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Clinical Cancer Research Vol. 8, 2455-2462, July 2002
© 2002 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

A New Type of Antimetastatic Peptide Derived from Fibronectin1

Rina Kato, Tetsuya Ishikawa, Sadahiro Kamiya, Fumiko Oguma, Masaaki Ueki, Sumio Goto, Hiroshi Nakamura, Takashi Katayama and Fumio Fukai2

Departments of Patho-Physiology [R. K., T. I., S. K., F. O., T. K., F. F.] and Analytical Chemistry [R. K., S. K., H. N.], Faculty of Pharmaceutical Sciences, Science University of Tokyo, Tokyo 162-0826; Department of Applied Chemistry, Faculty of Science, Science University of Tokyo, Tokyo 162-8601 [M. U.]; and Research Center for Material Cycles and Waste management, National Institute of Environmental Studies, Tsukuba-shi, Ibaraki 305-0053 [S. G.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: We found previously that fibronectin (FN) has a cryptic functional site (YTIYVIAL sequence within the 14th type III repeat) opposing cell adhesion to extracellular matrix. A 22-mer FN peptide containing this site, termed FNIII14, inhibits ß1 integrin-mediated adhesion without binding to integrins. The present study shows that FNIII14 has the potential to prevent lymphoma cell metastasis.

Experimental Design: Antimetastatic effect of FNIII14 has been evaluated through in vitro or in vivo experiments.

Results: FNIII14 inhibited the integrin {alpha}4ß1-mediated B lymphoma Ramos cell adhesion to VCAM-1 on venule endothelial cells, as well as to FN. Murine T lymphoma L5178Y-ML25 cells, which are known to metastasize to liver and spleen, preferentially adhered to vitronectin (VN) and migrated toward VN concentration gradients. FNIII14 abrogated both the integrin {alpha}vß3-mediated adhesion and migration of L5178Y-ML25 cells. Inhibition of the {alpha}vß3mediated L5178Y-ML25 cell adhesion by FNIII14 was reversed by phenylarsine oxide, a protein tyrosine phosphatase inhibitor. In addition, FNIII14 abrogated the VN-stimulated tyrosine phosphorylation of intracellular signaling proteins, including focal adhesion kinase (p125FAK) and paxillin, suggesting that such a diversity of FNIII14 effects might be because of the negative regulation of p125FAK and paxillin tyrosine phosphorylation, which has been involved in adhesion signals transduced by different integrins. The in vivo experiment using a murine metastasis model showed that FNIII14 would inhibit liver and spleen metastases of L5178Y-ML25 cells at a dose much lower than that of RGDS.

Conclusions: FNIII14 might be applicable as a new type of antimetastatic agent distinct from integrin-binding peptides.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Most of the cancer patients die because of metastasis and invasion of tumor cells. Metastatic formation requires specific cell-to-cell and cell-to-ECM3 interactions that are mediated by several classes of adhesion molecules, such as cadherins, selectins, integrins, and immunoglobulin superfamily (1, 2, 3, 4, 5) . Particularly, integrin-mediated adhesion of tumor cells to ECM proteins and cell surface components occurs at several stages of metastasis and is therefore thought to be a crucial event in metastasis. Accordingly, prevention of tumor cell adhesion to ECM proteins has been a potential target for therapeutic intervention (so-called "antiadhesion therapy"; Refs. 6, 7, 8 ).

Attempts to realize "antiadhesion therapy" have been undertaken predominantly by using synthetic integrin-binding peptides derived from cell adhesive sites of ECM proteins, including FN and LM. A number of previous studies has proposed that synthetic peptides containing the RGD cell adhesion motif common in several ECM proteins, including FN, have the potential to prevent tumor metastasis (7 , 9, 10, 11) . Efficacy of the LM peptide, YIGSR, as an antimetastatic peptide has also been reported by many investigators (12, 13, 14, 15) . However, these previous studies have also indicated that high doses of peptides (~3 mg/mouse) without any chemical modifications would be needed for full inhibition of metastasis on the murine experimental models, although an LM peptide YIGSR had a considerably high ability to inhibit lung metastasis of B16F10 melanoma cells with a minimum chemical modification, i.e., amide form (12) . This would be explained by low binding affinity of parental ECM proteins for the corresponding receptor integrins (kDa = 10-6 ~ 10-8 M) and further decrease in the binding affinity because of cutting short ECM proteins into small peptides (16, 17, 18) . It is also presumed that competitive inhibition of tumor cell adhesion to ECM requires high doses of integrin-binding peptides to conquer the binding affinity of integrin to ECM. To overcome these difficulties, various chemical modifications of the integrin-binding peptides to enhance their binding affinity for integrins have been performed (9 , 10 , 14 , 15 , 19, 20, 21, 22, 23) .

FN, a major component of ECM, has several active sites that serve as scaffoldings for cell anchorage. We have found that FN also has a functional site opposing cell adhesion to FN (24, 25, 26) ; a synthetic peptide derived from the 14th type III module (residues 1835–1855 of FN, termed FNIII14) strongly weakens the integrin {alpha}5ß1-mediated adhesion of various cell types to FN substrate in a reversible manner. FNIII14 loses the antiadhesive activity when its COOH-terminal sequence YTIYVIAL is scrambled, indicating that the effects are specific in the YTIYVIAL sequence. This antiadhesive site is buried within the type III module structure at least in plasma FN but exposed either by interaction of FN with sulfated glycosaminoglycan, including heparin, or by processing of FN with matrix metalloproteinase-2 (27) . Unlike integrin-binding peptides, such as RGDS and YIGSR, FNIII14 can inhibit cell adhesion to FN substrate when immobilized on culture plates or when added into culture media as a soluble supplement (24 , 25 , 27) , suggesting the presence of a specific receptor that mediates the FNIII14 effects. Our recent results have shown that FNIII14 induces the conformation change of ß1 integrin from active to resting, as confirmed by flow cytometric analysis using an mAb (AG89) to an active ß1 integrin-specific epitope, in which FNIII14 specifically binds to a 55-kDa membrane protein but not to integrins (28) . It has been demonstrated that this AG89 reacts with human integrin ß1 chain, regardless of the {alpha} subunits (29, 30, 31) . This implies that FNIII14 has the potential to inhibit cell adhesion mediated by any of ß1 integrins. In addition, it has also been shown that FNIII14 is capable of preventing cellular interactions with VN and plasma and cellular FNs through {alpha}vß3 and {alpha}5ß1 (26 , 32) . Although a more precise mechanism by which FNIII14 expresses the antiadhesive activity remains elusive, such extended ECM specificity and potent anti-integrin activity prompted us to test the ability of our peptide as an antimetastatic agent.

In this study, we investigate whether FNIII14 can prevent lymphoma cell metastasis, using the in vitro and in vivo assay systems. The in vitro data show that FNIII14 is capable of inhibiting various types of adhesive interactions involved in metastatic formation. The in vivo study using the murine metastasis model demonstrates that FNIII14 inhibits liver and spleen metastases of L5178Y-ML25 murine T lymphoma cells at doses much lower than that of a RGD-containing peptide. This high efficacy of FNIII14 as an antimetastatic agent would be interpreted by its action mechanism totally different from those of integrin-binding peptides.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human plasma FN (33) and VN (34) were purified as described previously. A synthetic peptide RGDS was purchased from Sigma. Peptide FNIII14 corresponding to residues 1835–1855 of FN (25) and its analogous inactive peptide FNIII14scr were obtained from Sawady Technology (Tokyo, Japan). Antiphosphotyrosine (RC20), paxillin (clone 349), and integrin {alpha}5 (A5-PUJ2) mAb were from Transduction Laboratories. Antiphosphotyrosine mAb (P-Tyr-100) was from Cell Signaling Technology. Anti-integrin {alpha}4 mAb (SG/73) was from Seikagaku Corp. pAb recognizing the phosphorylation site pTyr (397) of FAK (p125FAK; anti-FAK[pY (397)] pAb) was from Biosource International. Antimouse integrin {alpha}v (H9.2B8) and ß3 (2C9.G2) mAb were obtained from BD PharMingen. Antihuman VCAM-1 mAb was from Upstate Biotechnology.

Cell Culture.
Murine T lymphoma cells L5178Y-ML25 and human Burkitt’s lymphoma cells Ramos were cultured with RPMI 1640, supplemented with 10% FBS. HUVECs were grown in MCDB-105 medium supplemented with 10% FBS and EGM-2 supplements and growth factors (Clonetics, Sanko Junyaku, Japan) on culture plates coated with gelatin.

Cell Adhesion Assay.
Cell adhesion to various ECM protein substrates was assayed as described earlier (25 , 26) . Assay of Ramos cell adhesion to venule endothelial cells was carried out as reported by Elices et al. (35) . Briefly, confluent monolayers of HUVECs were prepared in 96-well tissue culture plates coated with gelatin and incubated for 24 h with or without TNF-{alpha} (10 ng/ml). Ramos cells (2 x 105) suspended with RPMI 1640 were preincubated with various concentrations of FNIII14, RGDS, or anti-integrin mAb at 37°C for 1 h, added onto the HUVEC monolayers in the presence or absence of 0.1 mM Mn2+, and then cultured for 1 h. After washing nonadhered cells off, the number of Ramos cells attached to HUVEC layer was counted microscopically (27) .

Cell Motility Assay.
Cell motility was assayed in blind-well chambers as reported by Yun et al. (36) , with some modifications (37) . Briefly, polyvinyl pyrrolidone-free polycarbonate filters (3-µm pore; Nucleopore) were coated with EHS gel (BTI-Biomatrix I; 0.2 mg of protein/ml). L5178Y-ML25 cells (1.2 x 106) suspended in RPMI 1640 containing 10% FBS and Mn2+ (0.1 mM) were treated with various concentrations of FNIII14 or anti-ß3 mAb (2C9.G2; 20 µg/ml) at 37°C for 1 h and then added to the top chambers. Various concentrations of VN or FN in RPMI 1640 containing 10% FBS and Mn2+ (0.1 mM) were placed into the bottom chamber (200 µl). After incubation for 2 h, cells transmigrated to the bottom chamber were counted.

Protein Tyrosine Phosphorylation.
L5178Y-ML25 cells were deprived of serum for 10 h and preincubated in RPMI 1640 with or without PAO (20 µM) for 5 min. The cells were further incubated with latex beads (0.5–µm) coated with VN (50 µg/ml) and 0.1 mM MnCl2 in the presence or absence of peptide FNIII14 (200 µg/ml) for 20 min. The cells were lysed with lysis buffer [1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride in 20 mM Tris-HCl buffer (pH 7.4)]. Cell lysates were immunoprecipitated with antiphosphotyrosine antibody (P-Tyr-100) and antipaxillin mAb using protein G Sepharose, respectively. Immunoprecipitated proteins derived from the equal number of cells were separated on SDS-PAGE, followed by electroblotting to polyvinylidene difluoride membrane. p125FAK and paxillin with phosphorylated tyrosines were detected by anti-FAK[pY (397)] pAb-(POD-labeled antirabbit IgG antibody) and biotinylated antiphosphotyrosine mAb (RC20)-(POD-conjugated streptavidin), respectively, followed by chemiluminescence detection (Enhanced Chemiluminescence Plus; Amersham Pharmacia Biotech).

Experimental Liver and Spleen Metastases.
CDF1 mice (6 weeks old; 5 mice/group) were given i.v. injection of L5178Y-ML25 cells (2 x 105) with or without peptide FNIII14, control peptide FNIII14scr, or RGDS in PBS (200 µl). Sixteen days later, the mice were sacrificed under anesthesia, and the liver and spleen weights were recorded to evaluate metastasis (38 , 39) . The livers and spleens were fixed with formalin, and their 4–µm sections were stained with H&E to evaluate metastasis histologically.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Peptide FNIII14 Inhibits Integrin {alpha}4ß1-mediated Cell Adhesion to VCAM-1 as Well as FN Substrate.
Integrin {alpha}4ß1 is considered to play an essential role in leukocyte infiltration, lymphocyte homing, and even tumor cell invasion. We first examined the effects of FNIII14 on {alpha}4ß1-mediated cell adhesion using Ramos cells that express mainly {alpha}4ß1 as a ß1 integrin (35) . Ramos cell adhesion to FN substrate in the presence of an integrin activator Mn2+ was blocked by {alpha}4 function-blocking mAb (SG/73) but not by {alpha}5-inhibiting mAb (A5-PUJ2; Fig. 1ACitation ). FNIII14, but not RGDS, inhibited the Ramos cell adhesion in a dose-dependent manner (Fig. 1A)Citation . An analogous peptide FNIII14scr, in which the YTIYVIAL sequence of FNIII14 is scrambled, had no antiadhesive activity (Fig. 1A)Citation . Ramos cell adhesion to FN induced by other integrin activators, such as phorbol myristate acetate (40) and ß1 integrin-activating mAb AG89 (29, 30, 31) , were also inhibited by FNIII14 (data not shown), excluding the possibility that FNIII14 served as a scavenger of Mn2+.



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  		Fig. 1. Peptide FNIII14 inhibits integrin {alpha}4ß1-mediated Ramos cell adhesion to FN and HUVEC monolayer. A, adhesion to FN substrate. Ramos cell suspensions (2 x 105/100 µl) with or without Mn2+ (0.1 mM) were pretreated with mAb (25 µg IgG/ml) to integrin {alpha}4 (SG/73) or {alpha}5 (A5-PUJ2), FNIII14 (50 and 100 µg/ml), FNIII14scr (100 µg/ml), and RGDS (300 µg/ml), respectively, and then seeded to a 96-well plate coated with or without FN (0.5 µg/ml). B, adhesion to HUVEC monolayer. Ramos cell suspensions (2 x 105/100 µl) with or without Mn2+ were preincubated with FNIII14 (50, 100 µg/ml), FNIII14scr (100 µg/ml), RGDS (500 µg/ml), and mAb (25 µg IgG/ml) directed to {alpha}4 (SG/73), VCAM-1 (VC-1), or {alpha}5 (A5-PUJ2), respectively, and then seeded onto HUVEC monolayer. Ramos cells adhered to FN substrate or HUVEC monolayer were counted as described previously (26) . Data represent the mean ± SE of four determinations.

 
Integrin {alpha}4ß1 serves also as a receptor for VCAM-1, which is expressed on cytokine-activated endothelial cells (35) . Ramos cells adhered to HUVECs only when Ramos cells and HUVECs were pretreated with Mn2+ and TNF-{alpha}, respectively (Fig. 1B)Citation . This Ramos cell adhesion to HUVECs was prevented by either anti-{alpha}4 (SG/73) or anti-VCAM-1(VC1) mAb, demonstrating that Ramos cells adhered to HUVECs through {alpha}4ß1-VCAM-1 interaction. FNIII14 also inhibited this Ramos cell adhesion to HUVEC in a dose-dependent manner. Because this inhibition was reversed by removing FNIII14 from culture media and because viability of nonadhered cells in the presence of FNIII14 exceeded 90% (data not shown) as judged by trypan blue exclusion test, FNIII14 reversibly disrupted Ramos cell adhesion to HUVECs without affecting cell viability. FNIII14scr and RGDS showed no significant effect on Ramos cell adhesion to HUVECs (Fig. 1B)Citation .

Peptide FNIII14 Inhibits L5178Y-ML25 T Lymphoma Cell Adhesion and Migration Induced by Integrin {alpha}vß3.
We next assessed antiadhesive effects of FNIII14 using L5178Y-ML25 T lymphoma cells that highly metastasize to liver and spleen (38) . As shown in Fig. 2ACitation , L5178Y-ML25 cells adhered well to VN substrate, depending on Mn2+, slightly to FN substrate, and hardly to types I and IV collagen and LM substrates. L5178Y-ML25 cell adhesion to VN substrate was blocked by anti-{alpha}v (H9.2B8) and ß3 (2C9.G2) mAb. FNIII14, but not its control FNIII14scr, inhibited dose dependently L5178Y-ML25 cell adhesion to VN substrate (Fig. 2B)Citation . RGDS also inhibited adhesion of L5178Y-ML25 cells to VN substrate but required high concentrations in molar basis as compared with FNIII14 (Fig. 2B)Citation . FNIII14 did not affect nonspecific adhesion of L5178Y-ML25 cells to poly-L-Lys substrate (Fig. 2B)Citation . L5178Y-ML25 cells failed to adhere to HUVECs when treated with TNF-{alpha}, even in the presence of Mn2+ (data not shown).



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  		Fig. 2. Peptide FNIII14 inhibits L5178Y-ML25 cell adhesion to VN substrate. In A, L5178Y-ML25 cell suspension (2 x 105) with or without Mn2+ (0.1 mM) was added to a 96-well plate coated with either BSA, LM, type I collagen (20 µg/ml), type IV collagen (20 µg/ml), FN (2 µg/ml), or VN (2 µg/ml). In B, L5178Y-ML25 cell suspension containing Mn2+ was pretreated with or without 25 µg/ml control IgG and function-blocking mAb to {alpha}v (H9.2B8) or ß3 (2C9.G2), respectively, and then seeded to a 96-well plate coated with VN (2 µg/ml) or poly-L-Lys (20 µg/ml) in the presence or absence of FNIII14 [22 and 44 µM (50 and 100 µg/ml)], RGDS [152 and 760 µM (100 and 500 µg/ml)], and FNIII14scr (88 µM), respectively. After the incubation at 37°C for 1 h, cells were fixed with formalin, and unadhered cells were washed off with PBS (26) . Cells adhered to substrates were stained with crystal violet. Dye bound to adhered cells was solubilized with 0.1% SDS, and absorbance at 590 nm was measured. Data represent the mean ± SE of four determinations.

 
Because both VN and FN are secreted by hepatocytes, the above data may point out the possibility that VN and FN attract L5178Y-ML25 cells to the liver. We then performed the cell motility assay using blind-well chambers. L5178Y-ML25 cells migrated across the EHS gel-coated filter into the bottom chamber in response to the increasing concentrations of VN (Fig. 3)Citation , which was prevented by anti-ß3 mAb (2C9.G2), indicating that ligation of {alpha}vß3 induced the migration of L5178Y-ML25 cells. FNIII14 inhibited dose dependently the VN-induced, integrin {alpha}vß3-mediated migration of L5178Y-ML25 cells. FNIII14scr did not affect the migration of L5178Y-ML25 cells (data not shown). FN was incapable of attracting L5178Y-ML25 cells (Fig. 3)Citation .



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  		Fig. 3. Peptide FNIII14 inhibits L5178Y-ML25 cell migration toward VN. Cell motility assay was carried out in blind-well chambers. The bottom chambers were filled with the indicated concentrations of VN (200 µl). L5178Y-ML25 cells (5 x 105) treated with either FNIII14 (50, 100 µg/ml), control IgG, or anti-ß3 mAb (2C9.G2; 20 µg/ml) were added to the top chambers. After the 2-h incubation, the number of cells migrating across the EHS gel-coated filter into the bottom chamber was counted as described in "Materials and Methods." Data represent the mean ± SE of three determinations.

 
Peptide FNIII14 Down-Regulates Protein Tyrosine Phosphorylation of Focal Adhesion Proteins.
The present data, taken together with previous data, show that FNIII14 is capable of inhibiting cell adhesion mediated by different integrins, such as {alpha}5ß1, {alpha}4ß1, and {alpha}vß3. On the other hand, protein tyrosine phosphorylation of focal adhesion proteins is considered to play a crucial role in integrin-mediated signaling events. To explain the extended integrin specificity of FNIII14, we examined whether antiadhesive effect of FNIII14 could be involved in modulation of protein tyrosine phosphorylation. First, we tested the effect of PAO, a protein tyrosine phosphatase inhibitor (41) , on the antiadhesive activity of FNIII14. As shown in Fig. 4ACitation , inhibitory effect of FNIII14 on L5178Y-ML25 cell adhesion to VN substrate was reversed by the addition of PAO, although PAO itself had no effect on L5178Y-ML25 cell adhesion. Next, we observed tyrosine phosphorylation of focal adhesion proteins involved in adhesion signaling (Fig. 4, B and C)Citation . When L5178Y-ML25 cells were stimulated with VN-coated latex beads in the presence of Mn2+, stimulation of p125FAK and paxillin tyrosine phosphorylation were detected (Fig. 4, B and C)Citation . When stimulation of L5178Y-ML25 cells with VN-coated beads was carried out in the presence of FNIII14, tyrosine phosphorylation of p125FAK and paxillin remained only at basal levels. The decreased tyrosine phosphorylations of p125FAK and paxillin were restored by the addition of PAO (Fig. 4, B and C)Citation . A band of ~90 kDa in lane 3 of Fig. 4BCitation may be because of a nonspecific binding of the secondary antibody, because this band was irregularly detected in other lanes when incubated exceedingly with the secondary antibody (data not shown).



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  		Fig. 4. Implication of peptide FNIII14 action in tyrosine phosphorylation of focal adhesion proteins. A, effect of PAO on the antiadhesive activity of FNIII14. Adhesion of L5178Y-ML25 cells to VN substrate was assayed under the indicated conditions as described in Fig. 1Citation . B and C, protein tyrosine phosphorylation. L5178Y-ML25 cell suspensions with Mn2+ were incubated with VN-coated latex beads in the absence or presence of FNIII14 (100 µg/ml) at 37°C for 20 min under the indicated conditions. Cell lysates were immunoprecipitated with antiphosphotyrosine (B) or antipaxillin (C) mAb using protein G-Sepharose. After the SDS-PAGE, phosphotyrosyl proteins electroblotted to polyvinylidene difluoride membrane were visualized by anti-FAK[pY (397)] pAb (POD-labeled antimouse IgG; B) or biotinylated antiphosphotyrosine mAb (POD-conjugated streptavidin; C), followed by the detection by enhanced chemiluminescence, as described in "Materials and Methods."

 
Peptide FNIII14 Inhibits Experimental Metastasis of L5178Y-ML25 T Lymphoma Cells.
On the basis of these in vitro data, we finally examined the effect of FNIII14 on liver and spleen metastases of L5178Y-ML25 T lymphoma cells (38) . When L5178Y-ML25 cells were injected i.v. into CDF1 mice, liver and spleen weights of the mice increased 3–4-fold compared with those of normal mice (Table 1)Citation , suggesting metastases of L5178Y-ML25 cells to the liver and spleen. Changes in weights of other organs were not found (data not shown). Histological observation also indicated massive invasion and propagation into the liver and spleen of mice injected with L5178Y-ML25 cells (Fig. 5, A and DCitation , tissues from normal mice; B and E, tissues from mice inoculated with L5178Y-ML25 cells). Then, effects of coinjection of the antiadhesive peptides at high doses with L5178Y-ML25 cells on metastasis were examined. FNIII14 [700 µg (300 nmol)/mouse] inhibited almost completely the increases in liver and spleen weights (Table 1)Citation . The control peptide FNIII14scr, inactive in cell adhesion, had only a negligible effect on the increases in these organ weights. RGDS [1000 µg (1520 nmol)/mouse] inhibited significantly, but not completely, the increases in liver and spleen weights (70 and 76% inhibitions of liver and spleen weights, respectively). Histological observation showed no significant invasion and propagation of L5178Y-ML25 cells throughout the liver (Fig. 5C)Citation and spleen (Fig. 5F)Citation sections, when L5178Y-ML25 cells were inoculated with FNIII14. The areas of metastatic foci were compared in these liver and spleen sections between the mice administered with or without FNIII14 (Table 2)Citation . The data also showed the potent inhibitory effect of FNIII14 on the liver metastasis of L5178Y-ML25 cells, indicating the close correlation between the increases in the liver and spleen weights and metastases.


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  		Table 1 Inhibition of the liver and spleen metastases of L5178Y-ML25 cells by peptide FNIII14 and RGDS as judged by alteration of tissue weights

 


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  		Fig. 5. Liver and spleen metastases of L5178Y-ML25 cells. H&E staining of liver and spleen sections from normal mouse (A and D), mouse inoculated with L5178Y-ML25 cells alone (B and E), or mouse inoculated with L5178Y-ML25 cells in the presence of FNIII14 (700 µg; C and F). Photographs of the liver and spleen sections with x200 and x36 magnifications, respectively. In the liver section (B), massive invasion and propagation of the lymphoma cells were observed in the lobule. In the spleen section (E), within the pulp was displaced widely by the lymphoma cells, leaving a few white pulps.

 

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  		Table 2 Inhibition of the liver and spleen metastases of L5178Y-ML25 cells by peptide FNIII14, as judged by the area of metastatic foci

 
To compare the antimetastatic abilities of FNIII14 and RGDS, effects of decreasing doses (in molar basis) of these peptides on the increases in liver and spleen weights were examined (Fig. 6)Citation . RGDS at <305 nmol had only a little or no effect on the increases in liver and spleen weights: 20–25% inhibition at 305 nmol (200 µg) and no inhibition at 76 nmol (50 µg). In contrast, FNIII14 prevented the metastases strongly at 88 nmol (200 µg) and considerably (by 80% in liver and 84% in spleen) at 22 nmol (50 µg). The inhibitory effects of FNIII14 at 22 nmol was comparable or slightly superior to that of RGDS at 1520 nmol.



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  		Fig. 6. Comparison of antimetastatic effects between peptide FNIII14 and RGDS. L5178Y-ML25 cells were injected i.v. with the indicated doses of FNIII14 [22 nmol (50 µg) and 88 nmol (200 µg)] (open symbols) or RGDS [76 nmol (50 µg) and 305 nmol (200 µg)] (closed symbols) to CDF1 mice (n = 5). Ordinate represents the percentage of inhibition relative to the increases in the liver (•) and spleen ({blacktriangleup}) weights of control mice. Bars, SD. *, P < 0.001; **, P < 0.05 compared with the increases in liver and spleen weights (control minus normal).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrated that the antiadhesive peptide derived from FN, FNIII14, had the ability to prevent liver and spleen metastases of L5178Y-ML25 T lymphoma cells on the murine metastasis model. This high efficacy might be explained by functional properties of FNIII14 totally distinct from the integrin-binding peptides.

First, taken together with previous results (25, 26, 27 , 32) , FNIII14 is capable of disrupting various types of adhesive interactions mediated by different integrins, such as {alpha}5ß1, {alpha}4ß1, and {alpha}vß3. Of these results, inhibitory effects of FNIII14 on the {alpha}4ß1- and {alpha}vß3-mediated cellular interactions should be emphasized. FNIII14 inhibited tumor cell adhesion to HUVEC that could not be disrupted by RGDS. Although we failed to detect the L5178Y-ML25 cell adhesion to endothelial cells in this study, tumor cell tethering and subsequent tight adhesion to venule endothelial cells would be essentially needed for the metastases. With regard to the {alpha}vß3-mediated interaction, FNIII14 inhibited the tumor cell migration toward VN concentration gradient, as well as adhesion to VN substrate. RGDS also inhibited L5178Y-ML25 cell adhesion to VN substrate, whereas the ability to inhibit it was much lower than that of FNIII14. Nicolson et al. (36) have reported a crucial role of {alpha}vß3 in the liver-specific metastasis of RAW large cell lymphoma. It appears likely that L5178Y-ML25 cells are attracted to hepatic sinusoids and further into the space of Disse, where VN is being excreted. Additionally, it has been reported that human hairy cell leukemic cells expressing {alpha}vß3 as one of the major integrins predominantly colonize in spleen, as well as liver (42) . Prevention of the {alpha}vß3-mediated interactions by FNIII14 might inhibit the targeting of L5178Y-ML25 cells to the liver and spleen. Thus, FNIII14 is capable of disrupting a variety of adhesive interactions involved in metastatic formation. Such a multiple integrin specificity and potent anti-integrin activity might enable FNIII14 to efficiently prevent the liver and spleen metastases of L5178Y-ML25 cells.

Second, these characteristics of FNIII14 might be based on its unique action mechanism entirely different from those of integrin-binding peptides, such as RGDS and YIGSR. We have shown previously that FNIII14 inhibits cell adhesion to ECM, without binding to integrin, even when immobilized on culture plates, as well as when added as a soluble supplement (24, 25, 26) . In addition, FNIII14 has been shown to inhibit cell adhesion regardless of RGD cell adhesive motif. The present study showed that the antiadhesive effect of FNIII14 was blocked by a protein tyrosine phosphatase inhibitor PAO, indicating that the FNIII14 effect is involved in protein tyrosine phosphorylation. Supporting this, FNIII14 abrogated the tyrosine phosphorylation of signaling proteins, including p125FAK and paxillin, that has been presumed to play an important role in adhesion signalings transduced by different integrins (8 , 41 , 43, 44, 45, 46) . Taken altogether, it appears likely, although not completely proven, that FNIII14 abrogates tyrosine phosphorylation of focal adhesion proteins and consequently disrupts cell-to-ECM adhesion mediated by any of integrins {alpha}5ß1, {alpha}4ß1, and {alpha}vß3. Our recent results have shown that FNIII14 induces the conformation change of ß1 integrin from active to resting, as confirmed by flow cytometric analysis using an mAb (AG89) to active ß1 integrin-specific epitope. On the other hand, FNIII14 has been shown to specifically bind to a 55-kDa membrane protein but not to integrins (28) . The 55-kDa membrane protein is an important candidate for a receptor mediating the FNIII14 effects. However, a signaling pathway of FNIII14, in consequence of the binding to this putative membrane receptor, is still unclear. Moreover, despite the reliability of FNIII14 effects on tumor cell adhesive events, the actual action site(s) of the peptide awaits identification. Identification of the 55-kDa membrane protein might provide important information to define biological effects of FNIII14. It would be necessary to investigate the effects of FNIII14 on other cellular events involved in metastasis, such as tumor cell growth, angiogenesis, and platelet tumor cell embolic formation.

Saiki et al. (47 , 48) have investigated the effect of recombinant polypeptides with cell-binding domain (C-274) containing the RGD motif or with heparin-binding domain (H-271) composed of the 12th to 14th type III modules and their fusion polypeptide (CH-271) on the experimental metastases. They showed that H-271 and CH-271 were able to inhibit liver metastasis of L5178Y-ML25 lymphoma cells at much lower concentrations than that of C-274. A 33-kDa FN fragment derived from the same heparin-binding domain is well known to have functional sites promoting melanoma cell adhesion in a heparansulfate proteoglycan-dependent manner (13 , 47 , 49 , 50) . Therefore, they presumed that antimetastatic effects of H-271 and CH-271 were because of the intervention of tumor cell adhesion to FN substrate by the cell adhesion-promoting sites of heparin-binding domain. However, these recombinant polypeptides additionally contain the YTIYVIAL antiadhesive site in the 14th type III module. Antimetastatic effect of H-271 and CH-271 might be because of this antiadhesive site that is possibly exposed in these recombinant polypeptides. In any case, combination or fusion of our antiadhesive peptide FNIII14 with the RGD-related peptides would synergistically strengthen the antimetastatic effects of individual peptides. Some chemical modification and conjugation of inert carriers, which confer an extended vascular half-life, would also be needed.


   FOOTNOTES
 
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.

1 Supported by Grant-in-Aid for Scientific Research (11680615) provided by the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

2 To whom requests for reprints should be addressed, at Department of Patho-Physiology, 12 Ichigaya Funagawara-Machi, Shinjuku-Ku, Tokyo 162-0826, Japan. Phone: 81(3)3260-6725; Fax: 81(3)3268-3045; E-mail: fukai{at}ps.kagu.sut.ac.jp Back

3 The abbreviations used are: ECM, extracellular matrix; FN, fibronectin; LM, laminin; FBS, fetal bovine serum; EHS, Engelbreth-Holm-Swarm; VN, vitronectin; pAb, polyclonal antibody; VCAM-1, vascular cell adhesion molecule-1; mAb, monoclonal antibody; RGD, Arg-Gly-Asp; RGDS, Arg-Gly-Asp-Ser; HUVEC, human umbilical vein endothelial cell; FAK, focal adhesion kinase; TNF, tumor necrosis factor; POD, peroxidase; PAO, phenylarsine oxide; YIGSR, Tyr-Ile-Gly-Ser-Arg. Back

Received 11/20/01; revised 3/ 7/02; accepted 3/22/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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