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Adhesion of Gastric Carcinoma Cells to ... - Cancer Research

Adhesion of Gastric Carcinoma Cells to ... - Cancer Research

INTEGRIN-MEDIATED CELL

INTEGRIN-MEDIATED CELL ADHESION TO PERITONEUMFig. 4. Adhesion of 3 integrin-expressing cellsto peritoneum. K3-9 (3 integrin transfectant) cellswere labeled with CMTMR (red fluorescence), andKR (mock transfectant) cells were labeled withCMFDA (green fluorescence). The mixture ofthese cells was incubated with excised parietal peritoneumat 37°C for 40 min. After the nonadherentcells were removed by gentle washing, the adherentcells were observed under fluorescence microscope.The same field was observed with differentfilter units: A, CMTMR-labeled K3-9 cells; B, CM-FDA-labeled KR cells.(245, 193, and 113 bp) were obtained (15). On the other hand, thedigestion of the 330-bp product with ApaI yielded two fragments (223and 107 bp; Ref. 16). These results indicate that mesothelial cells fromboth the parietal peritoneum and the diaphragm express mRNA forlaminin-5 and laminin-10/11, which are most likely to mediate theinteraction of 3-expressing carcinoma cells with the peritoneum.We then examined the effects of antibodies against laminin isoformson the adhesion of MKN1 cells to murine peritoneum. In thepresence of anti-laminin-5 polyclonal antibody (120 g/ml), the celladhesion was decreased to 55% that obtained in the control experiment(Fig. 7). Anti-laminin-1 antibody, however, showed no significantinhibition of the adhesion of MKN1 cells. The inhibition by theanti-laminin-5 antibody was significant but moderate, probably due tothe possible contribution of laminin-10/11 to the interaction of carcinomacells. It is likely that the combination of antibodies againstlaminin-5 and laminin-10/11 would inhibit cell adhesion more efficiently.This should be examined once anti-laminin-10/11 antibodybecomes available, but the results of the present study suggest thatlaminin-5 plays at least a partial role in the adhesion of 3 integrinexpressingcells to the peritoneum.DISCUSSIONFig. 5. Adhesion of 3 integrin-expressing cells to peritoneal mesothelial cells. K3-9(3 integrin transfectant) and KR (mock transfectant) cells were labeled with a fluorescentdye (BCECF-AM) and incubated with a monolayer of mesothelial cells at 37°C for 40min. The fluorescence intensity associated with the adherent cells was measured with afluorescence spectrophotometer. The assays were carried out in triplicate, and the errorbars indicate the SDs.The attachment of carcinoma cells to the peritoneum is an importantstep in the initial process of metastatic dissemination of gastriccancer. In this study, we presented evidence that 31 integrin playsa crucial role in the adhesion of cancer cells to the peritoneum: (a)Fig. 6. Analysis of mRNA for laminin 3 and 5 chains by the reverse transcription-PCR method. RNA was isolated from murine peritoneal mesothelial cells and subjectedto reverse transcription-PCR by the use of a set of primers for laminin 3 chain (Lanes 1and 2) and laminin 5 chain (Lanes 3 and 4) under the conditions described in Materialsand Methods. Lanes 1 and 3, mesothelial cells from parietal peritoneum; Lanes 2 and 4,mesothelial cells from diaphragm.6068Fig. 7. Effects of antibodies against laminin isoforms on the adhesion of MKN1 gastriccarcinoma cells to parietal peritoneum. Fluorescently labeled cells were overlaid onexcised peritoneum that had been treated with antibody against laminin-5 or laminin-1 at0°C for 30 min. After the cells and peritoneum were incubated at 37°C for 40 min, thenonadherent cells were removed by gentle washing three times with a warm medium. Theadherent cells were then lysed with 1% NP40, and the fluorescence intensity wasmeasured with a fluorescence spectrophotometer (Ex 490 nm, Em 520 nm). Theassays were carried out in triplicate. The error bars indicate the SDs.

inhibition of gastric carcinoma cell adhesion to excised peritoneum byspecific monoclonal antibodies (Fig. 1); (b) suppression of i.p. growthof NUGC-4 cells by pretreatment with anti-3 integrin antibody(Table 1); and (c) enhancement of the adhesion of K562 cells to theexcised peritoneum and to a monolayer of peritoneal mesothelial cellsby cDNA transfection (Figs. 3–5). In peritoneal mesothelial cells, wedetected the expression of mRNA for laminin-5 and laminin-10/11,which have recently been identified as high-affinity ligands for 31integrin (Fig. 6). Furthermore, the pretreatment of excised peritoneumwith antibody to laminin-5 significantly inhibited the adhesion ofcarcinoma cells (Fig. 7). With these findings taken together, we cansay that 31 integrin is a strong candidate as the mediation source inthe initial attachment of gastric cancer cells during their peritonealdissemination processes. These results may provide a biochemicalbasis for observations in a previous clinical report. In that report,histochemical studies on clinical specimens of gastric cancer associatedthe expression of 31 integrin with peritoneal metastasis (8).Another report suggested the importance of 21 integrin by virtue ofits significant expression on a larger proportion of tumor cells inperitoneal metastases than in primary tumors (9). The present studyalso showed 21 integrin to play a role in adhesion, although to alesser extent. Taken together, these findings suggest that the attachmentof cancer cells to peritoneum is mediated by multivalent interactionswith various adhesion molecules, including 21 and 31integrins, and that the individual adhesion molecules responsible forthe interaction might depend on the type of carcinoma. Nishimura etal. (21) and Kawamura et al. (22) also reported that the expression of21 and/or 31 integrin on gastric carcinoma cells was correlatedwith their peritoneal metastasizing potentials, i.e., highly metastaticcells expressed higher levels of these integrins as compared with theless-metastatic cells. These results, along with those of the presentstudy, strongly suggest that the initial attachment of cancer cells toperitoneal lining mediated by 21 and/or 31 integrin is a key steptoward the subsequent process of metastasis formation. Recently, Ishiiet al. (23) demonstrated the inverse correlation between the expressionlevel of 64 integrin and the frequency of peritoneal disseminationof gastric cancer from an immunohistochemical study. Thefinding is of interest because the expression of 64 integrin and31 integrin apparently had opposite effects even though theseintegrins share a common ligand, laminin-5. One possible explanationis that the intracellular signaling events occurring after the ligand isbound to 64 might differ from those occurring after binding to31 integrins.Our previous reports showed that the expression of 31 integrin atboth the protein and mRNA levels was increased after the oncogenictransformation of fibroblasts by SV40 or polyoma virus (24, 25). Anumber of studies have demonstrated that the increased expression of31 integrin in various tumor cells is correlated with their malignantphenotypes, including invasion and metastasis (26–32). Furthermore,the treatment of melanoma and glioma cells with antibodies against3 integrin was shown to inhibit their ability to migrate and invade(11, 26, 33). These studies suggest that 3 integrin is also involved inthe invasion of gastric carcinoma cells into the peritoneum followingtheir attachment to the peritoneal lining and are in agreement with ourresult that the anti-3 integrin antibody suppresses the i.p. growth ofNUGC-4 cells (Table 1). Our preliminary experiment also showedthat the i.p. implantation of 3 integrin-expressing cells (K3-9 cells)in severe combined immunodeficiency mice resulted in a poor survivalrate as compared with the implantation of mock transfected cells(KR cells). 33 H. Takatsuki, S. Komatsu, R. Sano, and T. Tsuji, unpublished observation.INTEGRIN-MEDIATED CELL ADHESION TO PERITONEUM6069The integrin-extracellular matrix interaction facilitates the transductionof signals into cells. This leads to regulation of the productionof extracellular matrix-degrading enzymes. It has recently been reportedthat the treatment of invasive mammary carcinoma cells withan anti-3 integrin antibody reduced the production of matrix metalloproteinase(MMP)-9 (34). In human glioma and rhabdomyosarcomacells, anti-3 integrin antibodies enhanced their invasion through theMatrigel in association with the increased levels of MMP-2 secretion(35, 36), suggesting that the antibody acted as an agonist. In addition,laminin-5 was shown to induce MMP-9 from human melanoma cells(11). Laminin-5 was also found to be specifically cleaved by MMP-2and MT1-MMP and the modified laminin-5 induced migration ofbreast epithelial cells (37, 38). These observations suggest that theexpression of extracellular matrix-degrading enzymes is regulated, atleast in part, through the integrin-mediated pathways.Recently, we found that the Ets family of transcription factorsregulated the expression of 3 integrin (39). The Ets transcriptionfactors were also reported to be involved in tumor metastasis throughthe promotion of angiogenesis and the expression of MMPs (40–42).These observations suggest that 31 integrin and MMPs cooperativelypromote adhesion and invasion of cancer cells during themetastatic processes. In conclusion, the present study strongly suggeststhat the up-regulation of 31 integrin on gastric carcinomacells facilitates their adhesion to the peritoneum and the subsequentdevelopment of peritoneal dissemination.ACKNOWLEDGMENTSWe thank Miki Ogiwara, Kunie Tanaka, Manami Nishiwaki, and MasashiTakada for their technical assistance.REFERENCES1. Varner JA, Cheresh DA. Integrins and cancer. Curr Opin Cell Biol 1996;8:724–30.2. Ruoslahti E. Fibronectin and its integrin receptors in cancer. Adv Cancer Res1999;76:1–20.3. Orr FW, Wang HH, Lafrenie RM, Scherbarth S, Nance DM. Interactions betweencancer cells and the endothelium in metastasis. J Pathol 2000;190:310–29.4. Holly SP, Larson MK, Parise LV. Multiple roles of integrins in cell motility. Exp CellRes 2000;261:69–74.5. Felding-Habermann B. Integrin adhesion receptors in tumor metastasis. Clin ExpMetastasis 2003;20:203–13.6. Yashiro M, Chung YS, Nishimura S, Inoue T, Sowa M. Peritoneal metastatic modelfor human scirrhous gastric carcinoma in nude mice. Clin Exp Metastasis 1996;14:43–54.7. Nakashio T, Narita T, Akiyama S, et al. Adhesion molecules and TGF-1 areinvolved in the peritoneal dissemination of NUGC-4 human gastric cancer cells. IntJ Cancer 1997;70:612–8.8. Ura H, Denno R, Hirata K, Yamaguchi K, Yasoshima T. Separate functions of 21and 31 integrins in the metastatic process of human gastric carcinoma. Surg Today1998;28:1001–6.9. Matsuoka T, Yashiro M, Nishimura S, et al. Increased expression of 21 integrin inthe peritoneal dissemination of human gastric carcinoma. Int J Mol Med 2000;5:21–5.10. Takeuchi K, Tsuji T, Hakomori S, Irimura T. Intercellular adhesion induced byanti-3 integrin (VLA-3) antibodies. Exp Cell Res 1994;211:133–41.11. Tsuji T, Kawada Y, Kai-Murozono M, et al. Regulation of melanoma cell migrationand invasion by laminin-5 and 31 integrin (VLA-3). Clin Exp Metastasis 2002;19:127–34.12. Takada Y, Hemler ME. The primary structure of the VLA-2/collagen receptor 2subunit (platelet GPIa): homology to other integrins and the presence of a possiblecollagen-binding domain. J Cell Biol 1989;109:397–407.13. Izumi Y, Taniuchi Y, Tsuji T, et al. Characterization of human colon carcinomavariant cells selected for sialyl Le X carbohydrate antigen: liver colonization andadhesion to vascular endothelial cells. Exp Cell Res 1995;216:215–21.14. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidiniumthiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–9.15. Galliano MF, Aberdam D, Aguzzi A, Ortonne JP, Menehguzzi G. Cloning andcomplete primary structure of the mouse laminin 3 chain. Distinct expression patternof the laminin 3A and 3B chain isoforms. J Biol Chem 1995;270:21820–6.16. Miner JH, Lewis RM, Sanes JR. Molecular cloning of a novel laminin chain, 5, andwidespread expression in adult mouse tissues. J Biol Chem 1995;270:28523–6.17. Carter WG, Ryan MC, Gahr PJ. Epiligrin, a new cell adhesion ligand for integrin31 in epithelial basement membranes. Cell 1991;65:599–610.

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