Vol.1, No.2, 49-61 (2011)
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
Open Journal of Preventive Medicine
Anoikis induction by glycoprotein from Laminaria
japonica in HT-29 cells
Hiroe Go, Hye-Jung Hwang, In-Hye Kim, Taek-Jeong Nam*
Department of Food Science and Nutrition, Pukyong National University, Busan, Korea; *Corresponding author: namtj@pknu.ac.kr
Received 10 May 2011; revised 1 July 2011; accepted 20 July 2011.
We extracted a glycoprotein from the brown
alga Laminaria japonica (LJGP). We previously
demonstrated that LJGP induced apop- tosis in
HT-29 colon cancer cells via the Fas- and the
mitochondrial signaling pathway, and cell-cycle
arrest. However, its effect on the cell membrane
remained unknown. In this study, we identified
the involvement of matrix metalloproteinase
(MMP), integrin, and Epi- thelial (E)-cadherin in
LJGP-induced apoptosis in HT-29 cells. LJGP
treatment increased the expression and activity
of MMP-2 and MMP-9. Furthermore, LJGP de-
creased the expression of integrin αν, β3, β5, β6
and E- cadherin. Consistent with a decreased
expression of E-cadherin, LJGP inhibited the
Wnt signaling pathway. Moreover, activation of
downstream molecules of integrin, including
focal adhesion kinase (FAK), the Src family of
protooncogenic tyrosine kinases, extracellular
signal-related kinase (ERK), and phosphatidyl
inositol 3 kinase (PI-3K) were also decreased.
These findings suggest that LJGP-induced
apoptosis of HT-29 cells involves possible ECM
disruption and cell detachment, which are exe-
cuted principally through the activation of
MMPs and by a decrease of adhesion molecules,
contributing to a down-regulation of the PI-3K,
MAPK, and Wnt signaling pathways. Apoptosis
induced by ECM disruption or cell detachment
is also known as anoikis. We can say that LJGP
induces anoikis in HT-29 cells.
Keywords: Anoikis; Glycoprotein; Integrin;
Laminaria Japonica; MMP; Seaweed
Loss of cell anchorage, including cell-matrix anchor-
age and cell-cell anchorage, induces apoptosis. The
growth of most cells is anchorage-dependent. To main-
tain the survival of anchorage-dependent cells, contact
with components of the extracellular matrix (ECM) in
vivo or contact with hydrophilic, negatively charged sur-
faces in vitro are required. Therefore, in the absence of
ECM attachment, cell death is induced. This mode of
cell-death induction is known as detachment-induced
anoikis [1,2].
The term anoikis, a Greek word meaning “homeless-
ness,” is a specific type of apoptosis induced by detach-
ment from the ECM, loss of cell adhesion, or inappro-
priate cell adhesion [3-5]. When epithelial and endothe-
lial cells detach from the ECM, these cells normally un-
dergo apoptosis; thus, anoikis can suppress the expan-
sion of oncogenically transformed cells and allow the
maintenance of tissue-cell homeostasis [3-6]. Anoikis
has been demonstrated and studied in numerous genu-
inely adherent cell types [7]. In contrast to non-trans-
formed cells, cancer cells are notoriously able to resist
anoikis, and this property has been proposed to contrib-
ute to metastasis and new tumor growth beyond the
original environment of the cancer cells [8]. Unsurpris-
ingly, obtaining resistance to anoikis is a critical step in
the metastatic process [2]. Conversely, it may be said
that anoikis functions as a physiological barrier to me-
In this study, we focused on the action induced by
LJGP on the cell surface. On the cell surface, there are
cell-ECM and/or cell-cell anchorages, and these an-
chorages are dependent on the cell-surface receptors.
Integrins are cell-surface adhesion receptors composed
of heterodimeric complexes of noncovalently linked
subunits, which work alongside other proteins
such as cadherins, cell-adhesion molecules, and selectins
to mediate cell-cell and cell-matrix adhesive interactions
and communications [9]. The functional contribution of
integrins is essential for cell proliferation, survival, and
migration, as integrins recognize the major adhesive
ECM components, fibronectin and laminin [6]. Integrins
can sense mechanical forces arising from the ECM,
H. Go et al. / Open Journal of Preventive Medicine 1 (2011) 49-61
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
converting these stimuli to chemical signals capable of
modulating intracellular signal transduction to allow
rapid and flexible responses to changes in the environ-
ment [5].
Integrin-mediated extracellular signals stimulate a va-
riety of intracellular signaling events, including adaptor
proteins, such as focal adhesion kinase (FAK), integrin-
linked kinase (ILK), the Src family of protooncogenic
tyrosine kinases, and Shc, as well as mitogen-activated
protein kinase (MAPK) pathways, leading to extracellu-
lar signal-regulated kinase (ERK) activation [10]. Acti-
vated ERK translocates to the nucleus, where it activates
several transcription factors [11]. Binding of integrin to
ECM components also stimulates structural proteins,
such as phosphatidylinositol-3 kinase (PI-3K), leading to
activation of Akt and serves as a link between the ECM
and the cytoskeleton [11].
In several studies, degradation of the ECM by MMPs
has been shown to be a signal that can induce apoptosis
[12]. MMPs constitute a family of extracellular proteases
that degrade ECM components important under a variety
of normal and pathological conditions [13].
E-cadherin is a type I cadherin transmembrane glyco-
protein adhesion molecule that mediates the formation of
cell–cell adhesion junctions. In epithelial cells, E-cad-
herin is known to play an important role in the develop-
ment and maintenance of morphological integrity.
Therefore, loss of E-cadherin could have profound ef-
fects on epithelial-cell homeostasis [14]. E-cadherins
form intracellular complexes with members of the cat-
enin family, which link the E-cadherin to the actin cy-
toskeleton [15]. Besides playing an important role in
cell-cell adhesion, E-cadherin also plays a role in the
Wnt signaling pathway through association with β-cat-
enin. The Wnt signaling pathway plays important roles
during the development of many organ systems and tu-
morigenesis [15]. Deregulated Wnt signaling is a key
factor for the development of several human cancers,
including breast tumorigenesis and colorectal carcinoma
In the present study, we identified a novel action of
LJGP on the cell surface, especially in regulation of cell
anchorage. In addition, integrin-signaling pathways and
the Wnt-signaling pathway may be involved in LJGP-
induced apoptosis, that is, detachment-induced anoikis.
2.1. Preparation of LJGP
LJGP was prepared as described previously [16].
Briefly, L. japonica was purchased in the traditional
market of Gijang-gun, washed, and stored at –20˚C until
used. The sample (160 g) was cut into small pieces and
steeped in 1 L distilled water for 6 h at room temperature.
The aqueous extract was clarified by centrifugation to
remove insoluble materials. The extract was then filtered,
and three volumes of ethanol were added. The ethanol
extract was filtered, and ammonium sulfate was added to
precipitate the proteins. The filtrates were combined,
pelleted with ammonium sulfate, dissolved in distilled
water, dialyzed in distilled water overnight, and concen-
trated by rotary evaporation at 40˚C. A concentrated so-
lution was distributed into 1.5-ml tubes, lyophilized, and
stored at –70˚C until use. Proteins in the lyophilized
samples were separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) and
subjected to periodic acid-Schiff (PAS) staining (Gel-
Code®Glycoprotein Staining Kit; Pierce Co., Ltd.,
Rockford, IL) to determine the presence of glycoprotein.
The extracts were solubilized in double-distilled H2O for
use in the assays.
2.2. Cell Culture
HT-29 colon cancer cells (ATCC HTB-38) were ob-
tained from the American Type Culture Collection
(Rockville, MD). HT-29 cells were cultured in RPMI
1640 medium supplemented with 10% fetal bovine se-
rum (Hyclone, Inc., South Logan, UT), 100 U/ml peni-
cillin, and 100 mg/ml streptomycin and were maintained
in a humidified incubator at 37˚C under an atmosphere
of 5% CO2. The medium was replaced every two days.
2.3. Western Blot Analysis
Cells were cultured to 80% confluence at 37˚C. Cells
were then maintained in serum-free medium (SFM) for
12 h. After 12 h, cells were incubated in SFM containing
various concentrations (0 - 100 μg/ml) of LJGP for 24 h
and then harvested. To prepare whole-cell extracts, the
cells were washed with phosphate-buffered saline (PBS)
and suspended in a cell-signaling lysis buffer (20 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1% Triton X-100, 2.5 mM sodium pyrophos-
phate, 1 mM β-glycerophosphate) containing protease
inhibitor (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml
pepstatin A, 200 mM Na3VO4, 500 mM NaF, 100 mM
PMSF). Lysates were incubated at 4˚C for 30 min and
then clarified at 15,000 g for 10 min at 4˚C to remove
detergent-insoluble material. The soluble lysates were
recovered and used as whole-cell extracts.
For the preparation of cytosolic extracts, the cells
were washed with PBS, and then 1.0 ml trypsin-EDTA
solution was added until the cell layer dispersed. Cells
were harvested in PBS and resuspended in lysis buffer
(20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM
MgCl2) containing protease inhibitors. The solutions
were incubated for 30 min at 4˚C. During this period,
solutions were vortexed five times. The solutions were
H. Go et al. / Open Journal of Preventive Medicine 1 (2011) 49-61
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
centrifuged at 3500 g for 10 min at 4˚C. The super-
natants were transferred to new tubes and centrifuged at
1400 g for 15 min at 4˚C. The resulting supernatants
were the cytosolic fractions.
To prepare nuclear extracts, the cells were suspended
in 500 μl buffer A (10 mM Tris (pH 7.5), 5 mM MgCl2,
0.05% TritonX-100) containing protease inhibitors. After
incubation at 4˚C for 15 min, the mixtures were centri-
fuged at 12,000 g for 15 min. Released nuclei were re-
suspended in buffer B (10 mM Tris (pH 7.4), 5 mM
MgCl2) and buffer C (10 mM Tris (pH 7.4), 4 mM
MgCl2, 1 M NaCl) containing protease inhibitors and
incubated for 30 min at 4˚C. After centrifugation at
12,000 g for 15 min, 80% glycerol was added to the su-
pernatants to a final concentration of 20%.
To prepare cell membranes, cells were suspended in
extraction buffer (50 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 1% Triton X-100) containing protease inhibitors
and stored overnight at –70˚C. The cells were then de-
frosted and placed on ice. To lyse the cells, the suspen-
sion was repeatedly aspirated and expelled. Finally, the
lysates were incubated at 4˚C for 30 min and then clari-
fied by centrifugation at 30,000 g for 30 min at 4˚C. The
resulting supernatants were the cell membrane fractions.
Protein concentration was determined using a bicin-
chonic acid protein assay kit (Pierce Biotechnology).
The proteins in the extracts (100 g) were separated us-
ing 7.5% - 15% SDS-PAGE and transferred to a polyvi-
nylidene fluoride membrane (Millipore, Billerica, MA).
The membranes were blocked with 1% bovine serum
albumin in TBS-T (10 mM Tris–HCl, 150 mM NaCl [pH
7.5], 0.1% Tween 20) and then incubated with the indi-
cated antibodies: anti-MMP-9 (H-129; 1:1000), anti-
MMP-2 (H-76; 1:1000), anti-TIMP-1 (H-150; 1:1000),
anti-GAPDH (FL-335; 1:1000), anti-integrin αν (H-75;
1:1000), anti-integrin β3 (H-96; 1:500), anti-integrin β5
(H-96; 1:1000), anti-integrin β6 (H-110; 1:1000), anti-E-
cadherin (H-108; 1:1000), anti-
-catenin (C-18; 1:1000),
anti-c-myc (C-19; 1:1000), anti-cyclin D1 (H-295; 1:
1000), anti-phospho-ERK1/2 (E-4; 1:1000), anti-phos-
pho-p38 (D-8; 1:500), anti-JNK (D-2; 1:1000), anti-
phospho-JNK (G-7; 1:1000), anti-p38α/β (H-147; 1:
1000), anti-
-actin (C-2; 1:500), anti-PI 3-kinase p85α
(B-9; 1:1000), anti-phospho-Akt1/2/3 (Ser473-R; 1:1000),
anti-Akt1 (C-20; 1:1000), anti-c-Src (SRC2; 1:1000),
and anti-FAK (C-20; 1:1000) antibodies from Santa Cruz
Biotechnology (Santa Cruz, CA) and anti-ERK1/2 (1:
1000), anti-phospho-FAK (Tyr397; 1:1000), and anti-
phospho-Src Family (Tyr416; 1:1000) antibodies from
Cell Signaling Technology (Beverly, MA). The mem-
branes were then incubated overnight at 4˚C with gentle
shaking. The secondary antibody was a peroxidase-
conjugated goat anti-mouse or -rabbit antibody (1:10,000;
GE Healthcare Bioscience, Piscataway, NJ) and horse-
radish peroxidase-conjugated goat ExactaCruzTM A
(1:10,000) (Santa Cruz). The antibody-bound proteins
were visualized using an enhanced chemiluminescence
Western blotting kit (Amersham Biosciences, Piscataway,
2.4. Immunoprecipitation Analysis
Formation of E-cadherin and β-catenin was analyzed
in cytosolic extracts prepared as follows. Cells were
cultured to 80% confluence at 37˚C and then maintained
in SFM for 12 h. After 12 h, the cells were incubated
with SFM containing various concentrations of LJGP (0
- 100 μg/ml) for 24 h. Cytosolic extracts (1000 μg) were
incubated overnight with primary antibodies (anti-E-
cadherin) with gentle agitation at 4˚C. Protein A-agarose
beads (Sigma, St. Louis, MO, USA) were then added,
and the mixture was incubated for 2 h at 4˚C with gentle
agitation to capture the immunocomplex. The beads
were collected by centrifugation (2 min at 10,000 g) and
washed four times with buffer A. The beads were resus-
pended in 1 sample buffer and boiled to elute the im-
munocomplex. The eluted proteins were analyzed by
SDS-PAGE followed by Western blotting with anti-E-
cadherin (H-108; 1:1000) or anti-
-catenin (C-18; 1:
1000) antibodies (Santa Cruz, CA).
2.5. Gelatin Zymography
MMP-2 and MMP-9 activities were analyzed using
SDS-substrate gels by adding gelatin (1 mg/ml) to the
10% acrylamide separating gel. Cells in 6-well plates
were treated with SFM containing various concentra-
tions (0 - 100 μg/ml) of LJGP for 24 h. Tumor-condi-
tioned medium (TCM) was mixed with substrate gel
sample buffer (13.3% SDS, 40% glycerol, 42 mM Tris-
HCl, pH 6.5, 0.013% bromophenol blue) and loaded
onto the gel without prior boiling. Following electro-
phoresis, the gels were washed three times in 2.5% Tri-
tonX-100 for 10 min at room temperature to remove
SDS. The gels were then incubated at 37˚C overnight in
incubation buffer (50 mM Tris-HCl (pH 8.0), 5 mM
CaCl2). The gels were stained with 0.5% Coomassie
brilliant blue G-250 in 40% methanol and 7% acetic acid
for 1 h at room temperature, and de-stained in the same
solution without Coomassie blue. Gelatin-degrading en-
zymes were identified as clear bands against the blue
background of the stained gel.
3.1. Effects of LJGP on Cell Morphology
We previously revealed that L. japonica extracts con-
tain a glycoprotein of approximately 10-kDa mass by
PAS staining, and we named it LJGP [16]. LJGP inhib-
H. Go et al. / Open Journal of Preventive Medicine 1 (2011) 49-61
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
ited cell viability in a dose-dependent manner in HT-29
cells and induced apoptosis through the Fas-signaling
pathway, mitochondria pathway, and cell-cycle arrest
[17]. In this study, we focused on the action of LJGP on
the cell surface of HT-29 cells.
First, we examined the effect of LJGP on the mor-
phology of HT-29 cells. As shown in Figure 1, micro-
scopic analysis confirmed that LJGP treatment caused a
dose-dependent change in the morphology. LJGP-treated
cells appeared to lose contact with the plates as well as
with neighboring cells. These morphological changes are
features of anoikis in cells. The maintenance of survival
in anchorage-dependent cells such as endothelial cells
(ECs) is significantly dependent on cell-to-cell and cell-
to-ECM contacts [1]. During cell death, observed mor-
phological changes result, in part, from effects on cell-
to-cell and cell-to-ECM contacts. In cell death of rat
small-intestinal epithelial cells induced by both ischemia
and ischemia/reperfusion injury, the disruption of epithe-
lial cell–matrix interactions may play an important role
[18]. Therefore, we hypothesized that LJGP may disrupt
cell-to-cell and cell-to-ECM adhesive interactions and
inhibit anchorage-dependent cell proliferation, leading to
the anoikis.
3.2. LJGP Induces MMP Activation
MMPs, which belong to a large family of extracellular
proteases, are able to cleave ECM components, includ-
ing proteoglycans, laminin, fibronectin, gelatin, collagen,
entactin, tenascin, vitronectin, and many others [13]. The
degradation of the ECM proteins has been shown to be
important in a variety of normal and pathological condi-
tions, including cancer, by altering the integrity of the
ECM. Moreover, MMPs have been shown to affect the
anoikis of endothelial cells and epithelial cells [1]. We
presumed that MMP activation might play a role in
LJGP-induced anoikis. To confirm whether HT-29 cell
detachment after LJGP treatment was mediated by MMP
activation, we evaluated the expression level and enzy-
matic activity of MMP-2 and MMP-9 by Western blot-
ting and gelatin zymography, respectively. LJGP treat-
ment increased the expression level of MMP-2 and
MMP-9 in a dose-dependent manner (Figure 2(a)). By
Figure 1. Effects of LJGP on cell morphology. HT-29 cells
were treated with various concentrations of LJGP for 24 h.
After 24 h, the morphologies were photographed using phase-
contrast optics. Photographs were taken at a magnification of ×
contrast, expression of one of the major negative regula-
tors of MMP, the tissue inhibitors of metalloproteinases-
1 (TIMP-1), decreased in a dose-dependent manner
(Figure 2(a)). Next, we performed gelatin zymography
to assess the activities of MMP-2 and MMP-9 after
LJGP treatment. The results demonstrated that the en-
zymatic activity of MMP-2 and MMP-9 was increased
by LJGP in a dose-dependent manner (Figure 2(b)).
3.3. LJGP Reduces the Expression of
Integrin αν, β3, β5, β6 in HT-29 Cells
Both tumor epithelial cells and endothelial cells re-
quire attachment to the ECM for cell survival, cell pro-
liferation, and migration. Upon loss of adhesion, cells
undergo detachment-induced cell death. Integrins con-
tribute functionally to these essential processes [7]. The
anchorage of cells to the ECM is mainly mediated by
integrins. Because they transmit signals between cells
and the environment, integrins are central to a number of
Figure 2. LJGP induced activities of MMP-2
and MMP-9 in HT-29 cells. (a) Effects of
LJGP on the expression levels of MMP-2,
MMP-9, and TIMP-1. Cells were treated with
various concentrations of LJGP for 24 h.
Whole-cell extracts were prepared and ana-
lyzed by Western blot analysis using anti-
MMP-2, anti-MMP-9, and anti-TIMP-1 anti-
bodies. The quantitative analysis was based
on densitometry of each band. (b) Stimulation
of MMP-2 and MMP-9 secretion in HT-29
cells treated with LJGP. Cells were treated
with various concentrations of LJGP for 24 h.
Conditioned medium (CM) was prepared and
analyzed by zymography assay.
H. Go et al. / Open Journal of Preventive Medicine 1 (2011) 49-61
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biological processes, including intercellular adhesion,
maintenance of cell morphology, cell migration, regula-
tion of cell growth and differentiation, and progression
through the cell cycle [7,13,19].
Integrins consist of two transmembrane glycoprotein
, that are non-covalently bound. The
receptors always contain one chain and one
In this study, we determined the expression level of
four types of integrin, integrin αν, integrin β3, integrin
β5, and integrin β6, after treatment of HT-29 cells with
LJGP. The αν integrins pair with multiple β subunits (β1,
β3, β5, β6, and β8).
Western blot analyses confirmed that the amount of
these proteins decreased in a dose-dependent manner
after treatment with LJGP (Figure 3). Especially, in-
tegrins αν and β3 significantly decreased, and integrin β6
slightly decreased.
3.4. LJGP Reduces the Phosphorylation of
FAK and Src in HT-29 Cells
Integrins do not themselves possess a kinase domain
or enzymatic activity, but rely on association with other
signaling molecules to transmit signals. There are some
important key signaling molecules in integrin-mediated
signal transduction. One of them is FAK, which is a
non-receptor protein tyrosine kinase that colocalizes
with integrins in focal adhesions. Upon integrin ligation,
FAK is phosphorylated on five different tyrosine resi-
dues (Y397, Y407, Y576, Y577, and Y925), with tyro-
sine 397 being the major autophosphorylation site [20-
21]. We determined whether activation of FAK occurs
after treatment of HT-29 cells with LJGP. The phos-
phorylation of tyrosine 397 of FAK has been used as a
marker of FAK activity. As shown in Figure 4, phos-
Figure 3. LJGP decreased αν, β3, β5, and β6
integrin expression in HT-29 cells. Cells were
treated with various concentrations of LJGP
for 24 h. Cell membranes were prepared and
analyzed by Western blot analysis using anti-
integrin αν, -integrin β3, -integrin β5, and -in-
tegrin β6 antibodies. The quantitative analysis
was based on densitometry of each band and is
reported as a fold increase over the control.
phorylation of FAK decreased in a dose-dependent man-
ner. However, there were no changes in total FAK pro-
tein levels.
Besides integrins, many down-stream molecules are
known to interact with the Y397 autophosphorylation
site of FAK, such as c-Src, Shc, Csk, and PI-3K [19]. It
has also been reported that abolishing FAK activity
alone is not sufficient to abolish apoptosis resistance in
colon cancer cells; a concomitant inhibition of Src is
also required to do this [22]. Src is a member of the SRC
family of kinases (SFK), which consists of nine mem-
bers: Blk, Fgr, Fyn, Hck, Lck, Lyn, Src, and Yrk [6].
Within the SFK, Src is the best-known protein and plays
a significant role during cancer progression [23]. We
determined the levels of phosphorylation of Src follow-
ing exposure of cells to LJGP. As shown in Figure 4,
LJGP inhibited Src phosphorylation in a dose-dependent
3.5. LJGP Affects the PI3K Pathway
Integrin-induced phosphorylation of FAK tyrosine
397 creates a high-affinity binding site for the SFK via
their SH2 domains and also for the p85 regulatory sub-
unit of PI-3K [24]. PI-3K is a heterodimer lipid kinase
composed of a 110-kDa catalytic subunit and an 85-kDa
regulatory subunit, which contains two SH2 domains
and one SH3 domain. PI-3K phosphorylates phosphoti-
dylinositol at the D3 position of the inositol ring in a
variety of phosphoinositide substrates, forming 3-phos-
phorylated phosphoinositides. The PI-3K signal trans-
duction pathway regulates cancer survival, apoptosis,
and a number of diverse cellular processes such as
growth, proliferation, cell transformation, metastasis,
membrane trafficking, and cell survival [25]. Increased
Figure 4. Effects of LJGP on the activities of
FAK and Src. HT-29 cells were treated with
various concentrations of LJGP for 24 h. Whole-
cell extracts were prepared and analyzed by
Western blot analysis using anti-phospho-FAK
(Tyr397), -FAK, -phospho-Src family, and -C-
Src antibodies. The quantitative analysis was
based on densitometry of each band and is re-
ported as a fold increase over the control.
H. Go et al. / Open Journal of Preventive Medicine 1 (2011) 49-61
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PI-3K activity in cancers evokes down-regulating signals
through Akt/PKB. Akt is phosphorylated at two sites,
Thr308 in the kinase domain and Ser473 in the regula-
tory tail, by 3’-phosphoinositide-dependent kinase 1/2
(PDK1/2) and is fully activated when both residues are
phosphorylated [26-28]. Phosphorylated Akt regulates
the function of a broad array of intracellular proteins,
including proapoptotic and apoptotic factors, mTOR,
glycogen synthase kinase-3 (GSK-3), and p53, among
others, which are involved in fundamental processes,
including cell proliferation, cell survival, cell growth,
cell death, cell motility/adhesion, cell transformation,
neovascularization, and inhibition of apoptosis [26-28].
To determine whether the PI-3K pathways are associ-
ated with LJGP-induced apoptosis, we examined the
activation levels of p85 and Akt by Western blotting. As
shown in Figure 5, LJGP treatment significantly de-
creased the expression level of p85 in a dose-dependent
manner. By contrast, the total Akt protein level was un-
affected by different concentrations of LJGP. The phos-
phorylation level of Akt at Ser473 was also down-regu-
lated in correlation with the levels of p85.
3.6. LJGP Affects the Phosphorylation of
ERK1/2 and p38 MAPK in HT-29 Cells
As noted above, we showed that LJGP-induced apop-
tosis occurred via inactivation of FAK/Src and down-
regulation of the PI-3K/Akt pathway. Some studies have
reported that FAK/Src signaling can stimulate the PI-3K/
Akt and MAPK pathways, either individually or in com-
bination, depending on the cell type analyzed [29]. The
MAPK signaling cascade begins at the plasma mem-
brane via non-receptor tyrosine kinases such as integrin
or membrane-associated receptor tyrosine kinases such
as epidermal growth factor receptor (EGFR) [30]. The
Figure 5. Effects of LJGP on the activities of
p85 and Akt. HT-29 cells were treated with
various concentrations of LJGP for 24 h. Whole-
cell extracts were prepared and analyzed by
Western blot analysis using anti-PI3-kinase
p85α, -phsopho-Akt (Ser473), and -Akt anti-
bodies. The quantitative analysis was based on
densitometry of each band and is reported as a
fold increase over the control.
importance of the MAPK signaling pathway in cancer
cells has been shown in some studies. Integrins can ac-
tivate the MAPK pathway by coupling either to FAK
(through the β integrin subunit) or to Fyn/caveolin/Shc
(through the α integrin subunit) [31].
We determined that the MEK/ERK pathways are also
related to LJGP-induced apoptosis in HT-29 cells.
MAPKs include the ERK, the c-Jun N-terminal kinase
(JNK), and p38 MAPK. We assessed the phosphoryla-
tion levels of these three MAPKs. As shown in Figure 6,
the phosphorylation level of ERK was decreased only at
the relatively high concentration of 100 μg/ml LJGP.
Moreover, the phosphorylation level of JNK did not
change substantially at any concentration. In contrast to
ERK, the phosphorylation level of p38 was increased at
concentration of 50 and 100 μg/ml.
3.7. LJGP Treatment Down-regulates
E-cadherin and β-catenin
Cell anchorage is a remarkably complex process in-
volving several adhesion molecules. As noted above,
anchorage to the cell-matrix is mediated by the integrin
family. In this study, we focused on the cadherins, which
have been known to mediate anchorage between cells.
Cadherin molecules are composed of five extracellular
domains, one transmembrane domain, and a cytoplasmic
domain [32]. Through interaction of the cytoplasmic
domain with the intracellular catenin anchor proteins (β-
catenin and α-catenin), cadherins are linked to the actin
cytoskeleton [33]. Therefore, cadherins are able to form
intracellular adhesive complexes that are essential for
the maintenance of tissue integrity [32]. Upon the loss of
Figure 6. LJGP affected the activities of
MAPK. HT-29 cells were treated with various
concentrations of LJGP for 24 h. Whole-cell
extracts were prepared and analyzed by West-
ern blot analysis using anti-phospho-ERK,
-p44/42 MAP kinase, -phospho-JNK, -JNK,
-phospho-p38, and -p38α/β antibodies. The
quantitative analysis was based on densitome-
try of each band and is reported as a fold in-
crease over the control.
H. Go et al. / Open Journal of Preventive Medicine 1 (2011) 49-61
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
anchorage, modulation of E-cadherin-cytoskeleton com-
plexes might be involved in the apoptosis-signaling cas-
Three major catenins are known: α-catenin, β-catenin,
and γ-catenin (plakoglobin). β-catenin and γ-catenin di-
rectly bind to cadherin in a mutually exclusive manner,
whereas α-catenin binds to β-catenin or γ-catenin and
makes an additional direct or indirect contact to the actin
cytoskeleton, leading to strong cell-cell adhesion [34].
First, we determined the expression levels of E-cad-
herin and β-catenin after LJGP treatment of HT-29 cells.
As shown in Figure 7(a), LJGP decreased the expres-
sion level of E-cadherin and β-catenin in a dose-de-
pendent manner.
β-catenin associates with E-cadherin at cell–cell junc-
tions. Therefore, we next examined the binding level of
E-cadherin and β-catenin after LJGP treatment of HT-29
cells. To test this, E-cadherin was immunoprecipitated,
followed by immunoblotting with anti-β-catenin and E-
Figure 7. LJGP suppressed cell-cell anchorage.
(a) E-cadherin and β-catenin levels during
LJGP-induced anoikis. HT-29 cells were treated
with various concentrations of LJGP for 24 h.
Whole-cell extracts were prepared and ana-
lyzed by Western blot analysis using anti-E-
cadherin and anti-β-catenin antibodies. The
quantitative analysis was based on densitome-
try of each band and is reported as a fold in-
crease over the control. (b) LJGP interferes with
E-cadherin-β-catenin linkage formation. HT-29
cells were treated with various concentrations
of LJGP for 24 h. Whole-cell extracts were
prepared and immunoprecipitated with E-cad-
herin. Immunocomplexes were analyzed by
Western blot analysis using anti-E-cadherin
and β-catenin antibodies. The quantitative ana-
lysis was based on densitometry of each band
and is reported as a fold increase over the con-
cadherin antibodies. The level of E-cadherin-associated
β-catenin was reduced after 24 h of LJGP treatment in a
dose-dependent manner (Figure 7(b)). Furthermore, de-
creased amounts of E-cadherin protein were also de-
tected in the immunoprecipitates. Thus, β-catenin disso-
ciated from E-cadherin in response to the LJGP treat-
3.8. LJGP Down-Regulates Wnt Signaling
Cadherins not only provide anchorage between neigh-
boring cells, but they are also elements in the complex
network of the Wnt/Wingless survival-signaling pathway.
E-cadherin and β-catenin act as downstream components
of this pathway and play a crucial role [33]. Accumula-
tion of β-catenin stimulates activation of the Wnt/Wing-
less signaling pathway and its interaction with members
of the lymphoid enhancer factor (LEF)/T-cell factor
(TCF) family of transcription factors, leading to cellular
proliferation and possibly neoplastic transformation [7].
Therefore, an understanding of the mechanism that
regulates the function and expression of E-cadherin and
β-catenin is critical for the understanding of invasion,
metastasis, and proliferation of cancer cells. The devel-
opment of colon cancer might begin by aberrant active-
tion of Wnt signaling. β-catenin is a multi-functional
protein that plays essential roles both at adherens junc-
tions and in Wnt signaling. In the Wnt signaling pathway,
β-catenin plays a central role as a cotranscriptional acti-
vator of genes in the nucleus, together with LEF/TCF
[33]. In Figure 7, we determined the participation of β-
catenin at adherens junctions during LJGP-induced
apoptosis in HT-29 cells. Next, we investigated its in-
volvement as a component of the Wnt signaling pathway.
To investigate whether LJGP affected the Wnt signaling
pathway, we investigated the translocation of β-catenin
from the cytosol to the nucleus, and the expression levels
of Wnt signal target proteins, such as c-myc and cyclin
D1. After treatment of the cells with LJGP, we measured
the amount of β-catenin in the cytosol and nucleus. As
shown in Figure 8, LJGP slightly increased the amount
of cytosolic β-catenin. By contrast, nuclear β-catenin
decreased in a dose-dependent manner after treatment of
cells with LJGP. These results suggest that LJGP
blocked the translocation of β-catenin from the cytosol
to the nucleus. We therefore hypothesized that the ex-
pression level of the β-catenin target gene might also be
decreased. To confirm this hypothesis, we investigated
the expression level of c-myc and cyclin D1 by Western
blotting. As shown in Figure 7, both c-myc and cyclin
D1 significantly decreased after treatment of cells with
LJGP. These results suggest that Wnt signaling is related
to LJGP-induced anoikis in HT-29 cells.
H. Go et al. / Open Journal of Preventive Medicine 1 (2011) 49-61
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
Figure 8. LJGP reduced translocation of β-cat-
enin and expression of target proteins of Wnt
signaling. HT-29 cells were treated with various
concentrations of LJGP for 24 h. Whole-cell ex-
tracts, cytosol cell extracts, and nuclear cell ex-
tracts were prepared and analyzed by Western
blot analysis using anti-β-catenin, -c-myc, and
-cyclinD1 antibodies. The quantitative analysis
was based on densitometry of each band and is
reported as a fold increase over the control.
Natural marine products, like seaweeds, have recently
become the focus of increased research interest due to
their potential pharmacological activities [35]. Marine
organisms have evolved various functions to adapt to a
wide variety of environmental changes, including salt
density, water pressure, drying at low tide, and ultravio-
let radiation. As a result, many of them contain various
physiologically active substances that are known to
benefit health.
The brown alga L. japonica is commonly consumed in
Korea, Japan, and China, and less so in Europe and the
United States [36]. It is known to have many biological
activities, including an antioxidant effect, anti-mutagenic
activity, and antibacterial activity [12,37]. We assumed
that its medicinal effects were mainly due to the carbo-
hydrate or protein components, as L. japonica contains
60.9% carbohydrate and 10.3% protein [38]. Previously,
we extracted a glycoprotein from L. japonica (named
LJGP), which we demonstrated had apoptotic effects on
HT-29 cells [16,17]. LJGP inhibited the growth of HT-29
cells by arresting the cell cycle and activating apop-
tosis-related pathways, including the Fas-signaling path-
way and mitochondrial pathway [17].
In the present study, we examined the actions of LJGP
on the cell-surface membrane during LJGP-induced
apoptosis. As shown in Figure 1, LJGP treatment caused
a dose-dependent increase in morphological changes that
are characteristic of anoikis in cells. Anoikis is a form of
apoptosis involving disruption of cell-to-cell or cell-to-
ECM adhesive interactions at the cell-surface membrane.
We hypothesized that the LJGP-induced apoptosis of
HT-29 cells was due to cell detachment-induced death;
that is anoikis. To examine the mechanism of anoikis
after treatment with LJGP, we studied the degradation of
the ECM and cell-anchorage-related signaling pathway.
There are some reports that cell detachment is related
to anoikis. Doxazosin induced anoikis and affected cell
morphology in prostate cancer cells [39]. In DU-145
prostate cancer cells, cell detachment was induced by
methyl selenium as a prerequisite for anoikis [40]. In
human osteosarcoma cells, receptor activator of nuclear
factor κβ ligand (RANKL) inhibited malignancy and
evoked caspase-3-mediated anoikis [41]. It was reported
that 15-deoxy-Delta (12,14)-prostaglandin J (2) decreased
the adherence rate of BEL-7402 hepatocellular carci-
noma cells, leading to anoikis [42].
MMP is associated with degradation of the ECM and
is known to induce cell death [43]. It was reported that
MMP induced apoptosis of human endothelial cells [44].
Moreover, FOXO3a induced apoptosis of endothelial
cells through activation of MMP [1]. Furthermore, some
studies have indicated that TIMP-1 functions as a potent
inhibitor of apoptosis in mammalian cells [45]. It was
reported that caspase-mediated apoptotic pathways in-
duced by a variety of stimuli, including anoikis, stauro-
sporine exposure, hydrogen peroxide, x-ray irradiation,
growth factor withdrawal, and adriamycin treatment,
were down-regulated by TIMP-1-activated cell survival
signaling [44]. Moreover, downregulation of TIMP-1
expression enhanced apoptotic cell death. In the present
study, we observed that LJGP increased the activities of
MMP-2 and MMP-9 and decreased the activity of
TIMP-1 (Figure 2). Down regulation of TIMP-1 may be
the cause of the increased MMP-2 and MMP-9 activities.
It may be that activation of MMP and suppression of
TIMP-1 induces anoikis in LJGP-treated HT-29 cells.
Signal transduction from the extracellular environ-
ment to the intracellular network is mediated by integrin-
activated signaling molecules, such as FAK, PI3-K, ILK,
SHC, ERK, MAPK, EGFR, and caveolin. These many
helper molecules are necessary for the cell to react spe-
cifically to varying cues from the environment and from
within the cell, and to precisely modulate the cell’s be-
havior [19]. Loss of integrin-mediated epithelial cell–
ECM interactions causes loss of phosphorylation of
these downstream effectors, which are known to mediate
cell susceptibility to anoikis and a variety of biological
processes [6]. In the present study, we showed that LJGP
treatment caused a dose-dependent decrease in the ex-
pression levels of integrin αν, integrin β3, integrin β5,
and integrin β6 (Figure 3).
Because integrins are a kind of cell-surface adhesion
receptor, their decreased expression after treatment with
LJGP would weaken the adhesion of cells to the ECM,
thus inducing cell death. These results are expected from
H. Go et al. / Open Journal of Preventive Medicine 1 (2011) 49-61
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
the fact that both MMP-2 and MMP-9 are known to de-
grade a wide variety of proteins, including chemotactic
molecules, adhesion molecules, proteinase inhibitors,
cell-surface receptors, blood clotting factors, latent
growth factors, and growth factor-binding proteins [46].
The decreased expression of integrin could be the result
of a degradation of the ECM due to an increase in MMP
activity (Figure 2).
FAK controls a variety of biological processes, in-
cluding cell survival, spreading, invasion, proliferation,
motility, migration, and cell-cycle progression [19].
Disruption of FAK signaling in cancer cells induces cell
death after the induction of cell rounding and detach-
ment from the basal stratum. FAK has also been reported
to become over-expressed in tumor cells, indicating that
FAK activity is critical for resistance to apoptosis [47]. It
also been reported that over-expression of FAK occurs in
a number of human malignancies and that the degree of
over expression is correlated with greater aggressiveness
of the cancer [48,49]. In spite of the loss of cell anchor-
age, over-expression of active FAK blocks anoikis in
cells, thereby supporting the role of this kinase in anoikis
regulation [5]. In the present study, LJGP decreased the
phosphorylation levels of FAK without changing the
total FAK level (Figure 4). LJGP may have the potential
to reduce the phosphorylation of FAK, thereby inducing
anoikis in HT-29 cells.
As shown in Figure 4, LJGP also decreased the phos-
phorylation level of Src. Upon cell adhesion, ECM-in-
tegrin-mediated signals activate Src through FAK, and
the loss of adhesion results in inactivation of Src, leading
to anoikis. Several studies have reported the inactivation
of Src through FAK. Garcinol and disruption of caveolae
decreased not only the phosphorylation of FAK, but also
that of Src during anoikis [47,50]. We hypothesize that
LJGP treatment, by reducing FAK phosphorylation, may
cause down-regulation of Src in HT-29 cells, thereby
inducing anoikis.
Alteration of the PI-3K/Akt pathway has been de-
tected in various human cancers. Many studies have re-
ported that the PI-3K/Akt pathway is constitutively acti-
vated and plays an important role in tumor formation in
several cancers, including gastric, renal cell, ovarian,
and lung cancers. Therefore, it may be effective to de-
velop potential treatment agents that target this pathway.
In fact, some small molecule inhibitors, including in-
hibitors of PI-3K, Akt, and mTOR, have been developed
and/or approved as treatment agents for various human
cancers [28]. As shown in Figure 5, LJGP decreased the
PI-3K/Akt pathway. It is known that integrin-mediated
signals activate the PI-3K/Akt pathway and its subse-
quent signaling functions. Furthermore, it has been re-
ported that integrins induce Akt phosphorylation through
PI-3K [25,51]. The suppression of integrin expression by
LJGP treatment may, therefore, induce the down-regula-
tion of FAK, Src, and PI-3K/Akt survival signaling in
HT-29 cells.
LJGP also affected the MAPK pathway (Figure 6).
The MAPK pathway is highly activated during the late
progression of colorectal cancer and in primary tumors
of diverse origins [30]. In intestinal epithelial cancer
cells, anoikis resistance is mediated by the activation of
MEK/ERK [29]. In colorectal carcinoma, the MAPK
pathway is aberrantly activated [52]. Because MAPKs
are believed to be responsible for the proliferation, sur-
vival, cell-cycle progression, and metastasis of cancer
cells, inhibition of this pathway is a potential therapeutic
approach. Indeed, highly potent inhibitors of MAPK
activation have been shown to be capable of inhibiting
human cancer growth in immune-deficient mice [30]. In
the present study, we found that integrin/FAK/Src sig-
naling down-regulated both the PI3K signaling pathway
and the MAPK signaling pathway in LJGP-treated HT-
29 cells. LJGP might, therefore, be a promising new
therapeutic agent for the treatment of colon cancer.
In embryonic tissue and primary mouse mammary
gland epithelial cells, anoikis induced by blockage of
cadherin after treatment with blocking antibodies was
inhibited when cell-to-cell anchorage was preserved,
despite loss of the cell-to-ECM anchorage [7]. Also,
inactivation of E-cadherin through mutations caused cell
death in mice embryo. These reports demonstrate the
importance of cadherins in cell survival. In epithelial
cells, physical integrity and polarity are maintained
through intercellular junctions consisting of adherens
junctions, tight junctions, desmosomes, hemidesmo-
somes, and gap junctions [53]. E-cadherin is an impor-
tant component of adherens junctions, where E-cadherin
interacts with catenin that mediates binding to the actin
cytoskeleton. E-cadherin indirectly interacts with the
actin cytoskeleton, and this interaction is essential for
the stabilization and strength of cell-cell junctions. Cad-
herin engagement has been shown to protect squamous
carcinoma cells and normal proximal tubular cells from
anoikis [54]. Disruption of cell-cell anchorage may also
play a role in anoikis, because expression of a dominant
negative N-cadherin mutant in the intestinal epithelium
was shown to increase the frequency of apoptosis in
cells, while perturbing migration and differentiation [54].
Upon the loss of anchorage, normal enterocytes executed
a strikingly rapid apoptosis program [54]. During induc-
tion of apoptosis in endothelial cells, degradation of
VE-cadherin and β-catenin occurred [12]. NIH3T3 cells
grown in serum-free media exhibited a reduction of
cell-cell contacts during apoptosis [54]. In the present
study, disruption of E-cadherin and β-catenin appeared
H. Go et al. / Open Journal of Preventive Medicine 1 (2011) 49-61
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
to reduce cell-cell contact during LJGP-induced anoikis
in HT-29 cells (Figure 7). Interaction between β-catenin
and E-cadherin is a prerequisite for cell adhesion due to
the role of β-catenin in protecting the cytoplasmic do-
main of cadherin from rapid degradation, enhancing the
efficiency of transport from the endplas- mic reticulum
to the cell surface, and recruiting α-catenin to the sites of
cell-cell contact [55]. Although anoikis has been known
to be induced by the disruption of integrin-mediated
signaling, the disruption of E-cadherin signaling also
affects anoikis. There appears to be a connection be-
tween these two pathways. One report suggests the pos-
sibility that β-catenin may lie downstream from the in-
tegrins, and several integrin-stimulated signaling path-
ways might lead to the induction of β-catenin signaling.
Because the anti-apoptotic kinase Akt is known to inhibit
the activity of glycogen synthase kinase 3-β, a serine
kinase that functions directly to reduce β-catenin signal-
ing, it is expected that Akt is a connection between in-
tegrins and β-catenin [56]. A report that activated Akt
could be localized at cell–cell contacts also supports the
existence of this connection. E-cadherin-blocking anti-
bodies, as well as the calcium chelator EGTA, induced
apoptosis and loss of Akt activity in granulose cells [57].
E-cadherin bound to renal epithelial cells and promoted
survival in a PI-3K-dependent fashion [58]. Down-
regulation of E-cadherin resulted in changes of ανβ5 and
ανβ1 integrin expression and migration toward tenascin
[59]. We showed here that LJGP induced anoikis via
both integrin-mediated and E-cadherin-mediated signals.
Based on these results, we conclude that LJGP intri-
cately affected both cell-cell and cell-matrix contacts,
leading to anoikis in HT-29 cells.
There are some data to support the notion that cad-
herin-associated β-catenin promotes the carcinogenesis
process. First, β-catenin associates with and is down-
regulated by the tumor suppressor APC. Second, β-cat-
enin transduces, at least partly, the oncogenic Wnt
growth factor signal to the nucleus. Third, β-catenin is
mutated in a significant number of human cancers, and
overexpression of an NH2 terminally truncated form of
β-catenin in the epidermis of transgenic mice produced
well-differentiated hair tumors [56]. A dynamic pool of
β-catenin in the cytosol and nucleus is responsible for
the transduction of Wnt signals. Wnt proteins play criti-
cal regulatory roles in many biological processes, in-
cluding the development, maintenance, differentiation,
and regulation of embryonic and adult stem cells. Fur-
thermore, deregulation of Wnt signaling is associated
with multiple diseases, including various cancers. In the
absence of a Wnt signal, cytosolic β-catenin is captured
and phosphorylated by the cytoplasmic protein complex
called the β-catenin-destruction complex; this complex
includes APC, axin, GSK-3β, and CKI, which catalyzes
the phosphorylation of N-terminal serines and threonine
in β-catenin, thus labeling it for ubiquitin-dependent
degradation by the proteasome. In the presence of a Wnt
signal, the disruption of the complex containing APC,
axin, GSK-3β, and β-catenin is triggered, which prevents
the phosphorylation-dependent degradation of β-catenin,
causing β-catenin to accumulate in the cytosol. Accu-
mulated β-catenin is recruited to the promoters of Wnt-
response genes through its interaction with members of
the Tcf/LEF family of DNA-binding proteins, and to-
gether, they turn on the transcription of a plethora of
genes [60]. β-catenin has been known to significantly
protect cells from anoikis and is known as a potent in-
hibitor of apoptosis. Similarly, it may be said that LJGP
is a potent anti-cancer treatment.
In the nucleus, β-catenin stimulates the expression of
target genes of Wnt signaling, such as cyclin D1 and c-
myc, which have been implicated as having key roles in
oncogenesis. Both the cyclin D1 and c-myc proteins
contain TCF binding sites and appear to be constitutively
active in several colon cancer cell lines. In Figure 8, we
show that LJGP inhibited the translocation of β-catenin
from the cytosol to the nucleus and, consequently, inhib-
ited the expression of cyclin D1 and c-myc. LJGP-in-
duced anoikis might be associated with Wnt signaling.
Some reports have indicated that Wnt signaling is asso-
ciated with apoptosis in cancer cells. Resveratrol sup-
presses human colon cancer cell proliferation and ele-
vates apoptosis via suppression of the Wnt signaling
pathway [61]. Lignans inhibit cell growth via inhibition
of Wnt/β-catenin signaling [62]. Quercetin inhibits hu-
man SW480 colon cancer growth through Wnt/β-catenin
signaling [52]. In acute myeloid leukemia cells, apop-
tosis is induced by small molecule inhibitors of Wnt
signaling [63].
In conclusion, LJGP could qualify as a promising, in-
Figure 9. Signal transduction following loss of
cell anchorage, leading to anoikis.
H. Go et al. / Open Journal of Preventive Medicine 1 (2011) 49-61
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJPM/
novative treatment for colon cancer. LJGP-induced anoikis
of HT-29 cells involved cell detachment and possibly
ECM disruption, which were executed principally
through the activation of MMPs and a decrease of adhe-
sion molecules, contributing to down-regulation of the
PI-3K, MAPK, and Wnt pathways (Figure 9).
This research was a part of the project titled “Development for novel
biofunctional protein source from marine algae produced in the coastal
area of Busan,” funded by the Ministry of Land, Transport and Mari-
time Affairs, Korea.
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