Anoikis induction by glycoprotein from <i>Laminaria japonica</i> in HT-29 cells

doi:10.4236/ojpm.2011.12008

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Vol.1, No.2, 49-61 (2011)

doi:10.4236/ojpm.2011.12008

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.

ABSTRACT

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

1. INTRODUCTION

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-

tastasis.

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



and



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

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

[14].

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. MATERIALS AND METHODS

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

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,

NJ).

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. RESULTS

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

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 ×

400.

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

(a)

(b)

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

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

subunits,



and



, that are non-covalently bound. The

receptors always contain one  chain and one



chain.

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

manner.

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

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

anchorage, modulation of E-cadherin-cytoskeleton com-

plexes might be involved in the apoptosis-signaling cas-

cade.

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-

(a)

(b)

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-

trol.

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-

ment.

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

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.

4. DISCUSSION

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

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

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

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).

5. ACKNOWLEDGMENTS

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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