Molecular Cancer Research Holland AM No Date
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Molecular Cancer Research 5, 1111-1123, November 1, 2007. doi: 10.1158/1541-7786.MCR-06-0289
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, B.-R.
Right arrow Articles by Lin, M.-T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, B.-R.
Right arrow Articles by Lin, M.-T.

Angiogenesis, Metastasis, and the Cellular Microenvironment

Cysteine-Rich 61 (CCN1) Enhances Chemotactic Migration, Transendothelial Cell Migration, and Intravasation by Concomitantly Up-Regulating Chemokine Receptor 1 and 2

Been-Ren Lin1, Cheng-Chi Chang2, Li-Ro Chen2, Ming-Hsun Wu1, Ming-Yang Wang1, I-Hsin Kuo2, Chia-Yu Chu2, King-Jen Chang1,3, Po-Huang Lee1, Wei-Jao Chen1, Min-Liang Kuo2,3 and Ming-Tsan Lin1,4

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

Requests for reprints: Ming-Tsan Lin, Department of Primary Care Medicine and 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: linmt{at}ha.mc.ntu.edu.tw


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cysteine-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 {alpha}vß3, but not {alpha}2ß1, effectively abolished Cyr61-elicited Src activation and the subsequent PI3K/Akt pathway. Antagonists toward integrin {alpha}vß3, 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 {alpha}vß3/Src/PI3K/Akt–dependent pathway. (Mol Cancer Res 2007;5(11):1111–23)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Gastric 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 ~12% 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 chemotherapeutic 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 neurotactin). 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 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.

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 {alpha}vß3/Src/phosphoinositide 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.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Enforced Expression of CYR61 Gene in Gastric Cancer Cells Lead to Up-Regulation of Chemokine Receptors CXCR1 and CXCR2
We previously found that CYR61 was highly expressed in more advanced gastric adenocarcinoma specimens, and overexpressed 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 overexpression 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 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).


Figure 1
View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 1. Induction of CXCR1 and CXCR2 chemokine receptors by Cyr61. A. Top, the transcripts of chemokine receptors in wild-type AGS cells and stably transfected with Cyr61-expressing plasmid, including mix population (AGS/Cyr61 mix), single clones (AGS/Cyr61-1, AGS/Cyr61-5, AGS/Cyr61-7), and control vector (AGS/neo) by RT-PCR analysis. ß-Actin was used as a loading control of RT-PCR. Bottom: Western blot analysis of total cell lysates probed with Cyr61, CXCR1, CXCR2, CXCR4, and {alpha}-tubulin. {alpha}-Tubulin was used as a loading and transferring control. B. Top, Western blot was used to detect the protein levels of Cyr61, CXCR1, and CXCR2 in different gastric cancer cell lines. {alpha}-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 transiently expressing Cyr61 in various dosages in AGS cells (left) and antisense Cyr61-expressing plasmids in MKN45 cells (right) for 48 h, total cell protein was extracted and Western blotting analysis was done. C. Top, the CXCR1 and CXCR2 mRNA expression by conditioned medium treatment of Cyr61 transfectants as detected by RT-PCR analysis. Cyr61 neutralizing antibody (10 µg/mL) or control IgG was placed with conditioned medium to deplete Cyr61 protein existence. Bottom, the protein levels of CXCR1 and CXCR2 were analyzed per sample, and {alpha}-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. {alpha}-Tubulin was used as internal control.

 
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 coexpressed CXCR1 and/or CXCR2 mRNA. In contrast, Cyr61 level in four normal counterparts is lower than that in most tumor parts (Fig. 2A ).


Figure 2
View larger version (84K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 2. CXCR1 and CXCR2 expression correlated with Cyr61 gene in primary gastric carcinomas. A. RNA was isolated from 20 cancerous tissues (T1-T20) and four normal tissues (N1-N4); cDNA was synthesized and subjected to RT-PCR for analysis of Cyr61, CXCR1, and CXCR2 mRNA expression. B. Immunohistochemical staining for Cyr61, CXCR1, and CXCR2 of formalin-fixed, paraffin-embedded gastric carcinomas. IgG staining was used as a negative control.

 
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 collectively, 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-Mediated Chemotactic 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 chemokines (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.


Figure 3
View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 3. Effect of recombinant IL-8 on Cyr61-overexpressing gastric cell chemotaxis. A. AGS/neo and AGS/Cyr61-C7 single clone were labeled with Fluo-3AM and exposed to rIL-8 (30 ng/mL) assessed intracellular calcium mobilization. B. Chemotactic migration ability of AGS/neo, AGS/Cyr61-C7, and AGS/Cyr61-M cells by stimulation of rIL-8. IL-6 was used as positive control. Phase micrographs showed crystal violet staining of migration of AGS transfectants. Values were compared with the ability of AGS/neo cells upon no stimulation. C. Inhibition of AGS/Cyr61 cells chemotaxis by neutralizing antibodies 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-C7 and AGS/Cyr61-M cells with no treatment. Columns, mean of three independent experiments; bars, SE. P values of <0.05 were considered statistically significant. 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 by Cyr61 transfectants in 96 h. The values of X-Y ({blacktriangleup}, AGS/Cyr61-7; •, AGS/neo) are as chemotaxis distance. Points, mean; bars, SE. All experiments were done in triplicate on separate occasions with similar results.

 
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 µg/mL) and/or CXCR2 (15 µg/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 IL-8–induced chemotaxis of Cyr61-expressing cells (either AGS/Cyr61-C7 or AGS/Cyr61-M) by ~40% 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 functionally 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 microchamber 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 µm/h during the first 48 h and then increasing to ~13 µm/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 Cell Transendothelial 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 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.


Figure 4
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 4. Effect of IL-8 secreted from the conditioned endothelial medium on transendothelial migration of Cyr61-overexpressing gastric cell. A. HUVEC cells were cocultured in lower chamber of Boyden transwell, and the conditioned medium was incubated with the absence or presence of anti–IL-8 (10 µg/mL) blocking antibody. AGS/Cyr61-7 cells (2.5 x 104) pretreated with CXCR1 (30 µg/mL) or CXCR2 (30 µg/mL) antibodies, respectively, were then seeded in the upper chamber allowed to migrate for 16 h. Values were compared with the ability of AGS/Cyr61 cells in the upper chamber without HUVEC cocultured condition. B. The inhibitory effects of CXCR1 and CXCR2 neutralizing antibodies on AGS/Cyr61 cell transendothelial migration. Endothelial cells were fully confluence preseeded in the upper chamber of transwell overnight; AGS/Cyr61-C7 and AGS/Cyr61-M cells were incubated above the HUVEC layer and allowed to transendothelial migrate with the CXCR1, CXCR2, or IL-8 blocking antibodies for 20 h. IgG treatment was used as control. Values were compared with the ability of AGS/CYR61 cells in the absence of antibody treatment. Each was placed in triplicate in experiment. Columns, mean; bars, SE. P values of <0.05 were considered statistically significant.

 
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 transmigrated 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.


Figure 5
View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 5. Effects of CXCR1 and CXCR2 on Cyr61-overexpressing gastric cancer cells spontaneously intravasation in the chick embryo model. A. Detection and quantitation of Cyr61-overexpressing AGS cells spontaneous metastasis in CAM assay. The 106 cells were suspended in serum-free RPMI medium in upper CAM chamber and allowed to intravasate for 1 to 6 d. Top, PCR amplification of Alu sequences in the mixture 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 antibodies against CXCR1 (30 µg/mL), CXCR2 (30 µg/mL), or control IgG in the upper CAM of the chicken embryo for 4 d. The human and chicken CAM DNAs from lower CAM were extracted and assessed by relative fold of DNA density compared with AGS/neo cells upon no stimulation. No tumor cells seeded chamber extracted DNA is used as negative control.

 
Cyr61-Mediated CXCR1/CXCR2 Up-Regulation and Chemotaxis 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 immunoblotting 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 µmol/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 µmol/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 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 µmol/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.


Figure 6
View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 6. Cyr61 overexpression activates PI3K signaling involved with CXCR1 and CXCR2 up-regulation. A. Phosphorylated Akt and ERK42/ERK44 and 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 µg protein 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 each extract. 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 µg) was resolved by SDS-PAGE and subjected to immunoblot analysis for phosphorylated Akt (top), phosphorylated ERK42/ERK44 (bottom), CXCR1, and CXCR2. Blots were reprobed with antibody for Akt, ERK42/ERK44. {alpha}-Tubulin was used to control protein loading and equal transfer. C. AGS cells were transiently transfected with 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 were determined by Western blot analysis. Results are representative of three independent experiments. D. AGS cells were incubated with the indicated concentrations 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.

 
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 {alpha}vß3, {alpha}vß5, {alpha}6ß1, 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 {alpha}vß3 antibody, but not anti-{alpha}2ß1 antibody, indicating a vital role of integrin {alpha}vß3 in Cyr61-induced expression of IL-8 receptors in human AGS cells.


Figure 7
View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIGURE 7. Integrin and Src signaling activation involved with Cyr61-induced CXCR1 and CXCR2 expression. A. Cells at 75% to 80% confluence were pretreated with integrin {alpha}vß3, {alpha}2ß1 neutralizing antibody or control IgG for 1 h and treated with 40 ng/mL rCyr61. Total protein (40 µg) was resolved by SDS-PAGE and subjected to immunoblot analysis for CXCR1 and CXCR2. Blots were reprobed with antibody for {alpha}-tubulin to control protein loading and equal transfer. 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 to control protein loading and transfer. Bottom, cells were pretreated neutralizing antibody against integrin{alpha}vß3, {alpha}2ß1 (10 µg) and control IgG for 1 h, and then treated with 40 ng/mL rCyr61. Total protein was resolved by SDS-PAGE and subjected to immunoblot analysis for phosphorylated Akt and phosphorylated Src. Blots were reprobed with antibody for Akt and Src to control protein loading and equal transfer. Results are representative of three independent experiments. 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 and subjected to immunoblot analysis for phosphorylated c-Src, phosphorylated Akt, CXCR1, and CXCR2. D. AGS cells were transiently transfected with dominant-negative Src (DN-Src) or vector control pcDNA3 and then treated with rCyr61 for 8 h. CXCR1 and CXCR2 expression and Src and Akt phosphorylation were determined by Western blot analysis. Blots were reprobed with antibody for Src, Akt, and {alpha}-tubulin to control protein loading and equal transfer. E. AGS cells were incubated with the indicated concentrations of Src protein inhibitor, PP1, and treated with IL-8 (30 ng/mL). The experiments were done in triplicates. Columns, mean; bars, SE. P values of <0.05 were considered statistically significant.

 
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 {alpha}vß3 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 phosphorylation. It was found that blockage of Src activity by PP1 led to inhibition of the Cyr61-induced CXCR1 and CXCR2 expression, 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 chemotaxis 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 {alpha}vß3/Src/PI3k/Akt pathway critically contributes to Cyr61-induced CXCR1 and CXCR2 expression, as well as IL-8–dependent chemotaxis and intravasation.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Accumulating studies have shown that Cyr61 plays a pleiotropic role and is substantially involved in the development 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 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 migration is a key event in cancer hematogenous metastasis (53). In the present study, we used specific neutralizing antibodies 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 melanoma 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 {alpha}vß3 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 {alpha}vß3 (28, 40, 54, 55), which has been implicated in the pathophysiology of malignant tumors. In addition, the level of integrin {alpha}vß3 is generally up-regulated in invasive tumors and distant metastases in a wide variety of cancers (56). Our unpublished data shows that the chemokine receptors CXCR1/CXCR2 were colocalized with integrin {alpha}vß3 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 {alpha}vß3. These findings are clearly relevant to previous studies published by Babic et al., showing Cyr61 could induce vascular endothelial cells chemotaxis through {alpha}vß3-dependent pathway (57). However, how the CXCR1/CXCR2 and integrin {alpha}vß3 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 {alpha}vß3 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 {alpha}vß3 integrins through an interaction with the C terminus of the ß3 subunit. The C terminus, including the NITY motif, is unique to the ß3 integrin subunit, which may explain the specificity of the interaction of c-Src with ß3 tails, but not ß1 or ß2 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 {alpha}vß3 to CXCR1/CXCR2 induction was mediated by Src kinase and its downstream PI3k-Akt pathway.

In conclusion, we, for the first time, showed that overexpression 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 transendothelial 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 Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Antibodies 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 antibody, 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–ß-actin antibody and anti–{alpha}-tubulin antibody, LY294002, and PD98059 were from Sigma. Function-blocking antibody against {alpha}vß3 (LM609); {alpha}2ß1 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. [{alpha}-32P]dCTP was obtained from Amersham Biosciences (Amersham International).

Cell Cultures and Tumor Samples
AGS, N87, MKN45, and TSGH human gastric adenocarcinoma 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 Clones
The 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 populations 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 Medium
At 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. Immunoprecipitation analysis was used to deplete CYR61 protein that existed in conditioned medium. Goat anti-CYR61 antibody (10 µg/mL) was added, the samples were rotated at 4°C overnight, and then protein A–Sepharose (Amersham Pharmacia; 30 µL 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-PCR
Total 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 µg) with Moloney murine leukemia virus reverse transcriptase and random hexamers (Promega). The reaction mixture was incubated at 37°C for 2 h and was terminated by heating at 95°C for 5 min. The primer sequences for PCR are as follows: CYR61, 5'-CGAGGTGGAGTTGACGAG AAAC-3' (F) and 5'-AGGACTGGATATCATGACGTTCT-3' (R); CXCR1, 5'-GCCACCTGCAGATGAAGATT-3' (F) and 5'-CAGCAGCCAAGACAAACAAA-3' (R); CXCR2, 5'-GTGAACCAGAATCCCTGGAA-3' (F) and 5'-AGACGCTCCTTCGGAAAAGT-3' (R); CXCR4, 5'-AATCTTCCTGCCCACCATCT-3' (F) and 5-GACGCCAACATAGACCACCT-3' (R); CCR7, 5'-ACATCGGAGA CAACACCACA-3' (F) and 5'-CATGCCACTGAAG AAGCTCA-3' (R); CCR10, 5'-GGGTTTCTCCTTCCACTCCT-3' (F) and 5-TATTCCCCACATCCTCCTTG-3' (R); ß-actin, 5'-GATGATGATATCGCCGCGCT-3' (F) and 5-TGGGTCATCTTCTCGCGGTT-3' (R). Primers were used at a final concentration of 0.5 µmol/L, and the thermal cycling conditions were as follows: 5 min at 95°C, followed by 32 cycles of 95°C for 60 s, 55°C for 60 s for CYR61 and ß-actin or 55°C for 60 s for chemokine receptor genes, and finally 72°C for 60 s. The reactions were done in a Biometra Thermaoblock (Biometra, Inc.). PCR products were separated on a 1.2% agarose gel containing ethidium bromide (0.5 µg/mL), visualized, and photographed, and relative intensity of the specific gene expression was determined using the Alpha-Image Analysis System (Alpha Innotech).

Western Blot Analysis
Cells 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 µg/mL leupeptin, and 1 µg/mL aprotinin] for 20 min on ice, and clarified by centrifugation at 14,000 rpm for 20 min at 4°C, and the supernatant was collected. Equal amounts of protein were loaded onto 10% gradient SDS polyacrylamide gel separated and electrotransferred onto polyvinylidene 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 membranes for 4°C 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 autoradiography film (Perkin-Elmer Life Sciences).

Under-Agarose Cell Migration Assay
A 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 µL) was placed in six-well tissue culture plates and allowed to solidify for 1 h at 4°C. Three holes were punctured 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 x 105 cells per milliliter. Gastric cells (2 x 103 in 6 µL) were placed in the middle hole, recombinant IL-8 chemoattractant (30 ng/3 µL) 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 37°C 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 Assay
Chemotaxis assays were done using modified Boyden chambers with filter inserts for 24-well dishes containing 8-µm 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 µL 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 x 104 in 100 µL 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 37°C 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/40x field was quantitated by counting five random fields.

Transendothelial Migration Assay
AGS/CYR61-C7, AGS/CYR61-M, and AGS/neo transfectant cells were labeled with calcein AM (Molecular Probes) 4 µg/mL at 37°C for 90 min in calcein-labeling buffer. Cells were then washed twice with HBSS and resuspended. Calcein AM–labeled cells (2.5 x 104) were added to confluent HUVEC-coated wells in the upper chamber of the transwell, incubated with medium alone or medium containing neutralizing antibody, and then allowed to migrate for 20 h at 37°C 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 Assay
This 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 38°C 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. Subsequently, 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 x 106 cells in 15 µL 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 –80°C. 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'-ACGCCTGTAATCCCAGCACTT-3' (F), 5'-TCGCCCAGGCTGGAGTGC-3' (R)], which produced 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 µg of genomic DNA as template and was done under the following conditions: polymerase activation at 95°C for 10 min followed by 36 cycles at 95°C for 30 s, 52°C for 30 s, and 72°C 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'-GAGGAAAGGTCGCCTGGTGGATCG-3' (F), 5'-GGTGAGGACAAGCAGTGAGGAACG-3' (R)] using the same PCR conditions described for alu.

Ca2+ Mobilization
Cells 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 37°C 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 Analysis
Formalin-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 x 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 monoclonal (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.

Statistics
For statistical analysis, P values were based on two-sided, parametric Student's t tests using Excel software from Microsoft. 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 {chi}2 test. Pearson correlation coefficients were obtained using SAS software (Release 6.12; SAS Institute, Inc.).


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
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 of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/ 7/06; revised 7/ 8/07; accepted 7/13/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001;94:153–6.[CrossRef][Medline]
  2. Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 1999;80:827–41.[CrossRef][Medline]
  3. Oliveira FJ, Ferrao H, Furtado E, et al. Early gastric cancer: report of 58 cases. Gastric Cancer 1998;1:51–6.[CrossRef][Medline]
  4. Lin MT, Kung SP, Yeh SL, et al. The effect of glutamine-supplemented total parenteral nutrition on nitrogen economy depends on severity of diseases in surgical patients. Clin Nutr 2002;21:213–8.[Medline]
  5. Lin MT, Chou TH, Lai PS, et al. A New Surgical Technique: Mini-invasive surgery for radical subtotal gastrectomy by gasless laparoscopy-assist for early cancer. Hepatogastroenterology 2006;53:843–4.[Medline]
  6. Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2002;2:563–72.[CrossRef][Medline]
  7. Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001;410:50–6.[CrossRef][Medline]
  8. Murphy PM. Chemokines and molecular basis of cancer metastasis. N Engl J Med 2001;354:833–5.
  9. Lee J, Horuk R, Rice GC, et al. Characterization of two high affinity human interleukin-8 receptors. J Biol Chem 1992;267:16283–7.[Abstract/Free Full Text]
  10. Campbell JJ, Butcher EC. Chemokines in tissue-specific and microenvironment-specific lymphocyte homing. Curr Opin Immunol 2000;12:336–41.[CrossRef][Medline]
  11. Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity 2000;12:121–7.[CrossRef][Medline]
  12. Sun YX, Wang J, Shelburne CE, et al. Expression of CXCR4 and CXCL12 (SDF-1) in human prostate cancers (PCa) in vivo. Cell Biochem 2003;89:462–73.
  13. Scotton CJ, Wilson JL, Milliken D, et. al. Epithelial cancer cell migration: a role for chemokine receptors. Cancer Res 2001;61:4961–5.[Abstract/Free Full Text]
  14. Geminder H, Sagi-Assif O, Goldberg L, et al. A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma. J Immunol 2001;167:4747–57.[Abstract/Free Full Text]
  15. Juarez J, Bendall L. SDF-1 and CXCR4 in normal and malignant hematopoiesis. Histol Histopathol 2004;19:299–309.[Medline]
  16. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004;4:540–50.[CrossRef][Medline]
  17. Murphy C, McGurk M, Pettigrew J, et al. Nonapical and cytoplasmic expression of interleukin-8, CXCR1, and CXCR2 correlates with cell proliferation and microvessel density in prostate cancer. Clin Cancer Res 2005;11:4117–27.[Abstract/Free Full Text]
  18. Varney ML, Li A, Dave BJ, et al. Expression of CXCR1 and CXCR2 receptors in malignant melanoma with different metastatic potential and their role in interleukin-8(CXCL-8)-mediated modulation of metastatic phenotype. Clin Exp Metastasis 2003;20:723–31.[CrossRef][Medline]
  19. Kuwada Y, Sasaki T, Morinaka K, et al. Potential involvement of IL-8 and its receptors in the invasiveness of pancreatic cancer cells. Int J Oncol 2003;22:765–71.[Medline]
  20. Li A, Varney ML, Singh RK. Expression of interleukin 8 and its receptors in human colon carcinoma cells with different metastatic potentials. Clin Cancer Res 2001;7:3298–304.[Abstract/Free Full Text]
  21. Lau LF, Nathans D. Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells. EMBO J 1985;4:3145–51.[Medline]
  22. Brigstock DR. The connective tissue growth factor/cycteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev 1999;20:189–206.[Abstract/Free Full Text]
  23. Brigstock DR. The CCN family: a new stimulus package. J Endocrinol 2003;178:169–75.[Abstract]
  24. Lau LF, Lam SC. The CCN family of angiogenic regulators: the integrin connection. Exp Cell Res 1999;248:44–57.[CrossRef][Medline]
  25. Perbal B. CCN proteins: multifunctional signaling regulators. Lancet 2004;363:62–4.[CrossRef][Medline]
  26. Kireeva ML, Mo FE, Yang GP, et al. Cyr61, a product of a growth factor-inducible immediate-early gene, promotes cell proliferation, migration, and adhesion. Mol Cell Biol 1996;16:1326–34.[Abstract]
  27. Xie D, Nakachi K, Wang H, et al. Elevated levels of connective tissue growth factor, WISP-1, and CYR61 in primary breast cancers associated with more advanced features. Cancer Res 2001;61:8917–23.[Abstract/Free Full Text]
  28. Xie D, Yin D, Tong X, et al. Cyr61 is overexpressed in gliomas and involved in integrin-linked kinase-mediated Akt and ß-Catenin-TCF/Lef signaling pathways. Cancer Res 2004;64:1987–96.[Abstract/Free Full Text]
  29. Holloway SE, Beck AW, Girard L, et al. Increased expression of Cyr61 (CCN1) identified in peritoneal metastases from human pancreatic cancer. J Am Coll Surg 2005;200:371–7.[CrossRef][Medline]
  30. Lin MT, Zuon CY, Chang CC, et al. Cyr61 induces gastric cancer cell motility/invasion via activation of the integrin/nuclear factor-kappaB/cyclooxygenase-2 signaling pathway. Clin Cancer Res 2005;11:5809–20.[Abstract/Free Full Text]
  31. Lin MT, Chang CC, Chen ST, et al. Cyr61 expression confers resistance to apoptosis in breast cancer MCF-7 cells by a mechanism of NF-{kappa}B-dependent XIAP up-regulation. J Biol Chem 2004;279:24015–23.[Abstract/Free Full Text]
  32. Eck M, Schmausser B, Scheller K, et al. Pleiotropic effects of CXC chemokines in gastric carcinoma: differences in CXCL8 and CXCL1 expression between diffuse and intestinal types of gastric carcinoma. Clin Exp Immuno 2003;134:508–15.[CrossRef][Medline]
  33. Kwak MK, Hur K, Park DJ, et al. Expression of chemokine receptors in human gastric cancer. Tumour Biol 2005;26:65–70.[CrossRef][Medline]
  34. Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood 1999;94:3658–67.[Abstract/Free Full Text]
  35. Ramjeesingh R, Leung R, Siu CH. Interleukin-8 secreted by endothelial cells induces chemotaxis of melanoma cells through the chemokine receptor CXCR1. FASEB J 2003;17:1292–4.[Abstract/Free Full Text]
  36. Yamaguchi H, Ishii E, Tashiro K, et al. Role of umblical vein endothelial cells in hematopoiesis. Leuk Lymphoma 1998;31:61–9.[Medline]
  37. Menendez JA, Vellon L, Mehmi I, et al. A novel CYR61-triggered CYR61-{alpha}vß3 integrin loop' regulates breast cancer cell survival and chemosensitivity through activation of ERK1/ERK2 MAPK signaling pathway. Oncogene 2005;24:761–79.[CrossRef][Medline]
  38. Kohn AD, Kovacina KS, Roth RA. Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase. EMBO J 1995;14:4288–295.[Medline]
  39. Kireeva ML, Lam SC, Lau LF. Adhesion of human umbilical vein endothelial cells to the immediate-early gene product Cyr61 is mediated through integrin {alpha}vß3. J Biol Chem 1998;273:3090–6.[Abstract/Free Full Text]
  40. Leu SJ, Lam SC, Lau LF. Pro-angiogenic activities of CYR61 (CCN1) mediated through integrins {alpha}vß3 and {alpha}6ß1 in human umbilical vein endothelial cells. J Biol Chem 2002;277:46248–55.[Abstract/Free Full Text]
  41. Grzeszkiewicz TM, Kirschling DJ, Chen N, et al. CYR61 stimulates human skin fibroblast migration through integrin {alpha}v-ß5 and enhances mitogenesis through integrin {alpha}v-ß3, independent of its carboxyl-terminal domain. J Biol Chem 2001;276:21943–50.[Abstract/Free Full Text]
  42. Stupack DG, Cheresh DA. Get a ligand, get a life: integrins, signaling and cell survival. J Cell Sci 2002;115:3729–38.[Abstract/Free Full Text]
  43. Tsai MS, Bogart DF, Li P, et al. Expression and function of CYR61, an angiogenic factor, in breast cancer cell lines and tumor biopsies. Cancer Res 2002;60:5603–7.
  44. Tong X, Xie D, O'Kelly J, et al. Cyr61, a member of CCN family, is a tumor suppressor in non-small cell lung cancer. J Biol Chem 2001;276:47709–14.[Abstract/Free Full Text]
  45. Sampath D, Winneker RC, Zhang Z. Cyr61, a member of the CCN family, is required for MCF-7 cell proliferation: regulation by 17ß-estradiol and over expression in human breast cancer. Endocrinology 2001;142:2540–8.[Abstract/Free Full Text]
  46. Richards BL, Eisma RJ, Spiro JD, et al. Coexpression of interleukin-8 receptors in head and neck squamous cell carcinoma. Am J Surg 1997;174:507–12.[CrossRef][Medline]
  47. Kitadai Y, Haruma K, Mukaida N, et al. Regulation of disease-progression genes in human gastric carcinoma cells by interleukin 8. Clin Cancer Res 2000;6:2735–40.[Abstract/Free Full Text]
  48. Miller LJ, Kurtzman SH, Wang Y, et al. Expression of interleukin-8 receptors on tumor cells and vascular endothelial cells in human breast cancer tissues. Anticancer Res 1998;18:77–82.[Medline]
  49. Mashino K, Sadanaga N, Yamaguchi H, et al. Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric carcinoma. Cancer Res 2002;62:2937–41.[Abstract/Free Full Text]
  50. Gunn MD, Kyuwa S, Tam C, et al. Tobacco, alcohol and the risk of gastric cancer. A population-based case–control study in Sweden. Int J Cancer 1994;57:26–31.[Medline]
  51. Nicolson GL. Metastatic tumor cell attachment and invasion assay utilizing vascular endothelial cell monolayers. J Histochem Cytochem 1982;30:214–20.[Abstract]
  52. Cohen MC, Bereta M, Bereta J. Effect of cytokines on tumor cell-endothelial interactions. Indian J Biochem Biophys 1997;34:199–204.[Medline]
  53. Nicolson GL, Irimura T, Nakajima M, et al. Metastatic cell attachment to and invasion of vascular endothelium and its underlying basal lamina using endothelial cell monolayers. Symp Fundam Cancer Res 1983;36:145–67.[Medline]
  54. Chen N, Leu SJ, Todorovic V, et al. Identification of a novel integrin {alpha}vß3 binding site in CCN1 (CYR61) critical for pro-angiogenic activities in vascular endothelial cells. J Biol Chem 2004;279:44166–76.[Abstract/Free Full Text]
  55. Schober JM, Chen N, Grzeszkiewicz TM, et al. Identification of integrin {alpha}(M)ß(2) as an adhesion receptor on peripheral blood monocytes for Cyr61 (CCN1) and connective tissue growth factor (CCN2): immediate-early gene products expressed in atherosclerotic le