Neuroscience Letters 448 (2008) 6266

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

The signaling cascades of Ganoderma lucidum extracts in stimulating

non-amyloidogenic protein secretion in human neuroblastoma SH-SY5Y
cell lines
Sirinthorn Pinweha a, , Payong Wanikiat a , Yupin Sanvarinda a,b , Porntip Supavilai a
a
Department of Pharmacology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
b
Center for Neuroscience, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Ganoderma lucidum (GL) is a medicinal mushroom that possesses various pharmacological properties
Received 23 July 2008 which are also documented in the ancient reports where GL is praised for its effects on the promotion
Received in revised form 6 October 2008 of health and longevity. In this study, we have investigated the effect of GL mycelia extracts on the non-
Accepted 8 October 2008
amyloidogenic protein secretion (sAPP) and the amyloid precursor protein (APP) expression in SH-SY5Y
neuroblastoma cells. In order to characterize the signaling pathway which mediates GL-enhanced sAPP
Keywords:
secretion, we used inhibitors of nerve growth factor (NGF) signaling pathways, phosphatidylinositol 3
Ganoderma lucidum
kinase (PI3K), phospholipase C1 (PLC1), protein kinase C (PKC) and extracellular signal-regulated kinase
Amyloid precursor protein
Nerve growth factor
(ERK1/2), to block GL-mediated sAPP secretion as well as ERK1/2 and PKC activation by using Western blot
Mitogen activated protein kinase analysis. Our results provided for the rst time evidence that GL mycelia extracts increased APP expression
Phospholipase C and promoted sAPP secretion. In addition, GL extracts activated ERK1/2 and PKC phosphorylation. The
Phosphatidyl inositol 3-kinase complex signaling cascades of PI3K and ERK may be responsible for GL-mediated sAPP secretion.
Protein kinase C 2008 Elsevier Ireland Ltd. All rights reserved.

Ganoderma lucidum (GL) has been considered a valuable medici-
nal mushroom in traditional Chinese medicine as a herbal tonic
that promotes longevity. Scientic information demonstrating
the effective delay in aging and extension of lifespan encom-
passes a variety of biological functions such as enhancing immune
function, antioxidation, cardiovascular support, lowering blood
pressure and serum cholesterol, antitumor and hepatoprotection
[31]. GL also has benecial effects on the central nervous sys-
tem. It has long been used for the treatment of insomnia and
neurasthenia in traditional Chinese medicine [31]. The presence of
neuroactive compounds in the extracts of GL that mediated the neu-
ronal differentiation of PC12 cells [7], neuroprotection from beta
amyloid (A) peptides exposure [15] or hypoxia/reoxygenation
injury [33] of rat cortical neuron has been investigated. Addi-
tionally, GL has benecial effects on the age-related impairment
of learning and memory in senescence-accelerated mice (SAMP8
strain) [29].
As an age-related disease, Alzheimers disease (AD) is the most
common form of degenerative dementia affecting the elderly cen-
tral nervous system. The disease involves abnormal processes of
the amyloid precursor protein (APP) with respect to the produc-
Corresponding author. Tel.: +66 2 997 2222x1413; fax: +66 2 997 2222x1417.
E-mail address: psirinthorn@hotmail.com (S. Pinweha).
tion, aggregation, deposition and toxicity of its A derivative [20].
Several factors, via multiple intracellular second messengers net-
work, are responsible for the regulation of APP processing [24].
Among these molecules, nerve growth factor (NGF) and its signaling
molecules play an important role [27]. NGF is also a crucial factor
in the functional impairments that occur in the aging brain and in
age-associated neurodegenerative disorders [32]. Extensive stud-
ies showed that the AD11 anti-NGF mice, an animal model for AD
based on the alterations of NGF signaling pathway, are a progressive
neurodegeneration closely resembling many features of AD [5].
The actions of NGF are mediated by Ras-extracellular signal-
regulated kinase (Ras/ERK), phospholipase C1 (PLC1) and
phosphatidylinositol 3 kinase (PI3K) pathways. Furthermore, pre-
vious studies demonstrated that protein kinase C (PKC) also has
important functions for growth factor-dependent events such as
gene regulation, growth control, and differentiation [8,10,12]. Acti-
vation of PKC is known to regulate APP processing, resulting in the
generation of non-amyloidogenic products (sAPP) via -secretase
activation [25]. Moreover, PKC has been shown to be involved in
learning and memory as well as in cognitive impairment [2]. Thus,
PKC might be considered an intracellular transduction that links the
cascade of abnormal events leading to AD. In the present study, we
proposed that GL extracts might possess NGF-like properties for the
processing of APP via an enhanced NGF signaling pathway. There-
fore, we determined the effect of GL extracts on expression and

0304-3940/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2008.10.028
 S. Pinweha et al. / Neuroscience Letters 448 (2008) 6266 63

metabolism of APP and explored downstream signaling cascade in

a SH-SY5Y neuroblastoma cell line.
The possible cytotoxic effect of GL extracts in SH-SY5Y was
examined rst. GL mycelia (G2 species: kindly provided by the
Royal Chitralada Projects) were cultivated by submerged cultivation
in potato dextrose broth (Difco Laboratories; Detroit, MI) at 28 C
for 28 days. The harvested mycelia were defatted with 95% alcohol
and extracted by autoclaving at 121 C for 20 min. The extract was
lyophilized and stored at 20 C for further experiments. Chemical
analysis revealed that GL extracts contained 69.4% carbohydrates
and 19.9% proteins. Our preliminary data showed that differentia-
tion of the SH-SY5Y cells induced by GL extracts was related to the
amount of proteins in the extracts. Therefore, the concentration of
GL extracts was based on the amount of proteins in each batch.
SH-SY5Y neuroblastoma cell lines (American Type Culture Col-
lection, Rockville, MD) were grown in equal parts of minimum
essential medium (MEM) and Hams F-12 supplemented with non-
essential amino acids, pyruvate and 10% heat-inactivated fetal
bovine serum (GIBCO Invitrogen corporation, Paisley, UK) at 37 C
in a 5% CO2 environment. Cells were treated with 1100 g/mL GL
extracts for 24 h. The numbers of viable cells were determined by
a colorimetric assay for mitochondrial function as described pre-
viously [22]. We found that GL extracts were not toxic to SH-SY5Y
cells in this concentration range (data not shown here).
We determined the effect of GL extracts-mediated sAPP secre-
tion by Western blotting of the proteins secreted into the medium.
Before any treatment, cells were kept in serum-free media for
1 h. After treatment of the cells with GL extracts in serum-free
medium for 2 h at 37 C, the secreted proteins were precipitated
[3], separated on 10% SDS-PAGE and electrotransfered to PVDF
membranes (Amersham Bioscience; Piscataway, NJ). After block-
ing with 5% non-fat dried milk for 30 min, membranes were washed
and then incubated overnight at 4 C with a monoclonal antibody
6E10 (Chemicon, Temecula, CA; 1:1000). The antigenantibody
complexes were detected by horseradish peroxidase-conjugated
goat anti-mouse IgG after exposure to HyperlmTM ECL using an Fig. 1. Effects of GL extracts on sAPP secretion and APP expression in SH-SY5Y cells.
enhanced chemiluminescence (ECL plus) detection system (Amer- Cells were treated with GL extracts (0.550 g/mL) for 2 h. (A) Western blot analysis
sham Bioscience; Piscataway, NJ). The intensity of the visual band of sAPP secreted into culture media. The relative density of immunoreactive bands
was quantitated by densitometric scanning (Image Master Total Lab was calculated as a percentage of the control after being normalized to the total cell
lysate protein concentration. (B) Western blot analysis of cellular full length APP.
version 1.0). Results were expressed as the percentage of control. The relative density of immunoreactive bands was calculated as a percentage of
Statistical analysis was performed by using the computer software control after being normalized to GAPDH level of each lane. H1 represents the high
program PRISM for Windows. One-way ANOVA with Dunnetts molecular weight of cellular APP. H2 represents the low molecular weight of cellular
multiple comparison test was used for the comparison of means APP. Nerve growth factor (NGF 100 ng/mL), benzolactam derivative (BL 1 M), and
retinoic acid (RA 10 M) were used as a positive control. Data are presented as
among the different groups. Differences were considered to be sig-
percent of the control. Values represent mean S.E.M. of four to six independent
nicant at p < 0.05. experiments (*p < 0.05, **p < 0.01, ***p < 0.001 compared to the control). Ctrl: Control.
As shown in Fig. 1A, Western blotting of conditioned media from
SH-SY5Y cells treated with GL extracts revealed an enhancement
of secreted sAPP. One micrograms per milliliter of GL extracts Protein samples (2030 g) were separated by SDS-PAGE (10%),
signicantly increased sAPP secretion to 250% of the basal level immunoblotted and detected with a polyclonal antibody specic
following 2 h of exposure. Under the same conditions, the positive to COOH-terminal of APP (Zymed Laboratories, South San Fran-
control benzolactam (BL: 1 M), which is known as a PKC activa- cisco, CA, 1:1000). The results showed two immunoreactive bands
tor and able to efciently enhance non-amyloidogenic APP [13], (Fig. 1B) with a molecular weight of 130 and 110 kDa, which repre-
caused stimulation of sAPP secretion by 825.89 166.12%. NGF sented the full-length APP 751/770 and APP 695 in SH-SY5Y cells
(100 ng/mL) and retinoic acid (RA: 10 M) also stimulated sAPP [30]. GL extracts signicantly increased the intensity of the two
secretion by 519.12 113.03% and 578.67 69.64%, respectively. bands of APP expression from 140% (1 g/mL; p < 0.05) to 170%
To explore whether GL-enhanced sAPP secretion was a result (50 g/mL; p < 0.01) in a concentration-dependent manner. The
of an increase in cellular APP expression, we detected the level degree of GL-mediated APP expression was about 160%, which
of APP from treated cells by Western blotting. The cells were was lower than that of GL-mediated sAPP secretion (250%). This
washed once with phosphate buffer saline, lysed with a lysis suggests that GL-enhanced sAPP secretion was attributed to the
buffer (Cell Signaling Technology, Beverly, MA) containing 1 mM activation of an -processing pathway rather than an enhance-
phenylmethylsulphonyluoride, 1 g/mL aprotinin and complete ment of APP expression. The level and pattern of APP expression
protease inhibitor cocktail (Roche Applied Science; Indianapolis, induced by GL extracts at the dose of 50 g/mL were compara-
IN) and centrifuged at 10,000 g for 5 min at 4 C. The amount of ble to those after 100 ng/mL NGF. However, BL (1 M) and RA
proteins in the supernatant was determined using a Bio-Rad DC (10 M) signicantly increased only the low molecular mass of APP
Protein Assay Kit. (p < 0.01).
64 S. Pinweha et al. / Neuroscience Letters 448 (2008) 6266

Fig. 2. Effects of GL extracts on the phosphorylation of ERK1/2 (A) and PKC (B) in SH-
SY5Y neuroblastoma cells. Cells were treated with GL-extracts (0.550 g/mL) for
2 h. The phosphorylation was detected in the cell lysates. Data are presented as the
percent of the control. Values represent a mean S.E.M. of four to six independent
experiments (*p < 0.05, **p < 0.01 compared to the control). pERK1 represents the
high molecular weight of pERK. pERK2 represents the low molecular weight of pERK.
Crtl: control, BL: benzolactam derivative, NGF: nerve growth factor, and RA: retinoic
acid.

The previous studies demonstrated that the ERK1/2 signal-

ing molecule regulated the secretion of sAPP, either by a
PKC-dependent or independent pathway [9,21]. Therefore, we
determined whether GL-enhanced sAPP secretion was mediated
by these signaling molecules. Activated ERK1/2 and total ERK were
detected with a polyclonal antibody specic for the phospho-
p44/p42 MAP kinase and anti-p44/p42 MAP kinase, respectively
(Cell Signaling, Beverly, MA; 1:2000). Activated PKC was detected by
phospho-pan PKC polyclonal antibody (Cell Signaling, Beverly, MA; Fig. 3. Effects of inhibitors on GL-enhanced sAPP secretion (A) and on the phos-
1:1000). The results showed that all positive controls (BL, NGF and phorylation of ERK1/2 (B) and PKC (C) in SH-SY5Y neuroblastoma cells. Cells were
RA) and GL extracts (0.550 g/mL) signicantly activated ERK1/2 preincubated for 30 min with indicated inhibitors, followed by 2 h in the presence
or absence of a 50-g/mL GL-extracts. The sAPP secretion was detected in the
(Fig. 2A), while only BL (1 M) and the highest concentration of
conditioned media. The phosphorylation was detected in the cell lysates. Data are
GL extracts (50 g/mL) signicantly activated PKC by 159.1 14.9% presented as percent of the control. Values represent a mean S.E.M. of four to six
and 153.1 10.5%, respectively (Fig. 2B). independent experiments (*p < 0.05, **p < 0.01 compared to the control: # p < 0.05,
##
To characterize whether GL-enhanced sAPP secretion is simi- p < 0.01 compared to the GL-treated cells). Crtl: control, PD: PD98059, Go: G
larly mediated via NGF signaling pathways, specic NGF signaling 6976, LY: LY249002, and U: U73122.

inhibitors were added 30 min before the addition of GL extracts.

The results showed that 10 M PD98059 (an ERK1/2 inhibitor)
and 30 M LY249002 (a PI3K inhibitor) signicantly decreased GL-
enhanced sAPP secretion by approximately 40%, whereas 5 M
U73122 (a PLC1 inhibitor) did not alter GL-enhanced sAPP secre-
tion. Surprisingly, G 6976 (a specic calcium dependent PKC
inhibitor) alone stimulated sAPP secretion to the same level as
GL treatment, whereas other inhibitors had no effect on the basal
sAPP secretion (Fig. 3A). To gain more information on the path-
way mediating the effect of GL, we determined the levels of ERK1/2
and PKC phosphorylation in the cells treated with NGF signaling
inhibitors. The results showed that U73122, which inhibits ERK1/2
phosphorylation, or G6976, which inhibits PKC phosphorylation,
 S. Pinweha et al. / Neuroscience Letters 448 (2008) 6266
did not decrease GL-enhanced sAPP secretion. On the other hand,
PD98059 or LY249002, which simultaneously inhibits ERK1/2 and
PKC phosphorylation, decreased the secretion (Fig. 3). We also
found that G 6976 activated ERK1/2 both in the basal condition
and under GL treatment (Fig. 3B), which correspond to its effect on
sAPP secretion.
The current experiment provided evidence for the rst time
that the extracts from GL mycelia promoted sAPP secretion in the
SH-SY5Y human neuroblastoma cell lines. The secretion was inhib-
ited by the inhibitors of PI3K and ERK1, but not PLC. In addition,
GL extracts activated ERK1/2 and PKC phosphorylation. Under our
experimental conditions, GL-enhanced sAPP secretion was not
only due to an increase in APP expression but also a result of the
-secretase activation. The complex signaling cascades of PI3K and
ERK may be responsible for GL-mediated sAPP secretion.
Among multiple signal transduction molecules, PKC is the rst
molecule that was identied to regulate APP metabolism. Surpris-
ingly, the inhibition of PKC by G 6976 increased the secretion
of sAPP both in the basal condition and under GL treatment.
These data do not support the role of PKC in GL-enhanced sAPP
secretion in our experiment. However, PKC is a heterogeneous
family of phospholipid-dependent kinases that can be divided, on
the basis of cofactor requirements, into three categories: calcium-
dependent PKC isoforms, calcium-independent PKC isoforms and
atypical PKC isoforms. Since G 6976 employed in this study is a
specic inhibitor for calcium-dependent PKC isoforms, other iso-
forms that are expressed in this cell line [14] may be involved in
GL-enhanced sAPP secretion. Support for this suggestion is pro-
vided by the observation that PKC, a calcium-independent PKC
isoforms, plays a role in the regulation of APP metabolism [16].
PKC is a downstream signaling molecule of diacylglycerol pro-
duced from PLC1 activation. Our results showed that the inhibition
of PLC1 did not signicantly antagonize GL-activated PKC phos-
phorylation and sAPP secretion, whereas the inhibition of PI3K
did. In addition, inhibition of PI3K also antagonized GL extracts
activated-ERK1/2 phosphorylation. These data suggest that the
action of GL extracts occur via PI3K signaling pathway. The results
also demonstrated the relative importance of the ERK1/2 signaling
pathway in GL-enhanced sAPP secretion. ERK1/2 is regarded as
the most important signaling molecule for the regulation of APP
processing that is regulated either by a PKC-dependent or inde-
pendent pathway [9,21]. It has been reported that the activation of
ERK1/2 alone is sufcient to promote sAPP secretion [26].
Our result showed that inhibition of ERK1/2 decreased GL-
activated ERK1/2 and PKC phosphorylation, and sAPP secretion.
Taken together, these data suggest that the action of GL extracts
may occur via complex signaling pathways of PI3K, PKC and
ERK1/2. These results are in agreement with other observations.
Inhibition of PI3K inhibited muscarine-enhanced ERK1/2 acti-
vation and sAPP secretion in SH-SY5Y cells [4] and inhibited
bradykinin-provoked calcium-independent PKC translocation and
ERK1/2 stimulation in MCF-7 breast cancer cells [11]. On the other
hand, it is important to mention that GL extracts may contain more
than one factor affecting sAPP secretion or these signaling path-
ways in different ways. Therefore, it remains uncertain whether
the concerted action of these signaling molecules is required for
the effects on sAPP secretion.
Several lines of evidence have demonstrated that RA and NGF
increase the expression of APP, and that activation of sAPP secre-
tion along with the induction of neurites extension are related
to the activation of PI3K, ERK1/2, and PKC signaling molecules
[1,18,28]. The physiological functions of APP and sAPP derivatives
are involved in neurotrophic and neuroprotective events [19] and
modulate functions such as synaptic formation and repair [17,23].
sAPP enhances the transdifferentiation of adult bone marrow pro-
65
genitor cells to neuronal phenotypes [6]. Moreover, a report showed
that GL extracts from mycelia could induce morphological changes
in PC12 cells during differentiation [7]. In this regard, we com-
pared the differentiation effect of GL extracts in SH-SY5Y cell with
those of NGF (100 ng/mL) or RA (10 M) by observing morpholog-
ical changes under a phase-contrast microscope during 7 days of
treatment. The result showed that the proportion of SH-SY5Y neu-
roblastoma cells with neurites appeared on day 4 with 50 g/mL
of GL extracts. The morphology was similar to NGF-treated cells. In
addition, we also observed a tiny branching that extended from
the GL-treated cells. However, cells did not markedly change in
morphologic differentiation compared to those observed in the RA-
treated cells (data not shown). These data suggest that GL might
possess a property that mimics the action of NGF.
The present results suggest a neurotrophic effect of GL extracts
that could be linked to some of the historical uses of GL in promot-
ing longevity with scientic data. Further studies on the isolation,
characterization and the health-promoting mechanism of the GL
extracts are necessary to further clarify this.
Acknowledgements
This work was supported by the National Science and Technol-
ogy Development Agency (NSTDA) and a Research Assistantship
from the Faculty of Graduate Studies, Mahidol University to SP.
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