Cysteine-Rich61(CCN1)EnhancesChemotactic … · (T2tumor; Fig. 2B) patterns of Cyr61 and...

Cysteine-Rich 61 (CCN1) Enhances ChemotacticMigration, Transendothelial Cell Migration, andIntravasation by Concomitantly Up-RegulatingChemokine Receptor 1 and 2

Been-Ren Lin,1 Cheng-Chi Chang,2 Li-Ro Chen,2 Ming-Hsun Wu,1 Ming-Yang Wang,1

I-Hsin Kuo,2 Chia-Yu Chu,2 King-Jen Chang,1,3 Po-Huang Lee,1 Wei-Jao Chen,1

Min-Liang Kuo,2,3 and Ming-Tsan Lin1,4

1Department of Surgery, 2Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology,College of Medicine, 3Angiogenesis Center, and 4Department of Primary Care Medicine,National Taiwan University, Taipei, Taiwan

AbstractCysteine-rich 61 (Cyr61; CCN1) plays an important role

in tumor development and progression in many kinds of

human malignancies. Here, we further show the

enforced expression of the Cyr61 gene or treatment with

recombinant Cyr61 protein enhanced expression of

chemokine receptors CXCR1 and CXCR2 in gastric

cancer AGS cells. Attenuation of Cyr61 levels in MKN-45

cells by transfecting with antisense Cyr61 significantly

reduced the level of CXCR1 and CXCR2. It is suggested

that Cyr61 tightly regulates the downstream genes

CXCR1 and CXCR2 in gastric cancer cells. Supportively,

reverse transcription–PCR and immunohistochemical

analysis of human gastric adenocarcinoma showed

that there was a high correlation between the

expression level of Cyr61 and CXCR1/CXCR2. The

up-regulated functionality of CXCR1 andCXCR2 in

Cyr61-overexpressing AGS cells could facilitate their

chemotactic migration toward interleukin-8, a

physiologic ligand of CXCR1 and CXCR2. In addition,

the Cyr61-mediated up-regulation of CXCR1/CXCR2 also

contributed to transendothelial migration, as well as

intravasation in a chick embryo model. Pharmacologic

and genetic approaches revealed that phosphoinositide

3-kinase (PI3K)/Akt, but not extracellular signal-regulated

kinase 1/2 or p38, signaling pathway is requisite for the

up-regulation of CXCR1/CXCR2 mRNA and protein

induced by Cyr61. Function-neutralizing antibody to

integrin AvB3, but not A2B1, effectively abolished

Cyr61-elicited Src activation and the subsequent PI3K/

Akt pathway. Antagonists toward integrin AvB3, Src

kinase, and PI3K/Akt not only suppressed CXCR1/

CXCR2 elevation but also blocked chemotactic

migration induced by Cyr61. In conclusion, we suggest

that Cyr61 promotes interleukin-8–dependent

chemotaxis, transendothelial migration, and

intravasation by induction of CXCR1/CXCR2 through

integrin AvB3/Src/PI3K/Akt–dependent pathway.

(Mol Cancer Res 2007;5(11):1111–23)

IntroductionGastric cancer is the second most common cause of cancer

mortality in the world behind lung cancer (1). It has been

estimated that there will have been >870,000 deaths from the

disease in the year 2000, accounting for f12% of all cancer

deaths (2). Approximately 65% of patients with gastric

carcinoma have regional or distant metastases at the time of

diagnosis. Despite the aggressiveness of surgical and chemo-

therapeutic treatment, the five-year survival rate in metastatic

and advanced stages is extremely poor, ranging from 5% to

15% (3). We have devoted to surgery and perioperative care for

gastric cancer patients, and have gained good perioperative

results (4, 5). However, a better understanding of the

progression of the disease would provide for relevant

development of therapies designed to prevent tumor invasion

and metastasis. Although a number of molecules have been

implicated in the metastasis of gastric cancer cells, the precise

mechanisms determining the directional migration and invasion

of cancer cells into specific organs remain to be established.

There is emerging evidence demonstrating the contribution of

chemokine receptors to organ-specific metastasis, which

provides important clues about why some cancers metastasize

to specific organs (6-8).

Chemokine receptors are G-protein coupled, seven-

transmembrane receptors (9). Based on the chemokine class

they bind, the receptors have been named CXCR1, 2, 3, 4 and

5 (bind CXC chemokines); CCR1 through CCR9 (bind CC

chemokines); XCR1 (binds the C chemokine, Lptn); and

CX3CR1 (binds the CX3C chemokine, fractalkine or neuro-

tactin). More than 50 chemokines have been discovered thus

far, and there are at least 18 human seven-transmembrane-

domain chemokine receptors (10, 11). Studies of human cancer

biopsy samples and mouse cancer models show that chemokine

Received 9/7/06; revised 7/8/07; accepted 7/13/07.Grant support: National Science Council of Taiwan (NSC 93-2320-B-002-019,93-2323-B-002-007, 95-2314-B-002-175).The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Ming-Tsan Lin, Department of Primary Care Medicineand Surgery, National Taiwan University Hospital, No. 7, Chung-Shan S. Road,Taipei 100, Taiwan. Phone: 886-2-23123456-5732; Fax: 886-2-23412969.E-mail: [email protected] D 2007 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-06-0289

Mol Cancer Res 2007;5(11). November 2007 1111on April 8, 2020. © 2007 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

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receptor expression of cancer cells is associated with increased

metastatic capacity. For example, CXCR4 and/or CCR7 are

commonly expressed in a wide variety of cancers and in cancer

cells, including breast (7), prostate (12) and ovarian cancer (13),

neuroblastoma (14), and certain types of leukemia (15). The

function of CXCR4 and CCR7 is required for tumor cell

invasion and metastasis (16). CXCR1 and CXCR2 are also

found to be expressed in prostate cancer (17), melanoma (18),

pancreatic cancer (19) and colorectal cancer (20), and their

expression levels are positively correlated with the more

aggressive and advanced stage of these cancers. Above all,

chemokine receptors might be involved in selective metastasis

of cancer cells to specific body sites with high-level expression

of their ligands.

FIGURE 1. Induction of CXCR1 and CXCR2 chemokine receptors by Cyr61. A. Top, the transcripts of chemokine receptors in wild-type AGS cells andstably transfected with Cyr61-expressing plasmid, including mix population (AGS/Cyr61 mix), single clones (AGS/Cyr61-1, AGS/Cyr61-5, AGS/Cyr61-7), andcontrol vector (AGS/neo) by RT-PCR analysis. h-Actin was used as a loading control of RT-PCR. Bottom: Western blot analysis of total cell lysates probedwith Cyr61, CXCR1, CXCR2, CXCR4, and a-tubulin. a-Tubulin was used as a loading and transferring control. B. Top, Western blot was used to detect theprotein levels of Cyr61, CXCR1, and CXCR2 in different gastric cancer cell lines. a-Tubulin was used as an internal control of protein loading and transferring.Bottom, the protein levels of Cyr61, CXCR1, CXCR2, and CXCR4 in Cyr61 or antisense Cyr61 (Cyr61-AS ) transient transfectants. After transientlyexpressing Cyr61 in various dosages in AGS cells (left ) and antisense Cyr61-expressing plasmids in MKN45 cells (right ) for 48 h, total cell protein wasextracted and Western blotting analysis was done. C. Top, the CXCR1 and CXCR2 mRNA expression by conditioned medium treatment of Cyr61transfectants as detected by RT-PCR analysis. Cyr61 neutralizing antibody (10 Ag/mL) or control IgG was placed with conditioned medium to depleteCyr61 protein existence. Bottom, the protein levels of CXCR1 and CXCR2 were analyzed per sample, and a-tubulin was used as internal control. D. Top,rCyr61 stimulated AGS cells for 12 h, and the CXCR1 and CXCR2 protein expression was detected by Western blot analysis. Bottom, rCyr61 (40 ng/mL)treated AGS cells as indicated time, and total cellular lysates probed with specific CXCR1 and CXCR2 antibodies. a-Tubulin was used as internal control.

Lin et al.

Mol Cancer Res 2007;5(11). November 2007

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Cysteine-rich 61 (Cyr61) is the first cloned member of

the CCN family (21), which comprises connective tissue

growth factor, Cyr61 (CCN1), nephroblastoma overexpressed

(Nov/CCN3), Wisp-1/elm1 (CCN4), Wisp-2/rCop1 (CCN5),

and Wisp-3 (CCN6). Most members of the CCN family share a

uniform modular structure and exhibit diverse cellular function,

such as regulation of cell division, chemotaxis, apoptosis,

adhesion, motility, and ion transport (22-24). Cyr61 has been

reported to mediate cell adhesion, stimulate chemostasis,

augment growth factor– induced DNA synthesis, foster cell

survival, and enhance angiogenesis (25, 26). Elevated Cyr61

expression is associated with advanced breast adenocarcinoma

pathogenesis, pancreatic cancer, and gliomas (27-29). Our

previous study showed that Cyr61 protein is highly expressed

in more advanced gastric adenocarcinoma specimens, and

overexpressed Cyr61 in human gastric cancer cell lines

significantly increased their invasion abilities (30). In the

present study, we further explored how Cyr61 promotes the up-

regulation of chemokine receptors CXCR1 and CXCR2

through the signaling pathway integrin avh3/Src/phosphoino-

sitide 3-kinase (PI3K)/Akt. Functional dissection showed that

the Cyr61-mediated expression of CXCR1/CXCR2 was

involved in gastric cancer cells, transendothelial migration,

and intravasation in a chick embryo model.

ResultsEnforced Expression of CYR61 Gene in Gastric CancerCells Lead to Up-Regulation of Chemokine ReceptorsCXCR1 and CXCR2

We previously found that CYR61 was highly expressed in

more advanced gastric adenocarcinoma specimens, and over-

expressed Cyr61 in human gastric cancer cell lines significantly

increased their migration and invasion abilities (30). Recently,

inherent chemokine receptors have had an important role in

determining distant organ dissemination of cancer cells (7, 8).

Here, we attempt to explore the hypothesis whether over-

expression of Cyr61 would affect the expression of chemokine

receptors in gastric cancer cells. Initially, we used several Cyr61

stable expression AGS clones, including Cyr61-C1, Cyr61-C5,

Cyr61-C7, and Cyr61-M, which have been established and

FIGURE 2. CXCR1 and CXCR2 expres-sion correlated with Cyr61 gene in primarygastric carcinomas. A. RNA was isolated from20 cancerous tissues (T1-T20) and fournormal tissues (N1-N4); cDNA was synthe-sized and subjected to RT-PCR for analysis ofCyr61, CXCR1 , and CXCR2 mRNA expres-sion. B. Immunohistochemical staining forCyr61, CXCR1, and CXCR2 of formalin-fixed,paraffin-embedded gastric carcinomas. IgGstaining was used as a negative control.

Cyr61 Promotes Invasiveness of Gastric Cancer

Mol Cancer Res 2007;5(11). October 2007

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well characterized previously (30, 31). Chemokine receptors,

such as CXCR1, CXCR2, CXCR4, and CCR7 (32, 33), have

been shown to be differentially expressed on gastric cancer

specimens that are associated with tumor aggressiveness.

Reverse transcription–PCR (RT-PCR) analysis was thus used

and showed that both CXCR1 and CXCR2 , but not other

chemokine receptors, mRNA were concomitantly elevated in

the Cyr61-expressing AGS cells compared with the neo control

cells (Fig. 1A, top). Moreover, there was a trend toward a

positive expression pattern for Cyr61 and CXCR1/CXCR2

mRNA in these stable clones (Fig. 1A, top). Consistently,

Western blot analysis revealed that the level of CXCR1 and

CXCR2 protein was significantly increased in the Cyr61-

expressing cells but not in the neo control cells (Fig. 1A,

bottom). Interestingly, the correlated expression of Cyr61 and

CXCR1/CXCR2 also existed in other gastric cancer cells, N87,

TSGH, and MKN45 (Fig. 1B, top). TSGH and MKN45 cells,

which have higher levels of Cyr61 protein, exhibited an

abundant level of CXCR1/CXCR2; in contrast, AGS and N87

cells, which expressed lower levels of Cyr61, displayed a trace

amount of CXCR1/CXCR2. When MKN45 cells were

transiently transfected with increasing amounts of plasmids

carrying the antisense orientation of Cyr61 , the level of CXCR1

and CXCR2 protein was effectively reduced in a dose-

dependent manner (Fig. 1B, bottom).

Because Cyr61 is a secreted protein, we collected 24-h

conditioned medium from Cyr61-overexpressing (AGS/Cyr61-

C7) and neo control cells. When parental AGS cells were

exposed to conditioned medium of AGS/Cyr61-C7, but not

control cells, the levels of CXCR1/CXCR2 mRNA and protein

were significantly elevated (Fig. 1C). The elevation was

strongly diminished if conditioned medium was before

immunodepletion of Cyr61 protein by anti-Cyr61 antibody

(Fig. 1C). In addition, we also used recombinant human Cyr61

protein to test its ability in induction of CXCR1/CXCR2 protein.

It was shown that the expression of CXCR1/CXCR2 protein

increased dose dependently by treatment with 10 to

80 ng/mL of recombinant Cyr61 (rCyr61; Fig. 1D, top). In

general, 40 ng/mL of rCyr61 could induce an evident increase of

CXCR1/CXCR2 protein in AGS cells. In induction kinetics, the

maximal expression of CXCR1/CXCR2 protein was observed at

8 h and sustained for 24 h of rCyr61 exposure (Fig. 1D, bottom).

The above findings strongly suggest that Cyr61 tightly regulates

the expression of the CXCR1/CXCR2 gene in human gastric

cancer cell lines.

To investigate the clinical significance of Cyr61 and

CXCR1/CXCR2 coexpression in gastric cancer, 20 gastric

cancer specimens (T1-T20) and the four adjacent nontumorous

gastric tissues of gastric cancer patients (N1-N4) were analyzed

by using RT-PCR. A total of 13of 20 (65%; samples T1, T2,

T3, T6, T7, T10, T11, T12, T13, T16, T17, T19, T20) of gastric

tumors showed a considerable amount of Cyr61 mRNA

expression. Among them, about 10 of 13 (77%) had coex-

pressed CXCR1 and/or CXCR2 mRNA. In contrast, Cyr61

level in four normal counterparts is lower than that in most

tumor parts (Fig. 2A).

The coexpression of Cyr61 and CXCR1 and/or CXCR2

protein was further shown by using immunohistochemical

analysis. The representative immunohistochemical staining

(T2 tumor; Fig. 2B) patterns of Cyr61 and CXCR1/CXCR2

from consecutive serial sections were near-identical in gastric

tumor tissues implying highly correlated expression. We also

found that CXCR1 and CXCR2 had strong staining at higher

levels in cancer tissue wherein the expression of Cyr61 was

highly localized, and these proteins were not expressed in the

adjacent normal gastric tissues (Fig. 2B). The CXCR1 or

CXCR2 protein seemed to be predominantly localized in the

membrane of tumor epithelial cells and also expressed in the

cytoplasm. Negative staining for gastric tumors was shown

when immunostaining used an IgG control. Taken collec-

tively, the staining data showed that Cyr61 expression was

tightly correlated with CXCR1 and CXCR2 receptors in

gastric carcinomas, implicating that chemokine receptor

induction by the Cyr61 gene plays a role in gastric cancer

progression.

Involvement of CXCR1/CXCR2 in Cyr61-MediatedChemotactic Migration

To clarify whether CXCR1 and CXCR2 receptors expressed

on Cyr61-overexpressing AGS cells are functional, we

stimulated human gastric cancer cells with recombinant

interleukin-8 (IL-8), a physiologic ligand for both CXCR1

and CXCR2. Binding of chemokines to their receptors produces

a characteristic increase in cytosolic calcium. This is one of the

earliest biochemical events that occur in response to chemo-

kines (34). To examine intracellular calcium flux, we labeled

Cyr61-overexpressing (AGS/Cyr61-C7) cells and control

(AGS/neo) cells with Fluo-3AM before adding chemokine

IL-8. Evaluation of the fluorescence of stimulated cells showed

that only AGS/Cyr61-C7 cells mobilized Ca2+ in response to

IL-8 (Fig. 3A). This result indicated that CXCR1 and CXCR2

expressed in Cyr61-expressing cells were the functional

receptors, which readily responded to its ligand.

We next explored if Cyr61-overexpressing gastric cells use

CXCR1 and CXCR2 to migrate in response to IL-8 by Boyden

chamber analysis. AGS/Cyr61-C7, AGS/Cyr61-mixed pool

(AGS/Cyr61-M), and AGS/neo cells were seeded into the

upper chamber of transwells, and their ability to migrate was

evaluated. The results showed that AGS/neo cells were not

responsive to IL-8, even when they are exposed to a high

concentration of the chemokine (Fig. 3B). A concentration of

30 ng/mL IL-8 was able to support the chemotaxis of AGS/

Cyr61-C7 cells as much as 3-fold over unstimulated cells, and

even low concentrations of the chemokine (10 ng/mL) was

sufficient to induce the Cyr61-overexpressing cells to migrate

more rapidly (Fig. 3B). AGS/Cyr61-C7 and AGS/Cyr61-M

cells did not respond to IL-6 ligand stimulation. These results

suggest that Cyr61-overexpressing cells induce CXCR1 and

CXCR2 expression and result in tumor cell chemotactic

migration in response to specific chemokine IL-8 stimulation.

The exact effect of CXCR1 and CXCR2 overexpression by

the Cyr61 gene on chemotactic migration of tumor cells was

further confirmed using neutralizing antibodies. To this end,

AGS/Cyr61-C7 and AGS/Cyr61-M cells were preincubated

with functional blocking antibodies against CXCR1 (15 Ag/mL)

and/or CXCR2 (15 Ag/mL) and then subjected to the upper

chamber of a transwell. The results showed that treatment

with either anti-CXCR1 or anti-CXCR2 antibody attenuated

Lin et al.


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IL-8– induced chemotaxis of Cyr61-expressing cells (either

AGS/Cyr61-C7 or AGS/Cyr61-M) by f40% to 50% and

exhibited over 80% inhibition with cotreatment of anti-CXCR1

and anti-CXCR2 (Fig. 3C). This implies that the chemotactic

activity of Cyr61-overexpressing cells toward IL-8 is function-

ally collaborated by CXCR1 and CXCR2.

To confirm a specific interaction between chemokine

receptors and chemokines in Cyr61-expressing cell migration,

rather than an aspecific interference with the Boyden micro-

chamber assay equipment, similar experiments were done in

the agarose assay (35). In this test system, the distance that cells

migrated toward IL-8 source was used as a variable for

chemotactic potency. Initially, cells lined up at the outer edge of

the center well and then flattened out, as they pushed

themselves underneath the agarose to migrate toward the

chemoattractant IL-8 (Fig. 3D, top). Micrographs taken at

higher magnifications showed that more numbers of cells often

migrated toward the source of the IL-8, and the chemotactic

migration of Cyr61-overexpressing cells was in a time-

dependent manner (Fig. 3D, bottom). Cyr61 cells migrated

toward the IL-8 at a rate of 7.8 Am/h during the first 48 h and

then increasing to f13 Am/h between 48 and 96 h. As a

control, AGS/neo cells migrated randomly and slowly. These

results indicate that overexpression of Cyr61 in gastric cancer

cells greatly and directionally enhances cell migration toward

chemoattractant IL-8.

Cyr61 Overexpression Enhanced Gastric Cancer CellTransendothelial Migration and Intravascular Invasion

Endothelial-derived chemokines, including IL-8, have a role

in attracting tumor cells migrating from a site of attachment to

the endothelium across the vessel wall (35). Indeed, consistent

with other findings (36), human umbilical vascular endothelial

cells (HUVEC) express substantial amounts of IL-8 (data not

shown). To explore whether endothelial-derived IL-8 would

also be a chemoattractant for Cyr61-overexpressing cells, we

have cocultured with tumor cells in the upper chamber and

HUVEC in the lower chamber of transwells. Interestingly, the

migratory potency of Cyr61-overexpressing cells was greatly

increased by 3-fold in the presence of HUVEC compared

with none (Fig. 4A). This endothelium-dependent chemotaxis

of Cyr61-expressing cells was effectively abolished by the

FIGURE 3. Effect of recombinant IL-8 on Cyr61-overexpressing gastric cell chemotaxis. A. AGS/neo and AGS/Cyr61-C7 single clone were labeled withFluo-3AM and exposed to rIL-8 (30 ng/mL) assessed intracellular calcium mobilization. B. Chemotactic migration ability of AGS/neo, AGS/Cyr61-C7, andAGS/Cyr61-M cells by stimulation of rIL-8. IL-6 was used as positive control. Phase micrographs showed crystal violet staining of migration of AGStransfectants. Values were compared with the ability of AGS/neo cells upon no stimulation. C. Inhibition of AGS/Cyr61 cells chemotaxis by neutralizingantibodies against CXCR1 and CXCR2. Cells in the upper chamber were pretreated with IgG, CXCR1, or CXCR2 alone or CXCR1 and CXCR2 antibodies,respectively, and recombinant IL-8 (30 ng/mL) was placed in lower chamber. After migrating for 16 h, values were compared with the ability of AGS/Cyr61-C7and AGS/Cyr61-M cells with no treatment. Columns, mean of three independent experiments; bars, SE. P values of <0.05 were considered statisticallysignificant. D. Time course experiment showing the X (the distance of cells moving to IL-8) and Y (the distance of cells moving to PBS control) migrated byCyr61 transfectants in 96 h. The values of X-Y (E, AGS/Cyr61-7; ., AGS/neo) are as chemotaxis distance. Points, mean; bars, SE. All experiments weredone in triplicate on separate occasions with similar results.



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addition of blocking antibodies to CXCR1, CXCR2, or IL-8

(Fig. 4A). The above results suggest that IL-8 might be the

important endothelial chemokine responsible for the stimulation

of Cyr61-expressing cell chemotaxis.

Transmigrating across the endothelial barrier in the primary

site is an important step for tumor cell invasion and metastasis.

To assess the role of the Cyr61 gene in transendothelial

migration, we used transwell chambers in which the upper and

bottom wells were separated by a HUVEC monolayer,

depositing calcein AM-labeled AGS/Cyr61 cells on top of a

monolayer of HUVEC. The coculture assay was fixed at 8 h,

and calcein-AM–labeled Cyr61 cells, showing a spreading

morphology under the endothelium, were scored as trans-

migrated cells by fluorescent microscopy. As shown in Fig. 4B,

neutralizing antibodies against CXCR1 and CXCR2 abrogated

transendothelial migration of Cyr61-overexpressing cells (either

AGS/Cyr61-C7 or AGS/Cyr61-M) by 40% to 50%. Treatment

with anti– IL-8 antibody seemed more effective (70-80%

inhibition) in inhibiting transmigration of Cyr61-expressing

cells. In contrast, control IgG had no effect on that (Fig. 4B).

The above results thus confirm the role of CXCR1 and CXCR2

in IL-8–dependent transendothelial migration of Cyr61-

expressing cells.

The ability of Cyr61 overexpression promoting tumor cells

across the endothelial barrier prompts us to investigate whether

Cyr61 could enhance the intravasation of tumor cells in vivo .

To address this, we used a chick embryo chorioallantoic

membrane (CAM) model in which the extent of tumor cells’

intravasation can be determined quantitatively by detection of

human Alu sequence. In a time-related experiment, Cyr61

expressing cells were initially detected at 2 days, steadily

increasing during 3 to 6 days of postinoculation (Fig. 5A). In

contrast, neo control cells were barely detected at 4 days, and

only little amounts appeared at 5 to 6 days of postinoculation

(Fig. 5A). The above results strongly suggest that Cyr61

expression effectively promotes gastric cancer cell intravasation

in a CAM model. Cyr61-expressing cells were pretreated with

neutralizing antibodies against CXCR1 or CXCR2, resulting in

significant inhibition to their intravasation ability (Fig. 5B).

This thus confirms the critical role of CXCR1/CXCR2 in

Cyr61-mediated tumor cell intravasation.

Cyr61-Mediated CXCR1/CXCR2 Up-Regulation andChemotaxis through the PI3K/Akt-Dependent Mechanism

We next studied the possible signaling pathway involved in

CYR61-induced CXCR1 and CXCR2 up-regulation and

chemotactic migration observed above. To this end, we exposed

cells to 40 ng/mL of rCyr61 for varying periods of time and

determined the activated signaling pathways by immunoblot-

ting using their specifically phosphorylated antibodies. As

shown in Fig. 6A, the phosphorylated form of both extracellular

signal-regulated kinase 1 (ERK1)/ERK2 and Akt, but not p38

or phosphorylated c-Jun-NH2-kinase, were evidently increased

after 10 min of treatment of rCyr61 and remained at substantial

levels for >2 h after exposure to rCyr61. These findings are

consistent with earlier studies that have shown the ability of

Cyr61 to strongly activate Akt in other cell systems (28, 37).

To determine the involvement of PI3K/Akt or Erk1/Erk2 in

Cyr61-induced up-regulation of CXCR1/CXCR2, their specific

inhibitors LY294002 and PD98059 were used. Western blot

analysis showed that pretreatment with 40 Amol/L LY294002,

a specific inhibitor of the p110 catalytic subunit of PI3K,

markedly decreased phosphorylation on Ser473 of Akt, as well

as the elevation of CXCR1/CXCR2 induced by Cyr61 (Fig. 6B,

top). In contrast, 25 Amol/L PD98059, a specific inhibitor of

ERK, completely abolished Erk1/Erk2 phosphorylation in

the absence of rCyr61, but failed to affect CXCR1/CXCR2

up-regulation in response to rCyr61 (Fig. 6B, bottom). To

further confirm that Akt, the downstream of PI3K, was indeed

involved in the Cyr61-induced elevation of CXCR1 and

CXCR2, we stably transfected a kinase-defective mutant of

Akt (Akt 179A), which can inhibit wild-type Akt in a

dominant-negative fashion (38), followed by stimulation of

40 ng/mL rCyr61. Western blot analysis revealed that stable

expression of dominant-negative Akt in AGS cells resulted in a

FIGURE 4. Effect of IL-8 secreted from the conditioned endothelialmedium on transendothelial migration of Cyr61-overexpressing gastriccell. A. HUVEC cells were cocultured in lower chamber of Boydentranswell, and the conditioned medium was incubated with the absence orpresence of anti – IL-8 (10 Ag/mL) blocking antibody. AGS/Cyr61-7 cells(2.5 � 104) pretreated with CXCR1 (30 Ag/mL) or CXCR2 (30 Ag/mL)antibodies, respectively, were then seeded in the upper chamber allowedto migrate for 16 h. Values were compared with the ability of AGS/Cyr61cells in the upper chamber without HUVEC cocultured condition. B.The inhibitory effects of CXCR1 and CXCR2 neutralizing antibodies onAGS/Cyr61 cell transendothelial migration. Endothelial cells were fullyconfluence preseeded in the upper chamber of transwell overnight; AGS/Cyr61-C7 and AGS/Cyr61-M cells were incubated above the HUVEC layerand allowed to transendothelial migrate with the CXCR1, CXCR2, orIL-8 blocking antibodies for 20 h. IgG treatment was used as control.Values were compared with the ability of AGS/CYR61 cells in the absenceof antibody treatment. Each was placed in triplicate in experiment.Columns, mean; bars, SE. P values of <0.05 were considered statisticallysignificant.

Lin et al.


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decrease in the rCyr61-stimulated increased level of CXCR1

and CXCR2 (Fig. 6C). Subsequently, we explored whether

PI3K/Akt pathway is involved in the Cyr61-induced IL-8–

dependent chemotaxis. Figure 6D reveals that 20 to 40 Amol/L

of LY294002 led to a dose-dependent inhibition of the IL-8–

induced chemotactic migration of AGS cells by stimulation of

rCyr61. Furthermore, the Erk inhibitor PD98059 had no

significant effect on Cyr61-induced chemotaxis toward IL-8.

The above results thus suggest that PI3k/Akt signaling pathway

is critically involved in Cyr61-mediated CXCR1/CXCR2 up-

regulation and chemotactic migration.

Integrin and Src Act as Upstream Regulator in the Up-Regulation of CXCR1 and CXCR2

It is well known that Cyr61 protein interacts with various

integrin receptors, such as avh3, avh5, a6h1, to transducer

downstream signaling cascades (39-41). Therefore, we want to

determine whether integrin receptor is necessary for Cyr61–up-

regulated CXCR1/CXCR2 expression. As shown in Fig. 7A,

Cyr61-induced CXCR1 and CXCR2 elevation is strongly

reduced by functional neutralizing antiintegrin avh3 antibody,

but not anti-a2h1 antibody, indicating a vital role of integrin

avh3 in Cyr61-induced expression of IL-8 receptors in human

AGS cells.

Integrins transmit signals directly through Src families (42),

the consequent downstream signals, especially via the mitogen-

activated protein kinase, and PI3K transduction cascades are

critical for regulation of biological activities. To address

whether c-Src is activated by CYR61 gene expression and that

it participates in CXCR1 and CXCR2 induction, we treated

AGS cells with rCYR61 (40 ng/mL) and did Western blot

analysis. The result showed that c-Src phosphorylation at Tyr416

was found at 10 min after rCYR61 stimulation, decreasing at

24 h (Fig. 7B, top). We also observed that c-Src and Akt

kinases phosphorylation decreased by inhibition of integrin

avh3 activity in the presence of rCyr61 (Fig. 7B, bottom),

implicating Cyr61 via binding to the integrin receptor

modulated Src and Akt activity. Moreover, to further examine

whether Src kinase is required for enhanced CXCR1 and

CXCR2 expression in Cyr61-overexpressing cancer cells, we

used a Src-specific inhibitor, PP1, to block c-Src phosphory-

lation. It was found that blockage of Src activity by PP1 led to

inhibition of the Cyr61-induced CXCR1 and CXCR2 expres-

sion, and Akt phosphorylation. We further confirmed the role

of Src in CXCR1 and CXCR2 up-regulation by dominant-

negative Src kinase. As expected, CXCR1 and CXCR2

expression were reduced by transfection with dominant-

negative Src of AGS cells (Fig. 7D). In addition, the chemo-

taxis activity was significantly decreased to a level compared

with that of the negative control untreated cells with treatment

of PP1 (Fig. 7E). Taken together, these results show that

integrin avh3/Src/PI3k/Akt pathway critically contributes to

Cyr61-induced CXCR1 and CXCR2 expression, as well as

IL-8–dependent chemotaxis and intravasation.

DiscussionAccumulating studies have shown that Cyr61 plays a

pleiotropic role and is substantially involved in the develop-

ment and progression of human malignancies, such as

melanomas, gliomas, and carcinomas of the lung, breast,

prostate, and bladder (28, 31, 37, 43-45). Our recent work also

showed that patients with gastric adenocarcinoma displayed

high expression of Cyr61, whose level was positively correlated

with aggressive lymph node metastasis, more advanced tumor

stage, and early recurrence (30), enforcing expression of Cyr61

in AGS gastric cells and directly promoting cell motility and

invasiveness. In this report, we further show that enforced

expression or treatment with Cyr61 increases chemokine

receptors CXCR1 and CXCR2 expression and promotes gastric

cancer cell migration toward CXCR1/CXCR2 native ligand,

IL-8. In addition, Cyr61-expressing cells have greater potency

FIGURE 5. Effects of CXCR1 and CXCR2 on Cyr61-overexpressinggastric cancer cells spontaneously intravasation in the chick embryomodel. A. Detection and quantitation of Cyr61-overexpressing AGS cellsspontaneous metastasis in CAM assay. The 106 cells were suspended inserum-free RPMI medium in upper CAM chamber and allowed tointravasate for 1 to 6 d. Top, PCR amplification of Alu sequences in themixture of human and chicken extracted CAM DNA from lower CAM.Bottom, the quantitation data of upper PCR amplification result. B. AGS/neo and AGS/Cyr61-7 cells were incubated with neutralizing antibodiesagainst CXCR1 (30 Ag/mL), CXCR2 (30 Ag/mL), or control IgG in theupper CAM of the chicken embryo for 4 d. The human and chicken CAMDNAs from lower CAM were extracted and assessed by relative fold ofDNA density compared with AGS/neo cells upon no stimulation. No tumorcells seeded chamber extracted DNA is used as negative control.



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in transendothelial migration and intravasation in vitro

compared with neo control cells. Blockage of CXCR1/CXCR2

by their specific neutralizing antibodies strongly impairs

Cyr61-mediated chemotaxis, transendothelial migration,

and intravasation. Supportively, immunohistochemical serial

section staining or RT-PCR in gastric tumor specimens revealed

that an evident correlation exists between the level of Cyr61

and CXCR1/CXCR2 receptor expression. Together, our

current data, for the first time, highlights a new function of

Cyr61 involved in promoting IL-8–dependent chemotaxis,

transendothelial migratioin, and intravasation via concomitant

up-regulation of functional CXCR1/CXCR2 in gastric cancer

cells.

CXCR1 and CXCR2 receptors are members of the seven-

transmembrane spanning, G protein–coupled receptor family.

CXCR1 and CXCR2 show a 77% overall sequence identity to

each other (9). Their transmembrane regions and intracellular

loops are highly similar, whereas the amino- and carboxyl-

terminal regions show striking differences. Both receptors are

expressed on polymorphonuclear neutrophil granulocyte

through ligand binding activation to regulate immune cell

migration. However, many clinical studies have revealed that

various carcinomas express both IL-8 receptors CXCR1 and

CXCR2 on the surface of human carcinoma cells by

immunohistochemical analysis, such as breast, colon, gastric,

pancreatic, and head and neck squamous carcinomas (18, 19,

46-48), suggesting an important role in tumor development

or progression. Recent studies reported that high levels of

expression of CXCR1, CXCR2, and CCR7 were found in

gastric cancer cells involved with carcinoma invasion and

metastasis (32, 47, 49). Among these chemokine receptors,

CCR7 played a role in metastasis of lymphoid organs by gastric

cancer cells (49), and the most abundant expression of both

chemokines CCL21/CCL6 and CCL19/ECL had been found in

the lymph nodes (7, 50). In contrast, the actual role of CXCR1/

CXCR2 in gastric cancer invasion and metastasis has yet to

be defined. Tumor-endothelial cell interaction is important for

tumor invasion and metastasis (51, 52), and transendothelial

FIGURE 6. Cyr61 overexpression activates PI3K signaling involved with CXCR1 and CXCR2 up-regulation. A. Phosphorylated Akt and ERK42/ERK44and p38 c-Jun-NH2-kinase (JNK) protein expression levels in rCyr61 treatment (40 ng/mL) for indicated time. Total cell lysates was collected, and 40 Agprotein were subjected to electrophoresis on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Immunoblots with anti –phosphorylated Akt, anti – phosphorylated ERK, anti –phosphorylated c-Jun-NH2-kinase, and anti-p38 show equivalent amounts of the proteins in eachextract. Blots were reprobed with antibodies for Akt and ERK to control for protein loading and transfer. B. Overnight serum-starved cells at 75% to 80%confluence were treated 1 h with vehicle, LY294002 or PD98059, followed by treatment with 40 ng/mL rCyr61 for 1 h. Total protein (40 Ag) was resolved bySDS-PAGE and subjected to immunoblot analysis for phosphorylated Akt (top ), phosphorylated ERK42/ERK44 (bottom ), CXCR1, and CXCR2. Blots werereprobed with antibody for Akt, ERK42/ERK44. a-Tubulin was used to control protein loading and equal transfer. C. AGS cells were transiently transfectedwith dominant-negative Akt (DN-Akt) or control vector pcDNA3 and then treated with 40 ng/mL rCYR61 for 8 h. CXCR1 and CXCR2 protein expression weredetermined by Western blot analysis. Results are representative of three independent experiments. D. AGS cells were incubated with the indicatedconcentrations of LY294002 or PD98059 and treated with IL-8 (30 ng/mL) and then subjected to chemotaxis. The experiments were done in triplicates.Columns, mean; bars, SE. P values of <0.05 were considered statistically significant.

Lin et al.


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migration is a key event in cancer hematogenous metastasis

(53). In the present study, we used specific neutralizing anti-

bodies against CXCR1 or CXCR2 that effectively blocked

Cyr61-overexpressing gastric cancer cell chemotactic migration

and intravasation in vitro . Therefore, we suggest that the

expression of chemokine receptors CXCR1 and CXCR2 on

gastric carcinoma cells may facilitate their hematogenous

metastasis. Consistent with our observations and notions,

Ramjeesingh et al. also showed CXCR1-overexpressing mela-

noma cells exhibiting enhanced activity in chemotaxis and

transendothelial migration toward endothelial-derived IL-8 (35).

Our current studies further suggest that organ specificity of

certain tumor metastases might depend on both the types of

chemokines secreted by the host endothelium or organ and the

expression pattern of chemokine receptors on the cancer cell.

Limited studies have focused on the regulatory mechanism

of chemokine receptors in tumor cells. Here, we show that

integrin avh3 acts as an innate receptor for Cyr61 protein to

transduce signaling for up-regulating CXCR1/CXCR2 mRNA

in gastric cancer cells. Actually, most of the Cyr61-promoted

effects, including cell adhesion, migration, and differentiation,

are mediated via its direct binding with the integrin receptor

avh3 (28, 40, 54, 55), which has been implicated in the

pathophysiology of malignant tumors. In addition, the level of

integrin avh3 is generally up-regulated in invasive tumors and

distant metastases in a wide variety of cancers (56). Our

FIGURE 7. Integrin and Src signaling activation involved with Cyr61-induced CXCR1 and CXCR2 expression. A. Cells at 75% to 80% confluence werepretreated with integrin avh3, a2h1 neutralizing antibody or control IgG for 1 h and treated with 40 ng/mL rCyr61. Total protein (40 Ag) was resolved by SDS-PAGE and subjected to immunoblot analysis for CXCR1 and CXCR2. Blots were reprobed with antibody for a-tubulin to control protein loading and equaltransfer. Results are representative of three independent experiments. B. Top, AGS cells were treated with 40 ng/mL of rCyr61 for indicated time.Immunoblots with anti –phosphorylated c-Src show equivalent amounts of the proteins in each extract. Blots were reprobed with an antibody for anti – c-Src tocontrol protein loading and transfer. Bottom, cells were pretreated neutralizing antibody against integrinavh3, a2h1 (10 Ag) and control IgG for 1 h, and thentreated with 40 ng/mL rCyr61. Total protein was resolved by SDS-PAGE and subjected to immunoblot analysis for phosphorylated Akt and phosphorylatedSrc. Blots were reprobed with antibody for Akt and Src to control protein loading and equal transfer. Results are representative of three independentexperiments. C. AGS cells were treated with vehicle PP1 for 1 h after treatment with 40 ng/mL rCyr61. Total protein was resolved by SDS-PAGE andsubjected to immunoblot analysis for phosphorylated c-Src, phosphorylated Akt, CXCR1, and CXCR2. D. AGS cells were transiently transfected withdominant-negative Src (DN-Src) or vector control pcDNA3 and then treated with rCyr61 for 8 h. CXCR1 and CXCR2 expression and Src and Aktphosphorylation were determined by Western blot analysis. Blots were reprobed with antibody for Src, Akt, and a-tubulin to control protein loading and equaltransfer. E. AGS cells were incubated with the indicated concentrations of Src protein inhibitor, PP1, and treated with IL-8 (30 ng/mL). The experiments weredone in triplicates. Columns, mean; bars, SE. P values of <0.05 were considered statistically significant.



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unpublished data shows that the chemokine receptors CXCR1/

CXCR2 were colocalized with integrin avh3 on the membrane

of Cyr61-overexpressing cells (data not shown). This suggests

that Cyr61-mediated chemotaxis and transendothelial migration

may be through coordination between CXCR1/CXCR2 and

integrin avh3. These findings are clearly relevant to previous

studies published by Babic et al., showing Cyr61 could induce

vascular endothelial cells chemotaxis through avh3-dependentpathway (57). However, how the CXCR1/CXCR2 and integrin

avh3 cooperate with each other to facilitate Cyr61-expressing

cells’ transendothelial migration needs further investigation.

Integrins might transmit signals through the activation of the

ERK42/ERK44 mitogen-activated protein kinase and PI3K/Akt

pathway (42). In the present study, we showed that Cyr61

elicited the activation of PI3k/Akt and Erk1/Erk2, but not p38,

signaling pathways. Indeed, the fact that Cyr61 can activate

both pathways is commonly observed in different kinds of cells

(28, 37). Utilization of pharmacologic inhibitors and genetic

inference shows that the PI3K/Akt, but not Erk1/Erk2, pathway

is required for Cyr61-induced CXCR1/CXCR2 up-regulation

and subsequent chemotaxis. We further found that c-Src was

necessary for Cyr61-induced PI3K/Akt activation and CXCR1/

CXCR2 production. c-Src belongs to the Src family of tyrosine

kinases, which represent important transducers during integrin-

mediated signaling. It has been reported that Src family of

tyrosine kinases are associated with the integrin avh3 and are

activated after integrin ligation in multiple cell types, including

osteoclasts (58), melanoma cells (59), and platelets (60). Src

family of tyrosine kinases are associated with avh3 integrins

through an interaction with the C terminus of the h3 subunit.

The C terminus, including the NITY motif, is unique to the h3

integrin subunit, which may explain the specificity of the

interaction of c-Src with h3 tails, but not h1 or h2 tails (61).

c-Src has been shown to directly phosphorylate many

downstream proteins, including FAK, Cas, paxillin, mitogen-

activated protein kinase, and PI3k-Akt signaling. This was

consistent with our findings that detailed signaling cascade

from integrin avh3 to CXCR1/CXCR2 induction was mediated

by Src kinase and its downstream PI3k-Akt pathway.

In conclusion, we, for the first time, showed that over-

expression of Cyr61 induced functional CXCR1 and CXCR2

receptors that play an important role in chemotactic migration

of human AGS cells toward IL-8. Under such a scenario,

Cyr61-expressing cells exhibited stronger potency in trans-

endothelial migration and intravasation in a CAM model via

a CXCR1/CXCR2-dependent mechanism. Collectively, our

current findings suggest that Cyr61 is possibly involved in

hematogenous invasion and metastasis of human gastric

adenocarcinoma. It also implicates that targeting Cyr61 or

CXCR1/CXCR2 in this disease may be an effective therapeutic

strategy in inhibiting the dissemination of gastric cancer after

local surgical control.

Materials and MethodsAntibodies and Reagents

Anti-CXCR1, CXCR2, and IL-8 monoclonal antibodies

were from R&D Systems; anti-CYR61 polyclonal antibody,

anti–phosphorylated Akt1/Akt2/Akt3 (Ser473)-R antibody,

anti–Akt-1 antibody, anti–phosphorylated ERK1/ERK2 anti-

body, anti-ERK1/ERK2 antibody, anti –phosphorylated Src

antibody, anti-Src antibody, anti–phosphoryalted p38 antibody,

and anti–phosphorylated c-Jun-NH2-kinase antibody were

obtained from Santa Cruz Biotechnology; anti–h-actin anti-

body and anti–a-tubulin antibody, LY294002, and PD98059

were from Sigma. Function-blocking antibody against avh3(LM609); a2h1 was purchased from Chemicon; Cyr61

neutralizing antibodies are a kind gift from Dr. Lester F.

Lau, and they do not cross-react with Fisp12/connective tissue

growth factor (57). Recombinant human IL-8 was purchased

from R&D Systems, and recombinant human Cyr61 was

purchased from Abnova Corporation. [a-32P]dCTP was

obtained from Amersham Biosciences (Amersham International).

Cell Cultures and Tumor SamplesAGS, N87, MKN45, and TSGH human gastric adenocarci-

noma cell lines were obtained from American Type Culture

Collection. All gastric carcinoma cell lines were grown in

RPMI 1640 (Invitrogen Corp.). HUVEC were isolated from

two to five umbilical cord veins, pooled, and established as

primary cultures in M199 containing 20% fetal bovine serum

(62). The surgical samples were obtained from the Department

of Surgery, National Taiwan University Hospital.

Transient Transfection and Established Stable ClonesThe expression vector CYR61 was constructed by placing

human CYR61 cDNA in the pcDNA3.1 eukaryotic expression

vector containing the neomycin gene under the control of the

same promoter. The CYR61-sense expression constructs were

transfected into AGS cells or CYR61-antisense constructs into

MKN45 cells using TransFast liposome (Promega). At 24 h

after transfection, the cells were serum-starved for 16 h and

lysed for analysis for transient transfection. Stable cell popu-

lations were selected by 0.8 mg/mL gentamicin (Invitrogen

Corp.). After G418 selection, we isolated one single clone,

AGS/Cyr61-C7, and a pool mixture, AGS/Cyr61-M.

Collection of Conditioned MediumAt full growth of cell, 90% of the confluence medium were

removed, and cells were washed and then incubated in serum-

free medium for 24 h. Conditioned medium was collected and

centrifuged to remove any cellular contaminants. Immunopre-

cipitation analysis was used to deplete CYR61 protein that

existed in conditioned medium. Goat anti-CYR61 antibody

(10 Ag/mL) was added, the samples were rotated at 4jC over-

night, and then protein A–Sepharose (Amersham Pharmacia;

30 AL of a 50% slurry) was added for 2 h. Followed by

centrifugation for 20 min at 14,000 rpm, the supernatant was

collected as conditioned medium that depleted CYR61.

RNA Isolation and RT-PCRTotal cellular RNA was isolated from gastric carcinoma cell

lines and patient tissue by using RNA TRIzol reagent

(Invitrogen Corp.) using the manufacturer’s directions. cDNA

was synthesized using total RNA (5 Ag) with Moloney murine

leukemia virus reverse transcriptase and random hexamers

(Promega). The reaction mixture was incubated at 37jC for

2 h and was terminated by heating at 95jC for 5 min. The

Lin et al.


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primer sequences for PCR are as follows: CYR61, 5¶-CGAGG-TGGAGTTGACGAG AAAC-3¶ (F) and 5¶-AGGACTGGA-TATCATGACGTTCT-3 ¶ (R); CXCR1, 5 ¶-GCCACCT-

GCAGATGAAGATT-3¶ (F) and 5¶-CAGCAGCCAAGA-

CAAACAAA-3¶ (R); CXCR2, 5¶-GTGAACCAGAATCCCTG-GAA-3¶ (F) and 5¶-AGACGCTCCTTCGGAAAAGT-3¶ (R);

CXCR4, 5¶-AATCTTCCTGCCCACCATCT-3¶ (F) and 5-GAC-

GCCAACATAGACCACCT-3¶ (R); CCR7, 5¶-ACATCGGAGACAACACCACA-3 ¶ (F) and 5 ¶-CATGCCACTGAAG

AAGCTCA-3 ¶ (R); CCR10, 5 ¶-GGGTTTCTCCTT-

CCACTCCT-3¶ (F) and 5-TATTCCCCACATCCTCCTTG-3¶(R); h-actin, 5¶-GATGATGATATCGCCGCGCT-3¶ (F) and

5-TGGGTCATCTTCTCGCGGTT-3¶ (R). Primers were used

at a final concentration of 0.5 Amol/L, and the thermal cycling

conditions were as follows: 5 min at 95jC, followed by 32

cycles of 95jC for 60 s, 55jC for 60 s for CYR61 and h-actinor 55jC for 60 s for chemokine receptor genes, and finally

72jC for 60 s. The reactions were done in a Biometra Ther-

maoblock (Biometra, Inc.). PCR products were separated on a

1.2% agarose gel containing ethidium bromide (0.5 Ag/mL),

visualized, and photographed, and relative intensity of the

specific gene expression was determined using the Alpha-

Image Analysis System (Alpha Innotech).

Western Blot AnalysisCells were harvested, lysed in radioimmunoprecipitation

assay buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl,

1% Triton X-100, 10% glycerol, 0.25% Na-deoxycholate, 1%

Nonidt P-40, 1 mmol/L EDTA, 1 mmol/L EGTA, 50 mmol/L

Na3VO4, 1 mol/L NaF, 50 mmol/L phenylmethylsulfonyl

fluoride, 1 Ag/mL leupeptin, and 1 Ag/mL aprotinin] for

20 min on ice, and clarified by centrifugation at 14,000 rpm for

20 min at 4jC, and the supernatant was collected. Equal

amounts of protein were loaded onto 10% gradient SDS poly-

acrylamide gel separated and electrotransferred onto polyviny-

lidene difluoride membrane (Immobilon-P membranes;

Millipore Corp.). The protein blot in the membrane was

blocked with 5% skim milk in PBS containing 0.1% Tween 20

(Sigma Chemical Co.), PBS-T, for 1 h at room temperature.

Primary antibodies as indicated were incubated with mem-

branes for 4jC overnight, and the membranes were washed in

PBS-T thrice, probed with horseradish peroxidase–conjugated

secondary antibodies (Santa Cruz Biotechnology) for 1 h

(1:5,000 dilution in PBS-T), and then washed thrice with

PBS-T. The antibody-bound protein bands were detected with

enhanced chemiluminescence reagent (Amersham Pharmacia

Biotech) and photographed with Kodak X-Omat Blue autora-

diography film (Perkin-Elmer Life Sciences).

Under-Agarose Cell Migration AssayA 2% (w/v) agarose solution in RPMI 1640 was mixed with

an equal volume of complete RPMI medium containing 1%

(w/v) bovine serum albumin (Sigma Chemical Co.). An aliquot

of this mixture (900 AL) was placed in six-well tissue culture

plates and allowed to solidify for 1 h at 4jC. Three holes werepunctured at equal distances apart in the agarose sheet by using

a hole puncher. AGS/CYR61-7 and AGS/neo gastric cells were

collected from culture dishes using 4 mmol/L EDTA in HBSS

and resuspended at a concentration of 3.33 � 105 cells per

milliliter. Gastric cells (2 � 103 in 6 AL) were placed in the

middle hole, recombinant IL-8 chemoattractant (30 ng/3 AL)was placed in the left hole, and control buffer (fresh medium)

was placed in the right hole. Solutions in all three holes were

replenished every 12 h. Cells were allowed to migrate under the

agarose sheet for 96 h in an incubator at 37jC and were then

fixed with 4% paraformaldehyde for 1 h. Coverslips were

stained with 0.5% crystal violet (Sigma) for 15 min and then

washed with PBS for visualization under a light microscope

(Leica Microsystems, type 090-135.001). The distance of

chemotactic migration was determined by subtracting migration

toward the chemoattractant (X) and random movement (Y)

toward the fresh medium or control buffer. The value of X-Y

was used as chemotaxis. Each experiment was done in

triplicates.

Transwell Chemotaxis AssayChemotaxis assays were done using modified Boyden

chambers with filter inserts for 24-well dishes containing

8-Am pores (6.5 mm; Corning Costar Corp.). The upper

compartment of the culture insert was coated with 1% gelatin

solution insert. A total of 900 AL of RPMI supplemented with

10% FBS and 10 or 30 ng/mL recombinant IL-8 (R&D

Systems, Inc.) was added to the lower chamber of the transwell

in a 24-well plate or coculture HUVEC grown 100%

confluence in the absence or presence of IL-8 neutralizing

antibody in the lower chamber. AGS/CYR61-C7, AGS/

CYR61-M, or AGS/neo cells (2.5 � 104 in 100 AL medium),

with or without pretreatment with neutralizing antibodies

against CXCR1 or CXCR2, were deposited into the upper

chamber and allowed to migrate for 16 h at 37jC in an

incubator. Cells on the upper side of the filters were removed

with cotton-tipped swabs, and the filters were washed in PBS,

fixed with 4% paraformaldehyde for 15 min, and were then

stained with 0.05% crystal violet for visualization under a light

microscope (type 090-135.001, Lieca Microsystems). The

number of cells in each lower chamber/40� field was

quantitated by counting five random fields.

Transendothelial Migration AssayAGS/CYR61-C7, AGS/CYR61-M, and AGS/neo trans-

fectant cells were labeled with calcein AM (Molecular Probes)

4 Ag/mL at 37jC for 90 min in calcein-labeling buffer. Cells

were then washed twice with HBSS and resuspended. Calcein

AM–labeled cells (2.5 � 104) were added to confluent

HUVEC-coated wells in the upper chamber of the transwell,

incubated with medium alone or medium containing neutral-

izing antibody, and then allowed to migrate for 20 h at 37jC in

an incubator. Nonadherent cells were removed by washing

HBSS thrice, and cells on the upper side of the filters were

removed with cotton-tipped swabs. The fluorescence was

quantitated with Millipore fluorescence plate reader (Millipore

Corp.) using an excitation wavelength of 485 nm and an

emission wavelength of 530 nm.

Chicken Embryo Metastasis AssayThis assay was modified by Zijlstra et al. (63), which was

used to study the intravasation ability of cancer cells. Fertilized

White Leghorn eggs were incubated in a rotary incubator at



1121



38jC with 60% humidity for 10 days. At this time, the CAM

was dropped by drilling a small hole through the eggshell into

the air sac and a second hole near the allantoic vein that

penetrates the eggshell membrane but not the CAM. Subse-

quently, a cutoff wheel was used to cut a square window

(1 cm2) encompassing the second hole near the allantoic vein to

expose the underlying CAM. After delivering 1 � 106 cells in

15 AL serum-free RPMI into the CAM windows, an artificial air

sac was subsequently created (‘‘upper CAM’’) then sealed, and

the eggs were returned to a stationary incubator. After 1 to

6 days of incubation, the lower half of the CAM (‘‘lower

CAM’’) was correlated and placed in a sterile 15-mL

polypropylene tube; the tissue was snap-frozen in liquid

nitrogen and stored frozen at �80jC. The frozen tissue was

analyzed for the presence of tumor cells by quantitative alu

PCR as described below. The two sections of the lower CAM

were combined, and genomic DNA was extracted from these

tissues using the Puregene DNA purification system (Gentra

System) according to the manufacturer’s specifications. To

detect human cells in the chick tissues, primers specific for

the human alu repeat sequence [5¶-ACGCCTGTAATCCCAG-CACTT-3¶ (F), 5¶-TCGCCCAGGCTGGAGTGC-3¶ (R)], whichproduced a band of 219 bp, were used to amplify the human alu

repeats present in genomic DNA that was extracted from chick

tissues. The PCR reaction mixture contained 1 Ag of genomic

DNA as template and was done under the following conditions:

polymerase activation at 95jC for 10 min followed by 36

cycles at 95jC for 30 s, 52jC for 30 s, and 72jC for 30 s. A

quantitative measure of amplifiable chick DNA as control was

obtained through amplification of the chick glyceraldehyde-3-

phosphate dehydrogenase genomic DNA sequence with

chGAPDH primers [5’-GAGGAAAGGTCGCCTGGTGGA-

TCG-3¶ (F), 5¶-GGTGAGGACAAGCAGTGAGGAACG-3¶(R)] using the same PCR conditions described for alu .

Ca2+ MobilizationCells were detached from the substratum with 2 mmol/L

EDTA/PBS (minus Ca2+ and Mg2+), washed twice with PBS,

then loaded with Fluo-3AM (Molecular Probes) for 30 min,

and warmed to 37jC before flow cytometry analysis. The

fluorescence intensity was followed kinetically after addition

of IL-8 (30 ng/mL) on a flow cytometer. Fluorescence-

activated cell sorting was done using a FACScan (Becton

Dickinson), and analysis was done using CellQuest 2.0

(Becton Dickinson).

Immunohistochemical AnalysisFormalin-fixed tissue sections were deparaffinized and

treated with proteinase K, whereas tissue sections were treated

with 0.01 mol/L citrate buffer followed by exposure to

microwaves (3 � 5 min). The sections were immersed for

30 min in 0.3% H2O2 in absolute methanol and then treated

with 5% fetal bovine serum for blocking nonspecific binding

sites at room temperature for 1 h. Overnight incubation with the

antihuman CYR61 polyclonal (Santa Cruz), CXCR1 monoclo-

nal (R&D Systems), CXCR2 monoclonal antibodies (R&D

Systems) at 1:100 dilution was followed by incubation with

biotinylated mouse anti-goat IgG or rabbit anti-mouse IgG and

biotin-streptavidin-peroxidase (Super Sensitive Multilink HRP

Detection System, Bio Genex) reaction that used 3,3¶-diaminobenzidine tetrahydrochloride solution as substrate.

Color reaction was developed in diaminobenzidine solution,

and counterstaining was done with Mayer’s hematoxylin

solution.

StatisticsFor statistical analysis, P values were based on two-sided,

parametric Student’s t tests using Excel software from Micro-

soft. A P value of <0.05 on the basis of at least three

independent sets of experiments was considered to be

statistically significant. The significance of the differences in

the CYR61 gene between CXCR1 or CXCR2 was analyzed

using the m2 test. Pearson correlation coefficients were obtained

using SAS software (Release 6.12; SAS Institute, Inc.).

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2007;5:1111-1123. Mol Cancer Res Been-Ren Lin, Cheng-Chi Chang, Li-Ro Chen, et al. Concomitantly Up-Regulating Chemokine Receptor 1 and 2Transendothelial Cell Migration, and Intravasation by Cysteine-Rich 61 (CCN1) Enhances Chemotactic Migration,

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