inhibition of the metastasis of murine malignant melanoma...

[CANCER RESEARCH 49. 38I5-.1822, July 15. 1989)

Inhibition of the Metastasis of Murine Malignant Melanoma by SyntheticPolymeric Peptides Containing Core Sequences of Cell-adhesive Molecules1

Ikuo Saiki, ' Joji lida, Jun Murata, Ryu Ogawa, N'oriÂ»Nishi, Kazuhisa Sugimura, Seiichi Tokura, and Ichiro Azuma

Institute of Immunological Science /I. S., J. !.. J. M., A'. S.. I. A.], and Department of Polymer Science, Faculty of Science [R. O., S. T., A/. /V./( Hokkaido Ã•'ntversity,

Kita-15, Nishi-7, Kita-ku, Sapporo 060. Japan

ABSTRACT

We investigated that the antimetastatic and antiadhesive activities ofpeptides based on Arg-Gly-Asp adhesive signal in fibronectin could beaugmented by their polymerization. Poly(Arg-Gly-Asp), which consistsof a repetitive sequence of Arg-Gly-Asp, inhibited lung mÃ©tastasesinC57BL/6 mice more effectively than Arg-Gly-Asp tripeptide was ableto do, when coinjected or separately injected with B16-BL6 cells. Theadhesion of tumor cells to fibronectin was sporificali) inhibited by addingpoly(Arg-Gly-Asp) but not unrelated peptides. In contrast, poly(Arg,Gly, Asp), in which three amino acids are randomly arranged, showedneither inhibition of lung mÃ©tastasesnor any adhesive ability to attachto tumor cells. The inhibitory effect of polymeric peptides containing theArg-Gly-Asp sequence on lung mÃ©tastasesdecreased according to thedecreasing repeat units of the Arg-Gly-Asp core sequence. Polymericpeptides with Arg-Gly-Asp entrapped within the liposome membranesalso caused a remarkable reduction of metastatic colonies. In a spontaneous metastasis model, multiple i.v. administrations of poly(Arg-Gly-Asp) after tumor inoculation caused the significant reduction of metastaticcolonies in the lung but did not affect the growth (size) of primary tumor.

We found that the polymerization (multivalency) of the Arg-Gly-Aspcore sequence was able to augment the inhibition of tumor lung mÃ©tastasesin experimental and spontaneous metastasis models as well as the cell-adhesive property more effectively than a monovalent unit of Arg-Gly-Asp peptide.

INTRODUCTION

Metastasis is one of the major causes of mortality in cancer.The pathogenesis of mÃ©tastasescan be subdivided into a varietyof sequential steps: Step 1, release from the primary tumor andinvasion of the surrounding tissues; Step 2, entry into vascularor lymphatic circulation; Step 3, transit in the circulation; Step4, arrest in the capillary bed of a distant organ; Step 5, extravasation from circulation; Step 6, growth at apparently selectivesites which are distant from the original tumor site (1-4). Fewcells in a primary tumor can complete all these steps necessaryto achieve metastasis. During the sequential steps of metastasis,metastasizing tumor cells encounter various host cells (platelet,lymphocytes, or endothelial cells) and/or extracellular matrixand basement membrane components (fibronectin and laminili)(1-4). As a result of adhesive interaction, this encounter mayresult in an embolus formation which can subsequently enhancethe survival, arrest, or invasiveness of tumor cells (2, 4-7).Specific incidents of tumor interaction with host cells or components are therefore fundamental events in the metastaticprocess. Consequently, both adhesion and detachment of cells

Received 3/28/88: revised 11/14/88, 4/7/89; accepted 4/19/89.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by Grants-in-Aid for Cancer Research from

the Japanese Ministry of Education. Science and Culture; from the JapaneseMinistry of Health and Welfare for Comprehensive 10-Year Strategy for CancerControl; for Scientific Research and for Developmental Scientific Research (No.62870023) from the Japanese Ministry of Education. Science and Culture; andfor Scientific Research from the Japanese Ministry of Education, Science andCulture; by the Osaka Foundation for Promotion of Clinical Immunology; byYamanouchi Foundation for Research on Metabolic Disorders; and by Grant-in-Aid for Special Project Research from Hokkaido University. Japan.

2To whom requests for reprints should be addressed.

are thought to be of prime importance in achieving control ofthe cellular functions of diverse cell types, including highlymetastatic cells. Rapid progress has been made in the analysisof those adhesion molecules which interact with cell surfaces.The primary structures of some cell adhesion proteins such asfibronectin (8), vitronectin (9), and laminin (10, 11) have beenidentified by recombinant DNA technology. Common or characteristic core sequences in molecules have been shown tocontribute to cell adhesion, spreading, and migration of cells(12-14); this suggests the existence of a gene superfamily ofcell adhesion molecules (13, 15). It has already been shown thatthe domain which recognizes cells for cell adhesion in fibronectin and other adhesive proteins is an Arg-Gly-Asp-Ser sequence ( 12, 13). Synthetic peptides which contain this sequencehave been found to promote cell attachment after surface immobilization and to inhibit the attachment of cells to fibronectinwhen added freely in solution. This indicates that Arg-Gly-Asp tripeptides reproduce the adhesion properties of fibronectin(12, 16-18).

Preincubation of metastatic murine melanoma cells withlaminin followed by i.v. injection stimulated the formation ofpulmonary mÃ©tastases,whereas a proteolytic fragment of laminin had the opposite effect (19). More recently, it has beenreported that the coinjection of pentapeptide containing Arg-Gly-Asp or a Tyr-Ile-Gly-Ser-Arg sequence in fibronectin orlaminin with melanoma cells is able to inhibit dramatically theformation of lung tumor colonies (20, 21). Adhesive interactionbetween cells and the extracellular matrix appears to be criticalfor the formation of metastatic tumors, while the receptor forcell adhesive molecules on the cell surface may also be involvedin these formations (22-24). Such evidence has prompted us tocarry out another attempt to more efficiently regulate themechanism involved in the adhesion of tumor cells during themetastatic process.

In this study, we synthesized some original polymeric peptideanalogues which consist of the repeated structure of the Arg-Gly-Asp core sequence, and we have tested these polymericpeptides in an in vitro cell adhesion assay and experimental andspontaneous models of metastatic lung tumor colonization.

MATERIALS AND METHODS

Mice. Specific-pathogen-free male C57BL/6 mice, 7-10 weeks old,were purchased from the Shizuoka Laboratory Animal Center, Hama-matsu, Japan. The mice were maintained in the Laboratory for AnimalExperiments, the Institute of Immunological Science, Hokkaido University, under laminar air flow conditions.

Cells. Highly metastatic B16-BL6 melanoma cells, obtained by an invitro selection procedure for invasion (25), were kindly provided by Dr.I. J. Fidler, M. D. Anderson Cancer Center, Houston, TX. B16-BL6melanoma was maintained as monolayer cultures in MEM1 supple

mented with 7.5% fetal bovine serum, vitamin solution, sodium pyru-vate, nonessential amino acid, and L-glutamine.

3The abbreviations used are: MEM, Eagle's minimal essential medium; PBS,Ca2*- and Mg2*-free phosphate-buffered saline: MLV, multilamcllar vesicle liposomes: Â«Ahx. (-aminocaproic acid: il., intralesionally: i.t.. intratumorully; BSA.bovine serum albumin.

3815

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CELL ADHESION AND THE INHIBITION OF METASTASIS

Synthetic Polymeric Peptide Analogues and Reagents. Polymeric pep-tides possessing an Arg-Gly-Asp sequence derived from fibronectinand their related analogues were prepared by the synthesis of a monomer peptide of the Arg-Gly-Asp sequence by the conventional methodand subsequent polymerization reaction. r-Butoxycarbonyl (t-Boc),mesitylenesulfonyl (Mts), and benzyl (Bzl) groups were used as theprotecting groups for Â«-aminoguanidino and 0-carboxyl groups. Thepurity of the peptides were confirmed by thin layer chromatographyand elemental analysis. Polymerization of monomer peptide was carriedout with diphenylphosphorylazide, as described elsewhere (26, 27).Polymeric peptide with a very low molecular weight was prepared byworking up the product in the course of the polymerization procedure.Removal of side chain-protecting groups from the resulting sequentialpolymeric peptide was carried out with methanesulfonic acid-anisolefollowed by the conversion of methanesulfonate to hydrochloride withAmberlite IRA 400 (chloride form) to give the final product. Completeremoval of the protecting groups was confirmed by IR. The finalproduct showed the typical pattern for polymeric peptides. All theamino acids used in this study were of the i. form. The structures ofthis polymeric peptide and similarly prepared other polymeric peptidesare indicated in Fig. 1. Poly(Arg-Gly-Asp) consists of a sequentialstructure of the Arg-Gly-Asp sequence, whereas poly(Arg, Gly, Asp)consists of a randomly arranged structure of three amino acids. Hence,in the sequence of poly(Arg-Gly-Asp), the glycyl residue is alwaysbetween arginyl and aspartyl residues, and the -Arg-Gly-Asp- sequence exists as a block. In the sequence of poly(Arg, Gly, Asp), on theother hand, these three amino acids are randomly arranged withoutrule and the probability of-Arg-Gly-Asp- sequence is statistically verysmall. Copoly(Arg-Gly-Asp, Lys) designates the polymeric peptiderandomly arranged by Arg-Gly-Asp tripeptide and Lys, in which the-Arg-Gly-Asp- sequence always exists as a block in the copoly(Arg-Gly-Asp, <Ahx). The polymeric peptides shown in Fig. 1 are estimatedto have an approximate average molecular weight of 5000, as assessedby viscometric measurements and sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and dissolved in PBS. Purified mouse fibronectin was purchased from Seikagaku Kogyo Co., Ltd., Tokyo, Japan.Arg-Gly-Asp and Arg-Gly-Asp-Ser were purchased from BACHEMFeinchemikauen AG, Switzerland. Arg-Gly-Glu-Ser was purchasedfrom Peninsula Laboratories Inc., CA. All reagents and media used inthis study were endotoxin free (approximately <1.0 ng/ml) as determined by the colorimetrie assay (Pyrodick; Seikagaku).

Lipids and the Preparation of Liposomes. Chromatographically pureegg phosphatidylcholine was purchased from Avanti Biochemicals (Birmingham, AL). Cholesterol was purchased from Nakarai Chemicals,Ltd. MLV were prepared from a mixture of phosphatidylcholine andcholesterol (1:1 molar ratio) by mechanical agitation on a vortex mixer(28). Poly(Arg-Gly-Asp, Â«Ahx)was dissolved in methanol:chloroform(1:1) and added to the phospholipids in chloroform. Untrapped materialwas removed by washing the MLV by centrifugation, and the entrappedamount was determined by amino acid analysis to be 87% of totalamounts added. The liposome preparations were adjusted to 25 ^moltotal lipid/ml in PBS.

Microassay for Cell Adhesion. This adhesion assay was carried outby the method described previously (29), with some slight modifications. B16-BL6 melanoma cells in an exponential growth phase were

Poly(Arg-Gly-Asp)

Poly(Arg , Gly , Asp)

Copoly(Arg-Gly-Asp , Lys)

CopOly(Arg-Gly-Asp . â‚¬¿�Ahx)

Poly(Arg-Gly)

Poly(Arg)

Poly(Asp)

Arg-Gly -Asp- Arg-Gly-Asp- Arg-Gly-Asp â€¢¿�

Arg-Asp-Arg-Gly-Asp-Gly-Asp-Arg-Gly â€¢¿�

Arg-Gly-Asp-Lys-Lys-Arg-Gly-Asp-Uys â€¢¿�â€¢

Arg-Gly-Asp-tAhx-Arg-Gly-Asp-eAhx â€¢¿�â€¢â€¢

Arg-Gly-Arg-Gly-Arg-Gly-Arg-Gly-Arg â€¢¿�â€¢

Arg- Arg- Arg- Arg- Arg- Arg- Arg- Arg- Arg â€¢¿�â€¢

Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp â€¢¿�â€¢â€¢

incubated for 24 h in MEM containing 5% fetal bovine serum supplemented with 0.3 iiCi/ml [I25l]iododeoxyuridine (specific activity, 2000

Ci/mmol; New England Nuclear, Boston, MA). The cells were washedtwice in warm PBS to remove unbound radiolabels, harvested by adding0.02% EDTA for 1 min at 37Â°C,and resuspended in cold serum-freeMEM to form a single suspension of cells. [':'I]Iododeoxyuridine-labeled tumor cells (2 x IO4)at a volume of 0.05 ml/well were added

to microculture wells precoated with synthetic polymeric peptides orfibronectin. The cultures were incubated at 37Â°Cfor 20 min and then

washed four times with PBS to remove any unattached cells. Theremaining substrate-bound tumor cells were lysed with 0.1 ml of 0.1 NNaOH. The lysate was absorbed on cotton swabs and monitored forradioactivity by gamma counting. The binding capacity (number of cellsbound/substrate) was expressed as

Binding capacity = cpm of targets bound to substratecpm of total tumor cells added

x total no. of tumor cells added

eAhx : t-Aminocaproic acid

Fig. 1. Structures of the polymeric peptides used in this study.

Labeling of Synthetic Polymeric Peptide. Copoly(Arg-Gly-Asp, Lys)was iodinated by Million l hinter reagent according to the conventionalprocedure. Briefly, this polymeric peptide (2 mg) was dissolved in 20n\ PBS and added to 1 mCi Bolton-Hunter reagent (Â¿V-succinimidyl-3-(4 hydroxy-3,5-['"I]diiodophenyl)propionate; specific activity 2000 Ci/

mmol; New England Nuclear) freshly dried from a solution in benzene.After agitation of the mixture at 4Â°Covernight, the reaction was

quenched by addition of 5 ^1 1 M glycine in borate buffer. Iodinatedpolymeric peptide was separated from byproduct by gel filtration onSephadex G-25 which was equilibrated and eluted with 0.05 M phosphate buffer (pH 7.5) containing 2% (w/v) gelatin. '"I-labeled poly

meric peptide thus obtained was confirmed by the absorbance at 230nm in spectrophotometer.

Procedure for Study of Clearance of '"I-Labeled Polymeric Peptidein Vivo. C57BL/6 mice were given i.v. injections of ':?I-labeIedpoly(Arg-Gly-Asp, Lys) (7 x 10" cpm/50 Mg)in a volume of 0.1 ml

PBS. After various times, mice were exsanguinated, and lung, liver,kidneys, spleen, and blood were collected and rinsed in 70% ethanol.The radioactivity in each organ was measured in a gamma counter.

Experimental and Spontaneous Lung MÃ©tastases.C57BL/6 mice weregiven i.v. injections (0.2 ml/mouse) of B16-BL6 melanoma (5 x IO4)

admixed with a various concentration of polymeric peptides in PBSunless otherwise mentioned. Fourteen days after the inoculation oftumor cells, mice were sacrificed and the number of lung tumor colonieswas recorded (experimental metastasis). Mice were given s.c. injectionsof BI6-BL6 melanoma cells (5 x 10 ) into the right hind footpad.Polymeric peptides were administered i.l. (i.e., i.t.) or i.v. on variousdays after tumor inoculation, and then primary tumors were surgicallyremoved by the amputation on Day 21. Mice were sacrificed 14 daysafter the amputation (spontaneous metastasis). The lungs were fixed inBouin's solution and the lung tumor colonies were counted under a

dissecting microscope.Statistical analysis. The statistical significance of differences between

groups was determined by applying Student's two-tailed r test.

RESULTS

Cell Adhesion to Polymeric Peptides Containing the Arg-Gly-Asp Sequence. Since metastatic cells presumably interact withextracellular matrix components (cell adhesion proteins) duringthe metastatic process (2, 4-7), we first tested the adhesion ofB16-BL6 melanoma cells to wells precoated with syntheticpolymeric peptides containing the Arg-Gly-Asp sequence derived from fibronectin. The results of a representative of severalexperiments are shown in Fig. 2. Poly(Arg-Gly-Asp),poly(Arg), and fibronectin promoted the adhesion of B16-BL6melanoma cells. However, few B16-BL6 cells attached themselves to the substrates coated with poly(Arg, Gly, Asp) inwhich three amino acids had been randomly arranged,

3816




Binding capacity(No. of cell bound/substrate) ; IO3

Coaled with: EDTA

Fibronectin

poly(Arg-Gly-Asp)

poly(Arg, Cly, Asp)

poly(Arg-Cly)

poly(Arg)

poly(Arg-Gly) *poly(Asp) -

poly(Asp)

BSA

Fibronectin 4-

poly( Arg-Gly-Asp) -}-

poly(Arg, Cly, Asp) +

m-

poly(Arg-Cly)

poly(Arg)

BSA

+

n-~Z}

m-

Fig. 2. Adhesion of B16-BL6 melanoma cells to polymeric peptide-coatedsubstrates. ['"Ijlododeoxyuridine-labeled B16-BL6 cells (2 x IO4)were added to

wells coated with 5 fig/ml fibronectin, 20 fig/ml polymeric peptides, or 1% BSAin PBS. After 20 min incubation, nonadherent cells were washed away and theadhered cells were counted.

poly(Arg-Gly), poly(Asp) or the mixture of poly(Arg-Gly) andpoly(Asp) as well as BSA. We also observe that other polymericpeptides containing Arg-Gly-Asp, including copoly(Arg-Gly-Asp, Lys) and copoly(Arg-Gly-Asp, eAhx) enhanced the adhesion of tumor cells (data not shown). Cell adhesion to fibronectin and poly(Arg-Gly-Asp) were decreased by the addition of5 mM EDTA, but the adhesion to poly(Arg) was not. Toinvestigate the specificity of cell adhesion to polymeric peptidescontaining an Arg-Gly-Asp sequence, we carried out a celladhesion assay in the presence of peptides with Arg-Gly-Asp(Fig. 3). Arg-Gly-Asp, Arg-Gly-Asp-Ser, and poly(Arg-Gly-

Asp) at various concentrations ranging from 10 to 1000 ng/m\were able to specifically inhibit the adhesion of cells topoly(Arg-Gly-Asp)- and fibronectin substrates in a dose-dependent fashion, but unrelated His-Gly-Gly and Arg-Gly-Glu-Ser peptides could not. B16-BL6 cells attached to thesubstrates coated with poly(Arg), which is well known as adhesion-associated substance, as well as to poly(Lys). In contrast,no inhibition of cell adhesion to poly(Arg) substrate was observed by the addition of Arg-Gly-Asp peptide. These resultsindicate that the cell adhesion-promoting activity of fibronectinor poly(Arg-Gly-Asp) at least depends on a mechanism specifically mediated by the Arg-Gly-Asp sequence in a Ca2*- orMg2+-dependent manner. Poly(Arg-Gly-Asp) could inhibit tu

mor cell adhesion to fibronectin substrate more effectively thanArg-Gly-Asp peptide on a weight basis. At the same time,cultures on the fibronectin- and poly(Arg-Gly-Asp)-coated substrates revealed a marked alteration in cell shape after celladhesion. After 2 h culture, B16-BL6 melanoma had spreadalmost completely over the tissue culture substrates precoatedwith 5 Mg/ml fibronectin or 20 Mg/ml poly(Arg-Gly-Asp),whereas cells on both poly(Arg, Gly, Asp)- and BSA-coatingsubstrates were round (data not shown).

Inhibition of Lung MÃ©tastases by Arg-Gly-Asp-containingPolymeric Peptides. The above results demonstrate that

Co-incubated Binding capacityCoated with: with: (No.ol cell bound/substrate) x 10

Q 2 4 6 8

poly(Arg-Gly-Asp)

Arg-Gly-Asp 100200500

1000

Arg-Gly-Asp-Ser 200

His-Gly-Gly 500

Fibtonectin

Arg-Gly-Asp 500

Arg-Gly-Asp-Ser 200

Poly(Arg-Gly-Asp) 10100200

His-Gly-Gly 500

Arg-Gly-Gju_-Ser500

poly(Arg)

BSA

Arg-Gly-Asp 200500

His-Gly-Gly 500

J

J-

/ig/ml

11Fig. 3. Inhibition of cell adhesion to polymeric peptide-coated substrates.-lododeoxyuridine-labeled B16-BL6 cells were added to wells coaled withml fibronectin, 20 ng/ml poly(Arg-Gly-Asp), or 20 <ig/ml poly(Arg) in thepresence of various concentrations of tri- or tetrapeptides. After 20 min incubation, nonadherent cells were washed away and the adhered cells were counted.

poly(Arg-Gly-Asp) is able to promote cell adhesion whenimmobilized in tissue culture wells and is also able to inhibitthe cell-adhesive function to a fibronectin substrate when addedfreely in solution more effectively than Arg-Gly-Asp peptideon a weight basis (Fig. 2). We next examined the effect ofpoly(Arg-Gly-Asp) on the experimental lung mÃ©tastasesbysystemic injection of B16-BL6 melanoma. Polypeptides dissolved in PBS were admixed with B16-BL6 melanoma cells and

the mixtures were immediately injected i.v. into C57BL/6 mice.Mice were sacrificed 14 days after coinjection. The results of arepresentative of three experiments are shown in Table 1,Experiment 1. Increasing doses of poly(Arg-Gly- Asp), 500 and1000 jig/mouse, led to significant decreases in the number oflung tumor colonies in a dose-dependent manner (P < 0.001).In contrast, poly(Arg, Gly, Asp), which is a random homologueof poly(Arg-Gly-Asp) and indicates no cell adhesion-promoting activity (Fig. 2), achieved no reduction of lung tumorcolonies. As Experiment 2 of Table 1 shows, tri- or tetrapeptides, Arg-Gly-Asp or Arg-Gly-Asp-Ser, induced a slight

reduction in the number of lung tumor colonies at the dose of500 Mg/mouse but this was not a significant inhibition ascompared with the untreated control. However, a significantreduction in the number of lung tumor colonies was observedat increased doses (3000 fig/mouse) of Arg-Gly-Asp (P <

0.001). The degree of inhibition of lung mÃ©tastasesby tripeptideis similar to that indicated in previous results, where a substantial inhibition of mÃ©tastasesusing B16-F10 melanoma cells wasobtained with 3 mg of Gly-Arg-Gly-Asp-Ser pentapeptide(20). A remarkable reduction of lung mÃ©tastaseswas achievedby a comparatively low dose of poly(Arg-Gly-Asp); this suggests that poly(Arg-Gly-Asp) is more active than the monomeron a weight basis and that the effect may be due to the sequential

3817




Table 1 Effect of poly(Arg-Gly-Asp) on experimental lung mÃ©tastasesofBI6-BI.6 melanoma

Five C57BL/6 mice per group were inoculated i.v. with BI6-BL6 (5x10*

cells) with or without the polymeric pcptides.

Administeredi.v.withExperiment

lControl (I'BS)Dose(jig/mouse)No.

of lungmÃ©tastaseson Day14Mean

Â±SD RangePÂ°166

Â±20 132-286

Poly(Arg-Gly-Asp)

Poly(Arg, Gly, Asp)

1000500100

20

5(MÂ»

42 Â±2227 Â±8

143 + 66170 Â±22

172 + 54

14-6821-3977-225

148-199

121-245

<0.001<0.001

Experiment 2Control(PBS)Poly(Arg-Gly-Asp)Arg-Gly-AspArg-Gly-Asp-SerPoly(Arg.

Gly. Asp)5003000

500100500500152

Â±2138

+531

+40123+ 22

137+1985+

15109

Â±7120-17633-462-88

98-158106-15372-99107-116<0.001<0.001

Â°Compared with untreated control (PBS) by Student's two-tailed / test.

Table 2 Effect of poly(Arg-Gly-Asp) anil its related peptides on experimentallung mÃ©tastasesof B16-BL6 melanoma

Five C57BL/6 mice per group were inoculated i.v. with B16-BL6 (5 x 10"

cells) with or without the agents indicated below.

No. of lungmÃ©tastaseson Day 14

DoseTreatment (ng/mouse) Mean Â±SD Range f

Untreated (PBS) 72 + 9 62-85Poly(Arg-Gly-Asp)

A/,5000 500 20 + 7 12-29 <0.001A/,1500 500 44 Â±22 16-57 <0.05

Gly-Arg-Gly-Asp-Ser-Gly- 500 66 Â±19 59-93Arg-Gly-Asp-Ser

Arg-Gly-Asp 500 74 Â±37 29-124Â°C'ompared with untreated control (PBS) by Student's two-tailed t test.

repetition of Arg-Gly-Asp core sequence in the polymericpeptide structures.

We therefore tested the effect of various polymeric peptidesrepresenting a different repetition (approximately 1-15 repeats)of the Arg-Gly-Asp sequence on lung mÃ©tastasesof B16-BL6cells. To do this, we used a 500-jjg dose of polymeric peptidesfor this comparative study because poly(Arg-Gly-Asp) at thedose of 500 ng showed the remarkable inhibition of lung mÃ©tastases as shown in Table 1. As Table 2 shows, poly(Arg-Gly-Asp) M, 5000 and M, 1500 significantly reduced the number oflung tumor colonies but deca- and tripeptide did not. Its inhibitory effects on lung mÃ©tastaseswere observed by increasing thenumber of Arg-Gly-Asp repeats in the peptide or polymericpeptide molecules at the doses of 500 yug-

In the next set of experiments, we examined the effect ofadministration protocol of poly(Arg-Gly-Asp) on the inhibition of lung mÃ©tastases. Poly(Arg-Gly-Asp) |50,>as well aspoly(Arg-Gly-Asp)5(XK>at a dose of 1000 pg inhibited significantly the lung mÃ©tastasesby its coinjection with B16-BL6 cells(P< 0.001, respectively, in Table 3, Experiment 1). In addition,the i.v. injection of poly(Arg-Gly-Asp)5oo<> following B16-BL6cells (separate sequential injections) was almost as effective asa means to reduce lung colonization as coinjection (admixing)of cells and poly(Arg-Gly-Asp)s,HH>. This indicates that the

inhibitory effects of polymeric peptide on tumor mÃ©tastasescannot be explained by simple cytotoxicity. The poly(Arg-Gly-Asp) used in this study did not affect any cytotoxicity directedagainst the cells, including B16-BL6 melanoma cells, mouseRBC, or splenocytes, or the cell growth (data not shown).Administrations of 500 ng poly(Arg-Gly-Asp)5<Mx>i.v. 30 minbefore or after tumor injection, however, inhibited the lungmÃ©tastasesless effectively than the coinjection of cells andpoly(Arg-Gly-Asp)5l)oo (Table 3, Experiment 2).

The results in Tables 1 and 2 show that i.v. injection ofsoluble poly(Arg-Gly-Asp) dissolved in PBS together withB16-BL6 cells dramatically inhibited the metastatic formationof tumors in the lung as compared with that of tripeptides oruntreated control. We also investigated the effect of variouspolymeric peptides containing Arg-Gly-Asp sequence on themetastatic lung colonization. For this purpose, we synthesizedcopoly(Arg-Gly-Asp, fAhx) and copoly(Arg-Gly-Asp, Lys), inwhich the Arg-Gly-Asp sequence and either eAhx or Lys wererandomly arranged (1:1 molar ratio) and the -Arg-Gly-Asp-sequence always exists as a block. As Table 4 shows, Arg-Gly-Asp-containing polymeric peptides, poly(Arg-Gly-Asp), co-poly(Arg-Gly-Asp, eAhx) and copoly(Arg-Gly-Asp, Lys), effectively reduced the lung mÃ©tastaseswhen compared with theuntreated control (P < 0.005, P < 0.001, and P < 0.001,respectively). Copoly(Arg-Gly-Asp, eAhx) incorporated withinMLV liposomes also inhibited lung mÃ©tastasesby the coinjection with B16-BL6 cells in a dose-dependent manner. However,liposomes alone were unable to inhibit the mÃ©tastases.Theseresults indicate that the inhibition of lung mÃ©tastasescan beinduced by coinjection with various polymeric peptides in whichArg-Gly-Asp sequences were sequentially or randomly arranged and -Arg-Gly-Asp- sequence always exists as a block,and even with a polymeric peptide entrapped (insolubilized)within liposome membranes. Thus, we used mainly sequentialpolymeric peptide, poly(Arg-Gly-Asp) in the following experiments.

Effect of Polymeric Peptides on the Spontaneous Lung MÃ©tastases. We also examined the therapeutic effect of systemicadministration of poly(Arg-Gly-Asp) on the lung metastasisof B16-BL6 melanoma in spontaneous metastasis model. Multiple treatments with polymeric peptides at the dose of 100 figwere performed on days 7, 9, 11, 13, 15, 17 and 19 after tumorinoculation. Primary tumor was surgically removed on Day 21and tumor colonies in the lung were monitored 14 days aftertumor excision. Table 5 shows that the i.v. administrations of100 ng poly(Arg-Gly-Asp) significantly reduced the number oflung tumor colonies (P < 0.001). Multiple treatments withpoly(Arg, Gly, Asp) or Arg-Gly-Asp tripeptide, however, didnot decrease the number of lung tumor colonies.

Retention of I25l-labeled Polymeric Peptide in the Circulation.

We next examined the behavior of polymeric peptide in thecirculation and various organs. To do this, I25l-labeled polymeric peptide (7 x IO6cpm/50 ng) in a volume of 0.1 ml was

injected into the lateral tail vein of C57BL/6 mice. Mice werekilled at various times after the injection, and blood sample andorgans were collected and monitored for radioactivity in agamma counter. Fig. 4 demonstrates that the clearance of '"!-labeled poly(Arg-Gly-Asp, Lys) from the circulation was bi-phasic and rapid during the early phase after the injection. Avery rapid initial decrease in polymeric peptide was obtainedwithin 5 min after the injection, implying that the initial decrease would probably be due to rapid equilibration or dilutioneffect in the body fluid. After initial equilibration, the clearanceof I25l-labeled polymeric peptide was rapid up to l h and

3818




Table 3 Effect of injection timing of poly(Arg-Gly-Asp) on the experimental lung mÃ©tastasesofB16-BL6 melanomaFive C57BL/6 mice per group were inoculated i.v. with B16-BL6 (5 x IO4)with or without the polymeric peptides indicated below.

No. of lungmÃ©tastases

TreatmentExperiment

1Untreated(PBS)Poly(Arg-Gly-Asp)500oPoly(Arg-Gly-Asp)â€žoâ€žExperiment

2Untreated(PBS)Poly(Arg-Gly-Asp)50ooTimingAdmixSeparate*AdmixAdmixSeparate'30

minbefore30min afterDose((ig/mouse)100010001000500500500Mean

Â±SD52

Â±106Â±620

Â±214Â±470

Â±123Â±156

Â±643Â±9Range40-602-1418-2010-1856-852-449-6532-54f<0.001<0.001<0.001<0.001<0.05<0.01

" Compared with untreated control (PBS) by Student's two-tailed t test.'' Polymeric peptide was administered i.v. just before the i.v. injection of B16-BL6 melanoma cells.' Polymeric peptide was administered i.v. 30 min before or after the i.v. injection of B16-BL6 melanoma cells.

Table 4 Effect of poly(Arg-Gly-Asp) and its related polymeric peptides onexperimental lung mÃ©tastasesofB16-BL6 melanoma

Five C57BL/6 mice per group were inoculated i.v. with B16-BL6 (5 X IO4)

with or without the agents indicated below.

No. of lung mÃ©tastaseson Day 14

TreatmentNone

(PBS)Poly(Arg-Gly-Asp)Copoly(Arg-Gly-Asp.Copoly(Arg-Gly-Asp,Copoly(Arg-Gly-Asp,inliposomesLiposomes

(empty).Ahx)

Lys)fAhx)Dose(jig/mouse)500

500500435*

8717Mean

Â±SD128

Â±1973 Â±2062 Â±1534 Â±1261 Â±1976 Â±31

107 Â±14126 Â±29Range109-147

44-8949-8215-4531-7630-9492-12694-153r<0.005

<O.OOI<0.001<O.OOI<0.02

Â»onipami wan umrtraieu cunÃ* Amount of polymeric peptide

calculated by amino acid analysis.iranes

thereafter the labeled polymeric peptide was gradually cleared.The respective half-life of this polymeric peptide during theearly and late phases was approximately 15 min and 6 h.

DISCUSSION

We have previously reported the antimetastatic and imimi-nopotentiating effects of a variety of synthetic immunomodu-lators in murine metastatic models and have examined nonspecific host defenses against metastatic tumors by effector cellssuch as tumoricidal macrophages (30-32). Since the adhesiveinteraction between tumor cells and host cells or extracellularmatrix presumably plays a crucial role in metastatic formationduring a series of complex events (1-4), we have now attemptedas distinct from the attempts mentioned above, to regulate the

mechanisms involved in cell adhesion during the metastaticprocess. To approach this attempt, we first synthesized somepolymeric peptides containing a common core sequence in celladhesion molecules. It is well known that the introduction ofplural peptides (for example, peptide hormones) into carrierproteins can sometimes augment the activity of the peptidehormone because of the cooperative interaction between themolecules, although at the same time this may reduce molecularflexibility and mobility and consequently lead to a decrease inthe affinity between the peptide and the specific receptors. Adrastically high activity of the polymerized functional moleculehas also been reported as a common phenomenon in the fieldof polymer catalyst or enzyme-model polymers and has cometo be called the "polymer effect" (33). The polymeric peptides

used in this study, therefore, consist of repeated units of acharacteristic Arg-Gly-Asp sequence derived from the cell-binding domain of fibronectin (12, 13). The present studydemonstrates that poly(Arg-Gly-Asp) consisting of sequentialrepeats of the Arg-Gly-Asp sequence has the ability to promotethe adhesion of B16-BL6 melanoma cells through the mediation of its recognition sequence and causes the inhibition oflung mÃ©tastasesmore effectively than monomeric Arg-Gly-Asp-containing peptide.

Poly(Arg-Gly-Asp), poly(Arg), and fibronectin promoted theadhesion of B16-BL6 melanoma cells when immobilized on thesurface of tissue culture wells, while tumor cell adhesion topoly(Arg-Gly-Asp) or fibronectin substrates were specificallyinhibited by the addition of Arg-Gly-Asp, Arg-Gly-Asp-Ser,or poly(Arg-Gly-Asp) in a dose-dependent manner and alsodecreased to the control level (BSA-coated substrate) in thepresence of 5 HIM EDTA (Figs. 2 and 3). In contrast, celladhesion to poly(Arg)-coated substrate was not affected by the

Table 5 Therapeutic effect of poly(Arg-Gly-Asp) on spontaneous lung mÃ©tastasesby intrafootpad administration ofB16-BL6 melanoma

Treatment"Untreated

(PBS)Arg-Gly-AspPoly(Arg-Gly-Asp)Poly(Arg. Gly, Asp)Dose

Timing (>ig)100

X 7on Day 7, 9, 11, 13, 15, 17, 19 100x7100x7Primary

tumorsize on Day 2 1

(mm Â±SD)10

Â±310 Â±29Â± 19Â±2No.

of lungmÃ©tastaseson Day35Mean

Â±SD69

Â±1572 Â±27I8Â±951 Â±9Range45-84

57-1176-21

36-60/>*<0.001" Five C57BL/6 mice per group were given 100 ng of polymeric peptide i.v. on Days 7, 9, 11, 13, 15, 17, and 19 after tumor inoculation. Primary tumors

(approximately 9 mm in diameter) were surgically removed on Day 21 and mice were sacrificed 2 weeks after tumor excision.Compared with untreated group by Student's two-tailed t test.

3819




10

10

ao

>5a>oâ€¢¿�oastr

10

310 L

O Blood (2 ml)

Lung

Kidney

-A-o

013 7Time atter injection (hours)

Fig. 4. Clearance of l;'l-labcled poly(Arg-Gly-Asp, Lys) from the circulation

in C57BL/6 mice. The level of radioactivities in blood, lung and kidney weredetermined as described in "Materials and Methods." The first samples were

collected afler S min. Each point represents the mean of a group of three mice.

addition of these peptides or EDTA. On the other hand,poly(Arg, Gly, Asp), which is a random sequence of three aminoacids, did not promote any adhesion of tumor cells. Our resultswith these polymeric peptides indicate that the cell adhesion topoly(Arg-Gly-Asp) or fibronectin is specifically mediated bythe molecular Arg-Gly-Asp sequence and is based on a Ca2+-or Mg:*-dependent manner; this suggests that cell surface receptors for adhesion can recognize the Arg-Gly-Asp sequencein fibronectin and poly(Arg-Gly-Asp). This finding is similarto previous accounts which have reported the attachment ofvarious cells to fibronectin in the different adhesion assay (12,16-18,34-37). Several investigators have shown that surrounding amino acids of Arg-Gly-Asp sequence, in particular car-boxyl-terminal amino acid residue, can play a role in the expression or cell adhesion activity in fibronectin and other proteinspossessing this sequence (17, 18). Although Arg-Gly-Asp wasreported not to inhibit the adhesion of normal rat kidney cellsto fibronectin substrate (17, 18, 35), this tripeptide could remarkably inhibit melanoma cell adhesion to fibronectin orpoly(Arg-Gly-Asp) substrates in our experiments (Fig. 3). Wealso observe that the peptide with the carboxyl terminal aminoacid residue (Ser), Arg-Gly-Asp-Ser, has antiadhesive activityof B16-BL6 melanoma cells to fibronectin more effectively thanArg-Gly-Asp tripeptide on a weight basis (Fig. 3). Humphrieset al. (34) have reported in the comparative study betweenmelanoma and fibroblastic cell adhesion to fibronectin thatGly-Arg-Gly-Asp (peptide without carboxyl-terminal aminoacid residue) and Gly-Arg-Gly-Glu-Ser did not inhibit theadhesion of baby hamster kidney fibroblastic cells but potentlyinhibited melanoma cell adhesion to fibronectin, thus suggesting that a Arg-Gly-Asp-type inhibitory pattern may reflect theprofile of cell-type specificity for the interaction with fibronectin. As Fig. 3 shows, Arg-Gly-Glu-Ser and His-Gly-Gly asnegative controls could not inhibit the adhesion of melanomacells to fibronectin substrate when added freely in solution inour adhesion assay. Further study will be needed to determine

the exact mechanism responsible for antiadhesive property byour polymeric peptide. One possibility is that -Arg-Gly-Asp-Arg- in our sequential polymeric peptide molecule may workin cell adhesion inhibition. The "Arg" in the fourth position is

what would happen in our polymeric peptide. The monomericpeptides, in which the fourth position (Xaa) in -Arg-Gly-Asp-Xaa was substituted with various amino acids except proline,have been shown to have the ability to inhibit cell attachmentto fibronectin or vitronectin (18). Consequently our resultsindicate that poly(Arg-Gly-Asp) is able to inhibit the celladhesion to fibronectin more effectively than Arg-Gly-Asptripeptide on a weight basis.

The coculture of tumor cells with purified laminin followedby an i.v. injection has enhanced the pulmonary mÃ©tastases,whereas a fragment of laminin has inhibited the mÃ©tastases(19). Synthetic Gly-Arg-Gly-Asp-Ser or Tyr-Ile-Gly-Ser-Arg pentapeptides respectively derived from fibronectin or laminin have been shown to possess similar inhibitory effects (20,21). The interaction of metastatic cells with extracellular matrixand basement membrane components may therefore be hindered by our synthetic polymeric peptides including poly(Arg-Gly-Asp). Indeed, the coinjection of B16-BL6 cells withpoly(Arg-Gly-Asp) caused a striking reduction in the numberof lung mÃ©tastases in a dose-dependent manner, whereaspoly(Arg, Gly, Asp) by itself did not (Table 1). Arg-Gly-Asptripeptides could induce the substantial inhibition of lung mÃ©tastases at comparatively high doses of 3000 ng when they werecoinjected with B16-BL6 melanoma cells (Table 1). The inhibitory effects of polymeric peptides containing the Arg-Gly-Aspsequence on pulmonary mÃ©tastasesdecreased according to thedecreasing repeat units of this sequence (Table 2). This indicatesthat multivalent units of the Arg-Gly-Asp core sequence areable to promote their capability to inhibit lung metastasis moreeffectively than a monovalent unit of this sequence. Otherpolymeric peptides with Arg-Gly-Asp sequence, copoly(Arg-Gly-Asp, tAhx) and copoly(Arg-Gly-Asp, Lys) at the dose of500 pg caused significant inhibition of lung tumor colonies aswell as poly(Arg-Gly-Asp) when coinjected with B16-BL6melanoma cells (Table 4). Furthermore, insolubili/ed polymericpeptide, copoly(Arg-Gly-Asp, tAhx) inserted within liposomemembranes, also resulted in the reduction of tumor colonization. Polymeric peptides used in this study are of similar lengthand possess the unique structures in which Arg-Gly-Asp sequences were sequentially repeated, or randomly arranged bythe separation with spacer compounds such as Â«Ahxor Lys butalways exist as a Arg-Gly-Asp block. This suggests that theinhibition of lung mÃ©tastasesby our polymeric peptides can becaused by a peptide sequence-mediated mechanism, even whenpolymeric peptides have been incorporated within the liposomemembrane. It has been reported that a recent attempt to augment antimetastatic activity by coupling the Arg-Gly-Asp andTyr-Ile-Gly-Ser-Arg (derived from laminin) within one peptide was not successful (21). However, it is possible that clustering of Arg-Gly-Asp tripeptides, e.g., poly(Arg-Gly-Asp),may have a higher affinity with or more appropriate conformation of the molecule to the adhesion receptor than tripeptide.We recently observed that poly(Tyr-Ile-Gly-Ser-Arg), whichconsists of sequential repeat units of this pentapeptide sequence, could dramatically inhibit the lung mÃ©tastasesof B16melanoma and Lewis lung carcinoma as compared with pentapeptide (38, 39). Finally we indicated that polymeric peptidewith Arg-Gly-Asp sequence is more active for the inhibitionof lung metastasis than monomeric peptide in the experimentaltumor metastasis model.

3820




ACKNOWLEDGMENTS

We thank Dr. Leo T. Furcht for his helpful comments and criticalreview of this manuscript and Mutsuko Araki for typing the manuscript.

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13.

In the spontaneous metastasis model, multiple i.v. administrations of poly(Arg-Gly-Asp) after tumor inoculation signifi- gcantly led to decrease the tumor colonization in lung, but Arg-Gly-Asp tripeptide or a random polymeric peptide, poly(Arg, 9Gly, Asp), did not (Table 5). The exact mechanism responsiblefor the inhibition of lung mÃ©tastasesby our synthetic polymericpeptide may be more complex than a simple blockage of cell I0adhesion. The clearance rate of i:5I-labeled polymeric peptide

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We have recently observed that the number of radiolabeledB16-BL6 cells arrested in the lung decreased remarkably over24 h after the intravenous injection of poly(Arg-Gly-Asp) with isthe cells as compared with untreated control.4 In addition, 16poly(Arg-Gly-Asp) inhibits tumor-induced platelet aggregation which in turn is responsible for the enhancement of tumorcell arrest in the capillaries (7,41-43) but does not provoke theaggregation of platelets (43). Some possibilities include theacceleration of the release of arrested tumor cells from the lung 18

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1989;49:3815-3822. Cancer Res Ikuo Saiki, Joji Iida, Jun Murata, et al. Cell-adhesive MoleculesSynthetic Polymeric Peptides Containing Core Sequences of Inhibition of the Metastasis of Murine Malignant Melanoma by

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