for neuronal derivatives
plasticity and had this ability
neuronal regeneration as well as
[15], and
anti-oxidant
these ability;
actions and are so, we thentoinvestigated
considered be importantthe forregulatory
developingmechanisms
therapies to of the neurite
promote outgrowth
neuronal
triggeredregeneration in the caseRegarding
by this compound. of nerve injury and neurological
the regulatory disordersat[16].
mechanisms playWein thus evaluated
neurite the from
outgrowth
ability of our carbazole compounds to promote neurite outgrowth from neuro2a cells.
neuronal cells (including not only neuro2a cells but also rat PC12 cells), various signal pathways have
As a result, we successfully found that one of our carbazole derivatives had this ability as well
been reported to be involved, such as extracellular signal-regulated kinase (ERK) [17–19], Akt and
as anti-oxidant ability; and so, we then investigated the regulatory mechanisms of the neurite
phosphatidylinositol
outgrowth triggered 3-kinase (PI3K)
by this [16,20].Regarding
compound. Thus, wethe investigated whether our
regulatory mechanisms at carbazole compound
play in neurite
had the outgrowth
ability to from
directly activate
neuronal cells(phosphorylate)
(including not only any signalcells
neuro2a transduction
but also rat molecule(s) and whether
PC12 cells), various
signal of
the blockage pathways
such signalhave been reported to be
transduction(s) involved,
would reducesuchthis
as extracellular signal-regulated kinaseneurite
carbazole compound-induced
(ERK) [17–19],
outgrowth from neuro2a cells.Akt and phosphatidylinositol 3-kinase (PI3K) [16,20]. Thus, we investigated whether
our carbazole compound had the ability to directly activate (phosphorylate) any signal transduction
molecule(s)
2. Results and whether the blockage of such signal transduction(s) would reduce this carbazole
and Discussion
compound-induced neurite outgrowth from neuro2a cells.
2.1. Effects of Carbazole Derivatives on H2 O2 -Induced Cell Death of Neuro2a Cells
2. Results and Discussion
First, we examined whether our carbazole derivatives (Figure 1) might exhibit a protective effect
2.1. Effects of Carbazole Derivatives on H2O2-Induced Cell Death of Neuro2a Cells
against H2 O2 -induced cell death of neuro2a cells cultured in normally used medium, namely, medium
containing 10% First,fetal
we examined
bovine serumwhether our carbazole
(FBS). derivatives (Figure
For this experiment, 1) might
cells were exhibit
seeded in awells
protective
of a 96-well
effect against H 2O2-induced cell death of neuro2a cells cultured in normally used medium, namely,
plate and maintained in this medium for 24 h. The cells were then treated with test compounds (10 µM
medium containing 10% fetal bovine serum (FBS). For this experiment, cells were seeded in wells of
concentration of each compound) for 1 h and then incubated for an additional 18 h in the presence of
a 96-well plate and maintained in this medium for 24 h. The cells were then treated with test
H2 O2 (30 µM). After
compounds (10 the exposure to of
µM concentration Heach
2 O2 ,compound)
the cell viability
for 1 h andwas significantly
then incubated for(*p < 0.05) reduced
an additional 18 to
about 80h%, butpresence
in the some ofofthe compounds
H 2O (3, the
2 (30 µM). After 5, 13, 21, and
exposure 2Ohad
22)
to H 2, the the
cell protective
viability waseffect on H2 O
significantly 2 -induced
(*p <
cell viability reduction
0.05) reduced in this80experimental
to about %, but some ofcondition and gave
the compounds (3, 5, similar cell22)
13, 21, and viability
had thein comparison to
protective
effect onand
that of control H2O50 2-induced cell viability reduction in this experimental condition and gave similar cell
µM vitamin E (V.E)-treated cells (Figure 2).
viability in comparison to that of control and 50 µM vitamin E (V.E)-treated cells (Figure 2).

Figure 1. Structures
Figure 1. Structuresof
of carbazole derivatives.
carbazole derivatives.
Molecules 2019, 24, 1366 3 of 10
Molecules 2019, 24, x FOR PEER REVIEW 3 of 10

Figure
Figure Effects
2. 2. ofof
Effects carbazole
carbazolederivatives
derivatives ononneuro2a
neuro2a cell viability.
cell Neuro2a
viability. Neuro2acells pretreated
cells pretreated with test
with test
compounds
compounds (compounds
(compounds at theatconcentration
1–221–22 of 10 µMofor10
the concentration with
µM vitamin E [V.E]
or with at the E
vitamin concentration
[V.E] at the
ofconcentration
50 µM) were exposed to 30
of 50 µM) H2 O2 . The
µMexposed
were results
to 30 µM H represent the mean ± SEM (n = 5, different culture).
2O2. The results represent the mean ± SEM (n = 5,
Significance
different culture). Significance difference in values between H
difference in values between the none-treated and 2 Onone-treated
the 2 -treated cells: * pH
and < 20.05 (Student’s
O2-treated cells:
t test).
* p < 0.05 (Student’s t test).
2.2. Effects of Carbazole Derivatives on Neuronal Differentiation of Neuro2a Cells
2.2. Effects of Carbazole Derivatives on Neuronal Differentiation of Neuro2a Cells
To investigate whether our carbazole derivatives could induce neurite outgrowth from neuro2a
To investigate whether our carbazole derivatives could induce neurite outgrowth from
cells, we first cultured the cells for 24 h in 24-well plates in a medium containing 10% serum. Then they
neuro2a cells, we first cultured the cells for 24 h in 24-well plates in a medium containing 10% serum.
were cultured in a low-serum (2% FBS) medium for 24 h to induce a transition from the proliferation
Then they were cultured in a low-serum (2% FBS) medium for 24 h to induce a transition from the
phase to the differentiation phase, after which the cells were incubated for 48 h in a low-serum medium
proliferation phase to the differentiation phase, after which the cells were incubated for 48 h in a
containing test samples (0.3–5 µM). As a positive control, N6 ,20 -O-dibutyryl- adenosine 30 ,50 -cyclic
low-serum medium containing test samples (0.3–5 µM). As a positive control, N6,2′-O-dibutyryl-
monophosphate (db cAMP), a membrane-permeable cAMP analog, was used at the concentration of
adenosine 3′,5′-cyclic monophosphate (db cAMP), a membrane-permeable cAMP analog, was used
5 mM.
at the concentration of 5 mM.
Figure 3A shows the untreated cells (none) having a round shape with few neurites and the db
Figure 3A shows the untreated cells (none) having a round shape with few neurites and the db
cAMP-treated ones apparently displaying long neurites. As shown in Table 1, one of the compounds
cAMP-treated ones apparently displaying long neurites. As shown in Table 1, one of the compounds
tested (13) at the wide range of concentrations apparently induced neurite extension from neuro2a cells
tested (13) at the wide range of concentrations apparently induced neurite extension from neuro2a
after a 48-h treatment; and some of the compounds (compounds 4, 5, 6, 8, 9, 10, 12, 18, 19, 20, and 22) at
cells after a 48-h treatment; and some of the compounds (compounds 4, 5, 6, 8, 9, 10, 12, 18, 19, 20,
the limited range of concentrations slightly induced neurite extension after the 48 h treatment, whereas
and 22) at the limited range of concentrations slightly induced neurite extension after the 48 h
others (compounds 1, 2, 4 and 12) were toxic during the incubation period. Figure 3A also shows
treatment, whereas others (compounds 1, 2, 4 and 12) were toxic during the incubation period.
the representative photographs of the cells cultured with 0.5 µM compound 13 for 48 h. Figure 3B
Figure 3A also shows the representative photographs of the cells cultured with 0.5 µM compound 13
shows that the % of neurite-bearing cells after the 48-h incubation with no compound, 5 mM db cAMP
for 48 h. Figure 3B shows that the % of neurite-bearing cells after the 48-h incubation with no
and 0.5 µM compound 13 was 56.8 ± 6.75 % (*** p < 0.001 vs. control [none]), and 23.1 ± 4.75 %
compound, 5 mM db cAMP and 0.5 µM compound 13 was 56.8 ± 6.75 % (*** p < 0.001 vs. control
(*** p < 0.001), respectively.
[none]), and 23.1 ± 4.75 % (*** p < 0.001), respectively.
2.3. Effects of Carbazole 13 on H2 O2 -Induced Cell Death of Neuro2a Cells
2.3. Effects of Carbazole 13 on H2O2-Induced Cell Death of Neuro2a Cells
As we revealed that compound 13 (3-ethoxy-1-hydroxy-8-methoxy-2-methylcarbazole-5-
As we had
carbaldehyde) revealed that compound
the protective effect on13H2(3-ethoxy-1-hydroxy-8-methoxy-2-methylcarbazole-5-
O2 -induced cell viability reduction (Figure 2) and
carbaldehyde)
neuritogenic had3)the
(Figure protective
activities effect
against on Hcells,
neuro2a 2O2-induced cell viability reduction (Figure 2) and
we then examined in detail the protective effect
neuritogenic (Figure 3) activities against neuro2a
of compound 13 against H2 O2 -induced apoptosis of neuro2a cells, we then
cells examined
cultured in detail medium.
in low-serum the protective
For
effect of compound 13 against H 2O2-induced apoptosis of neuro2a cells cultured in low-serum
this experiment, cells in wells of a 96-well plate were cultured in the normal-serum medium for 24 h
medium.
and then inForthe this experiment,
low-serum one forcells
24 in
h. wells
After of a 96-well
that the cellsplate
werewere cultured
treated in the normal-serum
with compound 13 at the
medium for 24 h and then in the low-serum one for 24 h. After
concentration of 1–10 µM or vitamin E at the concentration of 10–50 µM for 1 h, and thenthat the cells were treated with
incubated for
compound 13 at the concentration of 1–10 µM or vitamin E at the concentration
an additional 18 h in the presence of H2 O2 (30 µM). Figure 4 shows that the cells in the low-serum of 10–50 µM for 1 h,
and then incubated for an additional 18 h in the presence of H 2O2 (30 µM). Figure 4 shows that the
medium were more sensitive to oxidative stress than those in the normal-serum medium (Figure 2),
cells in the H low-serum medium were more sensitive to oxidative stress than those in the
namely, 30 µM 2 O2 reduced the cell viability to 36.1 ± 14.1 % (*** p < 0.001) of the none-treated group
normal-serum
after 18 h. On thismedium (Figure
condition, the2), namely, 30 µM
anti-oxidative H2O2 reduced
abilities of 1 or 5the
µMcell viability to
compound 13 36.1 ± 14.1
against 30%µM (***
p < 0.001) of the none-treated group after 18 h. On this condition, the anti-oxidative abilities of 1 or 5
µM compound 13 against 30 µM H2O2 were almost equal to those of 10 µM vitamin E, and that of 10
µM compound 13 against 30 µM H2O2 were almost equal to that of 50 µM vitamin E.
Molecules 2019, 24, 1366 4 of 10

H2 O2 were almost equal to those of 10 µM vitamin E, and that of 10 µM compound 13 against 30 µM

HMolecules
2 O2 were almost
2019, equal
24, x FOR toREVIEW
PEER that of 50 µM vitamin E. 4 of 10

Figure 3. Effects of compounds 13 and dibutyryl cAMP (db cAMP) on neurite outgrowth from neuro2a
Figure
cells. 3. Effects
Neuro2a cellsof compounds
were 13 test
treated with and compounds
dibutyryl cAMP
(5 mM(db cAMP)oron0.5neurite
db cAMP outgrowth
µM compound 13)from
for
48neuro2a cells.morphological
h, and then Neuro2a cellsimageswere were
treated with test
captured compounds (5 microscopy
by phase-contrast mM db cAMP or 0.5
(A). Cells µM
were
compound
randomly 13) for
chosen for48 h, and then
counting morphological
neurite-bearing cellsimages were
(n = more captured
than 100 cellsbyperphase-contrast microscopy
group) (B). Significance
(A). Cellsinwere
difference values randomly
between chosen for counting
the sample-treated and neurite-bearing cells
non-treated cells: ***(np <= 0.001
more(one-way
than 100ANOVA
cells per
group) (B).
followed Significance
by the Dunnett’sdifference in values between
multiple comparison test). the sample-treated and non-treated cells: *** p
< 0.001 (one-way ANOVA followed by the Dunnett’s multiple comparison test).
Table 1. Effects of carbazole derivatives on neurite outgrowth in neuro2a cells. Cells were treated with
carbazole derivatives at the indicated concentrations for 48 h.
Table 1. Effects of carbazole derivatives on neurite outgrowth in neuro2a cells. Cells were treated
with carbazole
Compounds derivatives
0.3 at
µM the indicated concentrations
0.5 µM for 48 h. 3.0 µM
1.0 µM 5.0 µM
1
Compounds −
0.3 μM 0.5 μM 1.0 μM cell
3.0death
μM 5.0 μM
21 − − cell
cell death
death
3 − −
2 − cell death
4 ± cell death
53 ± − −−
6 4 ± ± cell ±death
75 − ± −−
86 ± ± ± + +± ±
97 ± − −−
10 ± −
8 ± ± + + ±
11 − −
12 9 ± + + − cell death
1310 + ± + + +− +
1411 − − −−
1512 − + + − cell death
1613 − + + + −+ +
17 − −
14 − −
18 ± −
1915 − ± −
2016 ± − ±−
2117 − − −−
2218 − ± +−
(−) No cells with
19 neurite outgrowth; (±) cells with slight neurite
± outgrowth; (+) cells with apparent neurite
outgrowth; (cell20death) most of the ±
cells died. ±
21 − −
22 − +
(−) No cells with neurite outgrowth; (±) cells with slight neurite outgrowth; (+) cells with apparent
neurite outgrowth; (cell death) most of the cells died.
Molecules 2019, 24, 1366 5 of 10
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Figure
Figure 4.4. Effects
Effectsofofcompound
compound1313and andvitamin
vitaminE on neuro2a
E on neuro2acell cell
viability. Neuro2a
viability. Neuro2acells cells
pretreated with
pretreated
test compounds (compound 13 at the concentration of 1–10 µM or
with test compounds (compound 13 at the concentration of 1–10 µM or vitamin E at the vitamin E at the concentration of
10–50 µM) were exposed to 30 µM
concentration of 10–50 µM) were exposed H O
2 2 . The results represent the mean ± SEM (n = 5, different
to 30 µM H2O2. The results represent the mean ± SEM (n = culture).
Symbols areculture).
5, different significantly different
Symbols for the following
are significantly condition;
different vs. none-treated
for the following condition; cells
vs. (*** p < 0.001,
none-treated
Student’s t test), vs. 30 µM H O -treated cells ( ## p < 0.01, ### p < 0.001, one-way ANOVA followed by
2 2
cells (*** p < 0.001, Student’s t test), vs. 30 µM H2O2-treated cells ( p < 0.01, p < 0.001, one-way
## ###

the
ANOVADunnett’s multiple
followed comparison
by the Dunnett’stest).multiple comparison test).
2.4. Signaling Pathway Involved in Compound 13-Induced Neurite Outgrowth from Neuro2a Cells
2.4. Signaling Pathway Involved in Compound 13-Induced Neurite Outgrowth from Neuro2a Cells
Next, we characterized the cell signaling pathway functioning in the compound 13-induced
Next, we characterized the cell signaling pathway functioning in the compound 13-induced
neurite outgrowth. Over the past 2 decades, numerous studies have revealed the molecular
neurite outgrowth. Over the past 2 decades, numerous studies have revealed the molecular
mechanisms underlying the regulation of neurite outgrowth by various factors. The representative
mechanisms underlying the regulation of neurite outgrowth by various factors. The representative
pathway for neurite outgrowth is the activation of the PI3K/Akt and/or protein kinase A
pathway for neurite outgrowth is the activation of the PI3K/Akt and/or protein kinase A
(PKA)/mitogen-activated protein kinase (MAPK)/ERK signaling pathways [16–21]. We thus tested by
(PKA)/mitogen-activated protein kinase (MAPK)/ERK signaling pathways [16–21]. We thus tested
immunoblot analysis whether compound 13 had the ability to promote the phosphorylation of ERK1/2
by immunoblot analysis whether compound 13 had the ability to promote the phosphorylation of
and/or Akt in neuro2a cells. For this experiment, cells seeded in 6-well plates were maintained in a
ERK1/2 and/or Akt in neuro2a cells. For this experiment, cells seeded in 6-well plates were
normal-serum medium for 24 h and then in a low-serum medium for 24 h. Thereafter, the cells were
maintained in a normal-serum medium for 24 h and then in a low-serum medium for 24 h.
kept for 0.5–5 h in a low-serum medium containing 30 µM compound 13.
Thereafter, the cells were kept for 0.5–5 h in a low-serum medium containing 30 µM compound 13.
ERK1/2 is a component of the MAPK signaling cascade. This MAPK/ERK pathway is activated
ERK1/2 is a component of the MAPK signaling cascade. This MAPK/ERK pathway is activated
by a variety of extracellular agents, including growth factors, hormones, and also cellular stresses to
by a variety of extracellular agents, including growth factors, hormones, and also cellular stresses to
trigger cellular processes that include mainly proliferation and differentiation; and it also contributes
trigger cellular processes that include mainly proliferation and differentiation; and it also
to the control of a large number of other cellular processes, including synaptic plasticity, e.g., long-term
contributes to the control of a large number of other cellular processes, including synaptic plasticity,
potentiation (LTP) in hippocampal neurons [22,23]. As ERK2 (42 kDa), but not ERK1 (44 kDa), has been
e.g., long-term potentiation (LTP) in hippocampal neurons [22,23]. As ERK2 (42 kDa), but not ERK1
suggested to be the one mainly involved in neurogenesis and cognitive function [22], we analyzed the
(44 kDa), has been suggested to be the one mainly involved in neurogenesis and cognitive function
ratio of phosphorylated ERK2 (pERK2) to total ERK2 (ERK2) in the present study. Figure 5A shows
[22], we analyzed the ratio of phosphorylated ERK2 (pERK2) to total ERK2 (ERK2) in the present
that treatment with compound 13 apparently induced the phosphorylation of ERK2 in neuro2a cells
study. Figure 5A shows that treatment with compound 13 apparently induced the phosphorylation
when the cells were incubated in the presence of compound 13 (30 µM) for 3 h or more.
of ERK2 in neuro2a cells when the cells were incubated in the presence of compound 13 (30 µM) for
Akt, a serine/threonine-specific protein kinase, is known to be protein kinase B (PKB). Akt is
3 h or more.
a key downstream effector of PI3K, and the PI3K/Akt pathway is a signal transduction pathway
Akt, a serine/threonine-specific protein kinase, is known to be protein kinase B (PKB). Akt is a
that protects neurons against various forms of injury in the brain [24]. Phosphorylated Akt inhibits
key downstream effector of PI3K, and the PI3K/Akt pathway is a signal transduction pathway that
downstream targets, such as proapoptotic protein and caspases [25]. Figure 5B shows that treatment
protects neurons against various forms of injury in the brain [24]. Phosphorylated Akt inhibits
with compound 13 resulted in time-dependent phosphorylation of Akt in neuro2a cells when the cells
downstream targets, such as proapoptotic protein and caspases [25]. Figure 5B shows that treatment
were treated with compound 13 (30 µM).
with compound 13 resulted in time-dependent phosphorylation of Akt in neuro2a cells when the
cells were treated with compound 13 (30 µM).
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Figure 5. Effects
Figure of compound
5. Effects of compound 1313 ononactivation
activationofof extracellular signal-regulated
extracellular signal-regulated kinases
kinases (ERK)
(ERK) 1/2 1/2
(A) and Akt (B) in neuro2a cells. Neuro2a cells were treated with 30 µM compound 13 for 0.5, 3 5or 5 h;
(A)
Figure and5. Akt (B)
Effects in
of neuro2a
compound cells.
13 Neuro2a
on cells
activation were
of treated with
extracellular 30 µM compound
signal-regulated 13
kinasesfor 0.5,
(ERK) 3 or
1/2
h; and
and (A)
thenand then
equal equalin amounts
Akt amounts
(B) neuro2a of protein
cells.
of protein werewere
Neuro2a analyzed
cells
analyzed were by immunoblot
bytreated with 30 µM
immunoblot analysis. The results
compound
analysis. The results represent
13 for 0.5, 3 or 5 the
represent
h;the
andmean
then ± SEM
equal (n =
amounts 3, different
of protein culture).
were Significance
analyzed by difference
immunoblot in values
analysis. between
The
mean ± SEM (n = 3, different culture). Significance difference in values between compound 13-treated results compound
represent
13-treated
mean ± andSEMnon-treated (0 h) cells: * p <Significance
0.05, ** p <difference
0.01 (one-way ANOVA followed by the
and the
non-treated (0 h) (n = 3, different
cells: * p < 0.05, culture).
** p < 0.01 (one-way ANOVA in values
followed between
by compound
the Dunnett’s multiple
Dunnett’s and
13-treated multiple comparison
non-treated (0 h)test).
cells: * p < 0.05, ** p < 0.01 (one-way ANOVA followed by the
comparison test).
Dunnett’s multiple comparison test).
These results revealed that compound 13 had the ability to induce the phosphorylation of both
These results revealed that compound 13 had the ability to induce the phosphorylation of both
These
ERK1/2 results
and Akt. revealed
In order that compound
to confirm 13 had
whether the ability13
compound to induced
induce the phosphorylation
neurite of both
outgrowth through
ERK1/2
ERK1/2 andand
MAPK/ERK Akt. InInorder
Akt.
and/or ordertotoconfirm
PI3K/Akt signalingwhether
confirm whether
pathways, compound
compound 1313
we preincubated induced
induced neurite
the neurite
cells outgrowth
foroutgrowth
30 min withthroughthrough
specific
MAPK/ERK
MAPK/ERK and/or
inhibitors ofand/or
ERK1/2PI3K/Akt
PI3K/Akt signaling
signaling
(10 µM U0126), pathways,
pathways,
PI3K (10 µM we we preincubated
preincubated
LY294002) or PKA the the cells
(1 cells for
for 30and
µM H89) 30
minthenmin with
withincubated
specificspecific
inhibitors of
them with
inhibitors ERK1/2
of compound(10
ERK1/2 (10 13 µM U0126),
µMfor PI3K
48 h. PI3K
U0126), (10
These(10 µM
inhibitorsLY294002)
µM LY294002)themselves or
or PKA PKA
had (1
(1 no µM
µMeffect H89)
H89) on and
andthe
then then
formationincubated
incubatedof
themthem
withwith
neuritescompound
compound
(data 1313forfor
not shown). 4848h.h.6These
Figure These inhibitors
showsinhibitors themselves
themselves
that the blockade hadhad
of PI3K by no
no effect
effect
LY294002 on formation
on the
and the formation
the blockadeof of
of PKA
neurites
neurites (data by
(data
notH89
not reduced
shown).
shown). the neurite-promoting
Figure
Figure 6 6shows
showsthat
thatthe effect
the blockade
blockadeof compound
of
ofPI3K
PI3Kby 13. On theand
byLY294002
LY294002 other
and hand,
thethe
blockade the of
blockade
PKAofblockade
byPKA
H89by ofH89
reduced reduced
MAPK/ERK theU0126
by neurite-promoting
the neurite-promoting scarcelyeffect
affectedeffect
of the of compound
formation
compound 13.ofOn 13.
theOn
neurites. the hand,
These
other other hand,
results
the the
suggest
blockade of
that compound
blockade of 13-induced
MAPK/ERK by neurite
U0126 outgrowth
scarcely from
affected neuro2a
the cells
formation was
of being
neurites.
MAPK/ERK by U0126 scarcely affected the formation of neurites. These results suggest that compound regulated
These through
results suggestthe
that compound 13-induced
PI3K/Akt-mediated neurite
signaling outgrowth
pathway and not fromthe neuro2a cells was being
PKA/MAPK/ERK one.regulated through
Other various the
signal
13-induced neurite outgrowth from neuro2a cells was being regulated through the PI3K/Akt-mediated
PI3K/Akt-mediated
pathways such as c-jun signaling pathwaykinase
N-terminal and not(JNK) thehave
PKA/MAPK/ERK
been reportedone. to beOther various
involved signal
in neurite
signaling pathway and not the PKA/MAPK/ERK one. Other various signal pathways such as c-jun
pathways
outgrowthsuch as c-jun cells
of neuro2a N-terminal
[19,21].kinase
We will(JNK) have been
investigate reported
whether notto only
be involved in neurite
PI3K/Akt-mediated
N-terminal
outgrowthkinase
signaling of (JNK)
pathway,
neuro2a have
but alsobeen
cells other reported
We to
signaling
[19,21]. be investigate
involved
pathway(s)
will inwhether
neurite
underlie outgrowth
the compound of neuro2a
not only 13-induced cells [19,21].
neuronal
PI3K/Akt-mediated
We will investigate
signaling pathway,
differentiation whether not only PI3K/Akt-mediated signaling pathway,
but also other signaling pathway(s) underlie the compound 13-induced neuronal
or not. but also other signaling
pathway(s) underlie
differentiation the compound 13-induced neuronal differentiation or not.
or not.

Figure 6. Effects of compound 13 on activation of Akt in neuro2a cells. Neuro2a cells were treated
Figure 6. Effects
with
Figureeach of compound
inhibitor
6. Effects for 30 min
of compound onon
13and
13 activation
then ofofAkt
incubated
activation Aktinin
with neuro2a
0.5 cells.
cells.Neuro2a
µM compound
neuro2a cells
13 for 48
Neuro2a h. were
cells treated with
were treated
eachwith
inhibitor for 30 min
each inhibitor and
for 30 then
min andincubated withwith
then incubated 0.5 µM compound
0.5 µM compound forfor
1313 4848h.h.
Molecules 2019, 24, 1366 7 of 10

Many recent studies revealed that oxidative stress directly and/or indirectly results in neuronal
death [9] and neurodegeneration in the brain [26], and also plays a crucial role in various
neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple
sclerosis, and amyotrophic lateral sclerosis (ALS) [7,8]. In fact, edaravone, a free-radical scavenger, is a
clinical drug for the treatment of ischemic stroke [9].
On the other hand, recent studies also showed that factors regulating neurite outgrowth might be
targets for treating aging-induced or lesion-induced neurological dysfunctions, because aging/lesion is
associated with neuron atrophy and impaired sprouting [27]. Thus, attention has been recently drawn
to compounds promoting neurite outgrowth as being candidates of neuroregenerative drugs [28]. In
fact, compounds promoting neurite outgrowth reportedly enhance learning and memory of aged
subjects [21,29,30] or exhibit a potent antidepressant effect [31].
Taken together our findings suggest that compound 13 (3-ethoxy-1-hydroxy-8-methoxy-
2-methylcarbazole-5-carbaldehyde), by having both anti-oxidant ability together and neurogenetic
ability, might be beneficial for the treatment of neurological disorders. In fact, various series of
carbazole derivatives have been designed and synthesized as neuroprotective therapeutic agents for
AD [32], PD [33], ALS [34], ischemic stroke [35] or traumatic brain injury [36] and so on in recent years.
We are planning to investigate whether compound 13 has neuroprotective ability in vivo using various
disease model mice.

3. Materials and Methods

3.1. Chemical and Reagents

Carbazole derivatives were provided as follow; Compounds 2 (2-hydoxycaebazole: No. 322-55321)
and 4 (4-hydroxycarbazole: No. H1014) were purchased from FUJIFILM Wako Pure Chemical Corp.
(Tokyo, Japan) and Tokyo Chemical Ind. Co., Ltd., (Tokyo, Japan), respectively. Compounds 1,
3, 5, 6 (carazostatin), 20, 21, and 22 were synthesized by the method previously reported [6,37].
Compound 7 was synthesized by the method previously reported [38]. Compounds 8, 9, 10, 11, and 12
((±)carquinostatin A) were synthesized by the method previously reported [3]. Compounds 13, 14,
15, 16, 17, 18 (carbazomadurin A), and 19 ((S)-(+)-carbazomadurin B) were synthesized as reported
earlier [4].
All compounds were dissolved in dimethyl sulfoxide (DMSO) to yield 30 mM stock solutions.
LY294002 from Wako Pure Chemical Ind., Ltd. (Osaka, Japan), U0126 and H-89 from Calbiochem
Corp. (San Diego, CA USA), and vitamin E from Combi-Blocks Inc. (San Diego, CA, USA) were
also dissolved in DMSO. Db cAMP sodium salt was purchased from Sigma-Aldrich Company Ltd.
(St. Louis, MO, USA) and dissolved in phosphate-buffered saline (PBS).

3.2. Cell Culture

Neuro2a cells were maintained and cultured as previously described [39]. All cell culture materials
such as Dulbecco’s Modified Eagle Medium (DMEM), FBS, and antibiotics were purchased from
Thermo Fisher Scientific (Waltham, MA, USA). The final concentration of DMSO in all culture media
was below 0.1%.

3.3. Determination of Cell Viability

For the experiment described in Section 2.1, neuro2a cells were seeded in wells of a 96-well plate
at a density of 1 × 104 cells/well; whereas for the experiment described in Section 2.3, the cell density
was 2 × 103 cells/well. Cell viability was determined by the MTT assay as previously described [40].

3.4. Assessment of Neurite Outgrowth

Neuro2a cells were seeded in wells of a 24-well plate at a low density (5 × 103 cells/well).
Neurites were defined as cellular processes with lengths equivalent to 1 or more diameters of the
Molecules 2019, 24, 1366 8 of 10

cell body. The percentage of cells bearing neurites was calculated as the % of number of neurites
divided by the total number of cells examined. More than 100 cells were randomly chosen for counting
neurite-bearing cells.

3.5. Immunoblot Analysis

Neuro2a cells were seeded in wells of a 6-well plate at a density of 2.5 × 105 cells/well. The cell
extracts were prepared as previously described [39]. Equal amounts of proteins (20 µg) were separated
on SDS-polyacrylamide gels and electroblotted onto an Immuno-BlotTM PVDF membrane (Bio-Rad
Laboratories, Hercules, CA, USA). As primary antibodies, rabbit polyclonal antibodies against 44/42
ERK1/2, which recognize 44-kDa ERK1 and 42-kDa ERK2; phospho-44/42 MAPK (Thr202/Tyr204),
specific for phosphorylated ERK1/2 (pERK1/2); Akt; and phosphor-Akt (Ser473), which recognize
phosphorylated Akt (pAkt), were purchased from Cell Signaling Technology Inc. (Woburn, MA,
USA). As secondary antibody, alkaline phosphatase-linked anti-rabbit IgG (Cell Signaling) was used.
Immunoreactive bands were detected by use of the NCB/BCIP reagent (Roche Diagnotics GmbH,
Mannheim, Germany).

3.6. Statistical Analysis

All results were expressed as means ± SEM. Significant differences of experiments with two
groups were analyzed by the Student’s t-test. Experiments with three or more groups were subjected
to a one-way ANOVA followed by the Dunnet’s multiple comparison test. p < 0.05 was taken to be
statistically significant.

4. Conclusions
In conclusion, our present study revealed using neru2a cells that carbazole 13
(3-ethoxy-1-hydroxy-8-methoxy-2-methylcarbazole-5-carbaldehyde) had the ability to protect
neuro2a cells against H2 O2 -induced death and also to induce neurite outgrowth from these cells by
acting through, at least, the PI3K/Akt signaling pathway.

Author Contributions: Conceptualization, Y.F., A.S. and S.H.; Experiments, Y.F., A.S., M.Y., M.N.
(Makiko Nakaya), S.O. and M.N. (Mitsunari Nakajima); investigation, N.H.; resources, Y.H., T.C. and S.H.;
writing-original draft preparation, Y.F. and A.S.; writing-review and editing, Y.F., S.O., Y.H., T.C., N.H. and S.H.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 1–22 are available from the author T. Choshi.

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