Hidenori Takahashi, *,1 Satoru Ebihara, 1 Tatsuma Okazaki, 1 Masanori Asada, 1 Hidetada Sasaki & 1 Mutsuo Yamaya. Introduction. Sendai , Japan

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1 British Journl of Phrmcology (5) 16, & 5 Nture Pulishing Group All rights reserved /5 $3. A comprison of the effects of unfrctionted heprin, dlteprin nd dnproid on vsculr endothelil growth fctor-induced tumour ngiogenesis nd heprnse ctivity 1 Hidenori Tkhshi,,1 Storu Eihr, 1 Ttsum Okzki, 1 Msnori Asd, 1 Hidetd Sski & 1 Mutsuo Ymy 1 Deprtment of Geritric nd Respirtory Medicine, Tohoku University School of Medicine, Seiryo-mchi 1-1, Ao-ku, Sendi , Jpn Keywords: Arevitions: 1 Disseminted intrvsculr cogultion (DIC) is the most common compliction of solid tumours. In this study, the effectiveness of three polyscchride nticogulnts (PSAs), t therpeutic doses, t inhiiting solid tumour growth ws investigted. Mice with tumour xenogrfts were sucutneously injected with either unfrctionted heprin (; units kg 1 dy 1 ), dlteprin (75 units kg 1 dy 1 ) or dnproid (5 units kg 1 dy 1 ). At these concentrtions, these PSAs re equieffective t inhiiting lood cogultion ctivted fctor X. In mice with Lewis lung crcinom (LLC) tumours dlteprin nd, to lesser extent, inhiited oth tumour growth nd ngiogenesis, wheres dnproid did not. In contrst, in mice with KLN5 tumours, ll the PSAs inhiited tumour growth nd ngiogenesis. 3 All the PSAs significntly inhiited prolifertion, migrtion of endothelil cells nd vessel formtion in mtrigel plugs contining vsculr endothelil growth fctor (VEGF) nd there were no significnt differences etween these effects of the PSAs. The PSAs hd no effect on endothelil cell tuulr formtion in vitro. Although ll the PSAs inhiited VEGF production in KLN5 tumours in vivo nd cells in vitro, in LLC tumours nd cells only nd dlteprin inhiited VEGF production, wheres dnproid did not. 5 In oth LLC nd KLN5 tumours in vivo, heprnse ctivity ws inhiited y nd dlteprin, ut not y dnproid. 6 Hence, nd dlteprin my e more effective thn dnproid t inhiiting cncer progression in DIC ptients with solid tumours, due t lest in prt to their ility to suppress VEGF nd heprnse in tumours. British Journl of Phrmcology (5) 16, doi:1.138/sj.jp.763; pulished online 5 July 5 Heprins; ngiogenesis; vsculr endothelil growth fctor; heprnse; cncer DIC, disseminted intrvsculr cogultion; FX, ctivted cogultion fctor X; LLC, Lewis lung crcinom; LMWH, low moleculr weight heprin; PSA, polyscchride nticogulnt;, unfrctionted heprin; VEGF, vsculr endothelil growth fctor Introduction Disseminted intrvsculr cogultion (DIC) is one of the most common nd often ftl complictions of solid tumours (Sllh et l., 1). Conversely, solid tumours re the most common cuse of DIC (Okjim et l., ). Although the mechnism of the derngement of the cogultion system in ptients with cncer is uncler, the inhiition of lood cogultion ctivted fctor X (FX) is stndrd tretment for DIC in cncer s well s for DIC generlly. There is ccumulting evidence from oth niml nd humn studies tht polyscchride nticogulnts (PSAs) such s unfrctionted heprin () or low moleculr weight heprin (LMWH) cn ffect cncer progression (Bijsterveld et l., 1999; Hettirchchi et l., 1999; Smorenurg & vn Noorden, Author for correspondence; E-mil: s_eihr@gerit.med.tohoku.c.jp 1). Although mny mechnisms hve een postulted for the effects of PSAs on cncer, they re mostly independent of their nticogulnt effects. Since there is no dout tht the est wy of mnging DIC is to tret the underlying disorder (Levi & ten Cte, 1999), the possile effects of PSAs on cncer should e investigted to elucidte their mechnism of control of the growth of solid tumours. Blood supply is essentil for solid tumours nd tumour growth is highly dependent on ngiogenesis, the formtion of new cpillries from preexisting lood vessels (Crmeliet & Jin, ). Much evidence hs een otined suggesting tht nd LMWH cn ffect ngiogenesis (Smorenurg & vn Noorden, 1). In order to tret DIC in ptients with solid tumours, it is importnt to know which PSAs re effective t inhiiting tumour ngiogenesis nd, hence, reducing tumour growth. Therefore, in this study, we compred the effects of three PSAs, t therpeutic concentrtions equieffective t inhiiting FX, on vrious

2 33 H. Tkhshi et l Fctor X inhiition nd solid tumours spects of tumour growth, in order to elucidte their mechnisms of ction nd determine which PSA is most suitle for use in cncer ptients. Methods Animls Mle specific-pthogen-free C57Bl/6 or BDF1 mice (Cle Jpn Inc., Tokyo, Jpn) 6 9weeks of ge were used throughout the study. All procedures were performed ccording to Animls (Scientific Procedures) Act 1986 nd pproved y the locl ethics pnel t Tohoku University School of Medicine. The nimls were killed with n overdose of urethne ( g kg 1 ). FX evlution Blood ws collected into.13 M trisodium citrte from C57Bl/6 mice sucutneously injected with either ( units kg 1 dy 1 ), dlteprin (75 units kg 1 dy 1 ) or dnproid (5 units kg 1 dy 1 ) for 3 dys. The doses of PSAs used were similr to those given initilly to ptients with DIC. The FX cogultion fctor in the plsm ws evluted using cogultion nlyzer (Cogultion Anlyzer, SYSMEX CA 15, Dde Behring, Koe, Jpn). The plsm of ptient with fctor X (Biopool Interntionl, Venture, CA, U.S.A.) ws used s test regent for the FX. Results re expressed s percentge of the men vlue of the FX in lood from control mice tht were injected with 1 ml for 3 dys. (heprin sodium), dlteprin (frgmin s ) nd dnproid (orgrn s ) were purchsed from Tked Co. (Osk, Jpn), Kissei (Ngno, Jpn) nd Orgnon (Molenstrt, Netherlnd), respectively. Bleeding time C57Bl/6 mice were sucutneously injected with either ( units kg 1 dy 1 ), dlteprin (75 units kg 1 dy 1 ) or dnproid (5 units kg 1 dy 1 ) for weeks. The mice were then nesthetized with sodium thiopentl nd their tils trnsected 5 mm from the tip. Bleeding ws checked with filter pper every 3 s nd leeding time ws determined y mesuring the time until no lood ws detected on the filter pper. Cell culture Lewis lung crcinom cells (LLCs: ATCC, Mnsss, VA, U.S.A.) were cultured in high glucose Dulecco s modified Egle s medium (DMEM) contining 1% foetl clf serum (FCS), 1 mgml 1 knmycin. KLN5 (ATCC, Mnsss, VA, U.S.A.) cells, cell line of mouse squmous cell lung cncer, were cultured in minimum essentil medium (MEM) contining 1% FCS, 1% nonessentil mino cids nd 1 mgml 1 knmycin. Primry humn umilicl vein endothelil cells (HUVECs) were purchsed from Clonetics (Sn Diego, CA, U.S.A.), nd were cultured in endothelil cell growth medium (EGM- Bullet kit, Clonetics). To determine the effects of the PSAs on the prolifertion of LLC, KLN5 nd HUVEC in vitro, 1 cells were seeded into six-well dishes, nd then wshed with 1 h lter. Susequently, the dherent cells were stimulted y the ddition of culture medium contining vrious concentrtions of the PSAs. To estimte the prolifertion of HUVECs, fter 7 h of tretment with PSAs, the numer of HUVECs ws counted. To quntify VEGF in the superntnt of cultured LLC nd KLN5 cells, 1 5 cells were seeded into 1-mm dishes contining ech of the PSAs nd, 7 h lter, the superntnt ws collected nd stored t 81C. In vivo tumour models LLCs were injected (3 1 5 cells per niml) sucutneously into the centre of the ck of 6- to 9-week-old C57BL/6 mle mice on dy. KLN 5 cells were injected (5 1 5 cells per niml) sucutneously into the centre of the ck of 6- to 9-week-old BDF1 mle mice on dy. On dy 5, when ech tumour ecme plple, inoculted mice were rndomly llocted into four groups; mice in ech group received sucutneous injection of either (1 ml), ( units kg 1 ), dlteprin (75 units kg 1 ) or dnproid (5 units kg 1 ). Injections were strted from dy 5 nd given dily until the mice were killed. Tumour size ws quntified dily s width length.5. Immunohistochemistry nd determintion of microvessel density (MVD) When the dimeter of the tumour ecme B1 cm, tumour tissues were fixed in 1% formlin, emedded in prffin nd sectioned. The sections were locked with 1% norml got serum nd incuted with polyclonl nti-humn fctor VIIIrelted ntigen ntiody (DAKO, Crpinteri, CA, U.S.A.). Susequently, the sections were incuted with iotinylted got nti-rit IgG (Vector, Burlingme, CA, U.S.A.), nd then with ABC kit (Vector), nd were detected y 3-mino-9- ethylcrzole (Vector), counterstined with hemtoxylin. The intrtumorl MVD ws determined s descried previously (Weidner et l., 1991; Knd et l., 3). In rief, the vessels inside the tumour were stined with nti-humn fctor VIII-relted ntigen ntiody. The imge tht contined the highest numer of microvessels ws chosen for ech section from n initil scn t 1 mgnifiction. Then, the vessels were counted in the selected imge t mgnifiction. At lest four fields were counted for ech section, nd the highest count ws used. Two independent investigtors evluted the numer of vessels. Endothelil cell migrtion ssy To elucidte the effect of the PSAs on VEGF-induced endothelil cell migrtion, we used modified Boyden chmer (Chemotxicell, Kuro, Osk, Jpn). Humn recominnt VEGF ( ng ml 1 ), in the presence of ech PSA or, ws dded to the ottom well of the chmer t volume of 75 ml of EBM- (Sn Diego, CA, U.S.A.) contining.% FCS. Polycronte memrnes with 3 mm pores were coted with the ttchment fctor contining.1% geltin (Cscde Biologics, Portlnd, OR, U.S.A.) nd plced etween the test sustnces nd the upper chmers. HUVECs were hrvested t pproprite cell densities. Endothelil cells

3 H. Tkhshi et l Fctor X inhiition nd solid tumours 335 (1 1 5 ) were seeded to the inside of the upper chmer suspended in 5 ml of EBM contining.% FCS. The cells were incuted t 371C in5%co for 1 h. After incution, ll the nonmigrted cells were removed from the upper side of the filters with cotton ll. The filters were fixed with methnol, stined with Dif-Quick solution nd mounted onto microscope slides. The migrted cells were counted t mgnifiction using microscope. Endothelil cell tuulr formtion ssy Confluent HUVECs were detched with trypsin-edta nd suspended in EBM contining.5% FCS, 1 ng ml 1 humn recominnt VEGF (R&D Systems) nd 1 or 1 units ml 1 of ech PSA, to investigte the effect of the PSAs on VEGFinduced morphogenesis in vitro (Knd et l., 3). The cells were seeded into mtrigel-coted 1-well tissue culture pltes t density of 3 1 well 1 nd incuted t 371C. After 8 h, the cells were oserved using n inverted phse-contrst microscope (Nikon Eclipse TE3). Imges were cptured with lser scnning confocl imging system (Bio-Rd, Hercules, CA, U.S.A.). Tuulr formtion ws quntified y mesuring the length of tues in rndomly selected fields using the NIH imge progrm. In vivo mtrigel plug ssy The ngiogenic effect of ech PSA within.5 ml of mtrigel ws studied in 6- to 9-week-old C57Bl/6 mice. A mixture of mtrigel (Becton Dickinson Lwre) with either or one of the PSAs (1 units ml 1 ) ws injected into the dominl sucutneous tissue of mice long the peritonel midline. The mtrigel rpidly forms solid plug t ody temperture. After 1 dys, excised plugs were photogrphed nd their hemogloin content ws determined using Hemogloin Test Wko (Wko Pure Chemicl Industries, Osk, Jpn). In prllel experiment, VEGF-induced ngiogenesis in vivo ws ssessed s the growth of lood vessels from sucutneous tissue into solid mtrigel plug tht contined 5 ng ml 1 VEGF. Growth fctor-reduced mtrigel (Becton Dickinson Lwre), in liquid form t 1C, ws mixed with 5 ng ml 1 mouse recominnt VEGF (R&D Systems) nd injected (.5 ml). To investigte the effect of PSA tretment on VEGF-induced ngiogenesis in vivo, mice were sucutneously injected with either or ech PSA, t the sme doses used in the in vivo tumour experiments, into their cks every dy for 1 dys. The plugs were cut out y retining the peritonel tissues, fixed in 1% formlin nd emedded in prffin. Sections stined with hemtoxylin nd eosin were studied y light microscopy. The vessel re nd the totl mtrigel re were plnimetriclly clculted from the stined sections using the NIH imge progrm. Only those structures possessing ptent lumen nd contining erythrocytes were considered to e vessels. Results re expressed s percentge, clculted s the rtio of the vessel re to the totl mtrigel re. Quntifiction of VEGF protein y ELISA To quntify serum VEGF proteins, the inferior ven cv of the mouse ws punctured nd peripherl lood ws collected. To quntify tumour VEGF proteins,.3 g of the frozen tumour tissues were homogenized in 3 ml, centrifuged for min t 1, g t 1C nd the superntnt collected. The concentrtion of VEGF in ech smple ws determined using murine VEGF ELISA kit (R&D). Simultneously, the totl mount of protein in ech smple ws mesured y using Bio-Rd protein ssy (Bio-Rd, Hercules, C, U.S.A.). The VEGF concentrtion in tumour smples is expressed s pg mg 1 protein. Heprnse ctivity ssy Frozen tumours were homogenized in extrction uffer (.1 M,.15 M NCl, 1 mm PMSF, 1 g ml 1 leupeptin, 1% NP-) nd centrifuged t 1, g for 15 min t 1C. The protein concentrtion of the superntnt ws mesured y use of Brdford ssy (Bio-Rd, Richmond, CA, U.S.A.). The heprnse ctivity in the superntnt ws determined y mesuring heprn sulphte-degrding enzyme ctivity in the smple using Heprn Degrding Enzyme Assy Kit (Tkr Bio Inc., Otsu, Jpn) (Tkhshi et l., ). Quntifiction of VEGF nd heprnse RNA in LLCs LLCs were cultured in growth medi with or without PSAs for 7 h nd RNA ws isolted using RNAzol B regent (Tel-test, Inc., Friendswood, TX, U.S.A.). RNA expression of VEGF nd heprnse in LLCs ws determined y quntittive RT PCR using specific oligonucleotide primers nd TqMn proes purchsed from Applied Biosystems (Foster City, CA, U.S.A.) (Assy-on-Demnd Products: Mm373 for mouse VEGF nd Mm61768 for mouse heprnse). Mouse -ctin RNA ws simultneously ssessed s housekeeping gene using the following primers nd proes: forwrd, 5 -ACGGCCAGGTCATCACTATTG-3 ; reverse, 5 -CCAAGAAGGAAGGCTGGAAAA-3 ; TqMn proe, 5 -FAM-CAACGAGCGGTTCCGATGCCC- TAMRA-3. Rel-time RT PCR ppliction ws crried out in duplicte in 5 ml rection volumes contining ng totl RNA for ech specific rection using TqMn One-Step RT PCR Mster Mix Regents Kit (Applied Biosystems, CA, U.S.A.). Dt nlysis nd sttistics Dt re presented s men7s.e. Unless otherwise indicted, sttisticl comprisons etween groups were performed using Student s t-test or one-wy ANOVA with Fisher s lestsignificnt-difference test s post hoc test, s pproprite. Results FX inhiition nd leeding time in mice As shown in Figure 1, the levels of FX in mice injected with, dlteprin nd dnproid were significntly lower thn those in -treted mice (Po.1,.5 nd.1, respectively). There ws no significnt difference in FX

4 336 H. Tkhshi et l Fctor X inhiition nd solid tumours Fctor X (% control) 1 5 n.s. Dl Dn Bleeding time (min) 1 8 Dl Dn Tumor volume (x 1 3 mm 3 ) LLC in vivo Tumor volume (x 1 3 mm 3 ) KLN 5 in vivo 1 3 Dl Dn Dl Dn c Cell numer (x 1 5 ) 3 1 LLC in vitro Cell numer (x 1 5 ) 3 1 KLN 5 in vitro 8 7 Dl Dn 8 7 Dl Dn Figure 1 () Effects of PSAs on fctor X ctivities (left) nd leeding time (right). Mice were injected with (1 ml), ( units kg 1 ), dlteprin (Dl; 75 units kg 1 ) or dnproid (Dn; 5 units kg 1 ) for 3 dys. Fctor X ctivities re expressed s the percentge of the men of -injected mice (control). Po.5, Po.1 vs control y Student s unpired t-test. N.s. (not significnt) etween PSAs y one-wy ANOVA with Fisher s lest-significnt-difference test. The vlues represent the men7s.e. (n ¼ 5 7 per group). () The tumour volumes in LLC inoculted mice (left) nd KLN5 inoculted mice (right). The vlues represent the men7s.e. (n ¼ 7 1 per group). (c) Prolifertion curves for LLCs (left) nd KLN5s (right) in vitro in the mediums supplemented with PSAs (1 units ml 1 ). The vlues represent men7s.e. of triplicte wells. ctivity etween mice injected with, dlteprin nd dnproid. However, the mice injected with hd significntly longer leeding times thn those in the other three groups. There ws no significnt difference in leeding times etween the mice injected with, dlteprin nd dnproid. Effect of PSAs on tumour growth nd cncer cell prolifertion In mice inoculted with LLCs or KLN5s, ll the PSAs, t the doses used, hd similr inhiitory effects on FX. Wheres in LLC-inoculted mice, the tumour growth ws mrkedly inhiited y dlteprin nd, to lesser extent, y, ut dnproid did not inhiit tumour growth (Figure 1). In KLN5-inoculted mice, ll the PSAs inhiited tumour growth, the potency order eing dlteprindnproid. The prolifertion curves of LLCs nd KLN5s were investigted in vitro. None of the PSAs ffected either LLC or KLN5 prolifertion, suggesting tht PSAs do not inhiit tumour growth y directly ffecting the prolifertion of cncer cells. Effects of PSAs on tumour ngiogenesis The immunostining of fctor VIII-relted ntigen in tumours of similr size (B1 cm dimeter) showed tht there were decresed densities of vessels in - nd dlteprin-treted tumours compred with those treted with nd dnproid (Figure, left). The MVDs in - nd dlteprintreted tumours were lso significntly lower thn those in - nd dnproid-treted tumours (Figure, right). There ws no difference in MVD etween - nd dnproid-treted LLC tumours, nd etween - nd dlteprin-treted LLC tumours. In KLN5 tumours, the immunostining showed decresed densities of vessels in ll PSA-treted tumours compred with -treted tumours (Figure, left). The microvsculr

5 H. Tkhshi et l Fctor X inhiition nd solid tumours 337 Dl Dn Numer of vessels (HPF-1) 8 6 Dl Dn 5 Dl Dn Numer of vessels (HPF-1) Dl Dn Figure () Immunohistochemicl stining for fctor VIII-relted ntigen of the LLC tumours. Left: representtive photogrphs from ech group re shown (mgnifiction, 1). Right: the effects of the PSAs on the tumour microvessel densities. Ech vlue is the men7s.e. (n ¼ 5 7 per group) of vessel counts oserved with high-power field (mgnifiction, ). Po.5 mong PSAs y one-wy ANOVA with Fisher s lest-significnt-difference test. Po.1 compred with group y Student s unpired t-test. () Immunohistochemicl stining for fctor VIII-relted ntigen of KLN5 tumours. Left: representtive photogrphs from ech group re shown (mgnifiction, ). Right: the effects of the PSAs on the tumour microvessel densities. Ech vlue is the men7s.e. (n ¼ 5 7 per group) of vessel counts oserved with high-power field (mgnifiction, ). Po.5, Po.1 compred with the group y the Student s unpired t-test. There ws no significnt difference etween the PSAs y one-wy ANOVA with Fisher s lest-significnt-difference test. densities in PSA-treted KLN5 tumours were significntly lower thn those in -treted KLN5 tumours (Figure, right). Effect of PSAs on in vivo ngiogenesis in mtrigel plugs To investigte the interction etween PSAs nd ngiogenesis, we performed mtrigel plug ssy using mtrigels contining 1 units ml 1 of ech of the PSAs. PSA-contining mtrigels showed loody ppernce, suggesting vsculriztion inside the plug, wheres -contining mtrigels did not (Figure 3, upper). Estimtion of hemogloin content in the mtrigel plug showed tht PSAs themselves re le to induce ngiogenesis (Figure 3, lower). We investigted the effect of sucutneous injections of the PSAs on VEGF-induced ngiogenesis using mtrigel plugs contining VEGF. Vessel formtion in mtrigels in PSAinjected mice did not look like tht in mtrigels in -injected mice (Figure 3, upper). Since the mtrigel itself did not contin the PSAs, it hd little hemogloin, so ngiogenesis ws estimted y clculting the re of vessels. All PSAs significntly inhiited VEGF-induced vessel formtion in mtrigel plugs compred with. There were no significnt differences etween the inhiitory effects of the PSAs on VEGF-induced vessel formtion. Direct effects of PSAs on endothelil cells in vitro In contrst to in vivo ngiogenesis, 1 U ml 1 of ech of the PSAs significntly suppressed HUVEC prolifertion (Figure ). Moreover, ll the PSAs significntly inhiited VEGF-induced migrtion of HUVEC in vitro (Figure ) nd there were no significnt differences etween the inhiitory effects of the PSAs on migrtion. In ddition, the effect of the PSAs on the morphogenesis of endothelil cells ws investigted y estimting the tuulr formtion of HUVEC. VEGF-induced tuulr formtion in the mtrigel ws not significntly ffected y the ddition of the PSAs (Figure c). Effect of PSAs on VEGF expression nd production Since VEGF is known to hve pivotl role in tumour ngiogenesis (Ferrr et l., 3) nd the ngiogenesis

6 338 H. Tkhshi et l Fctor X inhiition nd solid tumours H content (%Vol) Men re % induced y VEGF (Figures 3 nd ) ws inhiited y PSAs, we investigted the possile modultion of VEGF y PSAs. In mice with LLC tumours, the verge serum VEGF protein levels in the four groups were in the order of dnproiddlteprin. The serum VEGF protein level in dlteprin-treted mice ws significntly lower thn the other three groups (Figure 5), ut there were no significnt differences etween the, nd dnproid groups. The concentrtions of VEGF protein in LLC tumours were in Dl Dl n.s. Dn Dl Dn VEGF Dn Figure 3 () Mtrigel contining either or ech PSA (1 units ml 1 ). Upper: representtive plugs from ech group re shown (r, 1 cm). Lower: the hemogloin content of the mtrigels contining the PSAs. The vlues represent the men7s.e. (n ¼ 8 1 per group). Po.5 vs y Student s unpired t-test. Po.5 etween the PSAs y one-wy ANOVA with Fisher s lestsignificnt-difference test. () Vessel formtion in mtrigels contining VEGF. Upper: representtive microphotogrphs of mtrigel plugs contining VEGF treted with or PSAs s indicted (mgnifiction, ). Lower: the vessel re compred to the totl mtrigel re (n ¼ 5 7 per group). Po.5, Po.1 vs þ VEGF y Student s unpired t-test. There ws no significnt difference etween the PSAs y one-wy ANOVA with Fisher s lest-significnt-difference test. the sme order s those in serum (Figure 5). The concentrtion of VEGF protein in tumours in dlteprin-treted mice ws significntly decresed compred with tht in the other three groups (Figure 5). In vitro, VEGF protein levels in culture medium contining LLCs were reduced y nd dlteprin in dose-dependent mnner, ut not y dnproid (Figure 5). The inhiitory effect of dlteprin ws lwys greter thn tht of. We ssessed VEGF mrna in LLCs quntittively y rel-time PCR. Both nd dlteprin dose-dependently inhiited VEGF mrna (Figure 5d). In contrst, in mice with KLN5 tumours, ll the PSAs inhiited serum VEGF to similr extent (Figure 6) nd they significntly reduced tumour VEGF production (Figure 6). In vitro, ll the PSAs inhiited VEGF production in KLN5 cells in concentrtion-dependent mnner, with potency order of ¼ dlteprindnproid (Figure 6c). In ddition, ll the PSAs significntly inhiited the expression of VEGF mrna in vitro (Figure 6d). Effects of PSAs on heprnse ctivity nd mrna expression PSAs re well-known inhiitors of heprnse ctivity (Prish et l., 1987; Voldvsky & Friedmnn, 1) nd heprnse is potent ngiogenic fctor (Elkin et l., 1; Goldshmidt et l., ). Hence, we investigted the effects of the PSAs on heprnse ctivity in LLC nd KLN5 tumours in vivo. In LLC tumours, nd dlteprin significntly inhiited tumour heprnse ctivity, wheres dnproid did not t the dose used (Figure 7). In KLN5 tumours, dlteprin ws the only PSA tht inhiited heprnse ctivity (Figure 7). We lso ssessed heprnse mrna quntittively in LLCs treted with nd dlteprin in vitro; oth nd dlteprin inhiited heprnse mrna in dosedependent mnner (Figure 7c). Discussion In this study, we showed tht n LMWH, dlteprin, is more effective t inhiiting experimentl tumour growth thn nd dnproid t concentrtions tht produce the sme level of FX inhiition nd tht this effect is medited, t lest in prt, y inhiition of tumour VEGF nd heprnse production. Despite the incresing vilility of LMWHs, is still widely used s n ntithromotic drug for the initil mngement of DIC ecuse it is inexpensive (Hirsh et l., 1) nd dnproid is sometimes chosen ecuse of its potent nti-fx ctivity (Meulemn, 199). Since DIC is one of the most common complictions of cncer, the effect of these drugs on tumour growth during DIC tretment might e n importnt fctor in the choice of ntithromotic drug. Although the dose of the drug used to tret DIC will vry depending on the sitution, the initil doses of, dlteprin, nd dnproid re out 5 1 units kg 1 dy 1, 1 units kg 1 dy 1, nd 75 nti-fx units kg 1 dy 1, respectively. Since the hlf-life of is out four times less thn those of dlteprin nd dnproid, from the phrmcokinetics of sucutneous injections, doses of units kg 1 dy 1, 75 units kg 1 dy 1 nd 5 nti-fx units kg 1 dy 1, respectively, were chosen for the dily injections. The nti-fx level is the only method ville for estimting

7 H. Tkhshi et l Fctor X inhiition nd solid tumours 339 Numer of HUVEC (x1 ) 8 6 Numer of migrted HUVEC Dl Dn VEGF Dl Dn c d n.s. Reltive tuulr formtion % (u ml -1 ) 1 1 Control Dl Dn Figure () Numer of HUVECs fter 7 h tretment with PSAs (1 units ml 1 ). The vlues represent the men7s.e. of triplicte wells. Po.5, Po.1 vs y Student s unpired t-test. N.s. (not significnt), Po.5 etween PSAs y one-wy ANOVA with Fisher s lest-significnt-difference test. () VEGF-induced migrtion of HUVEC. Po.5, Po.1 vs VEGF þ, nd w Po.5 vs VEGF þ y ANOVA with Fisher s lest-significnt-difference test. (c) The effect of PSAs on HUVEC tuulr formtion in vitro. Representtive microphotogrphs of HUVEC morphogenesis to form cpillry-like structures in culture medium contining or PSAs ( 1). (d) Quntittive nlysis of the extent of tuulr formtion. Ech r indictes the men7s.e. of dt from four experiments performed in duplicte wells. There were no significnt differences etween control () nd PSA-treted HUVEC y Student s unpired t-test. clotting ctivities for ll these drugs. At the concentrtions used,, dlteprin nd dnproid were equieffective t inhiiting FX, suggesting tht their potency t treting DIC should e comprle. However, leeding time ws significntly prolonged in -treted mice (Figure 1). Severl studies hve descried oth stimultory nd inhiitory effects of heprins on tumour growth nd metstsis (Hejm et l., 1999). These diverse effects my e due to the ility of these drugs to ffect other processes, such s ngiogenesis, in ddition to their nticogulnt properties (Folkmn & Shing, 199). In this study, lthough none of the PSAs ffected the prolifertion of cncer cells, oth LLC tumour growth nd microvessel densities were significntly suppressed y nd dlteprin ut not y dnproid. In KLN5 tumours, ll the PSAs inhiited oth tumour growth nd ngiogenesis in vivo. These results suggest tht the suppression of tumour growth cn e ttriuted to the inhiition of tumour ngiogenesis. LMWH nd hve een found to hve different effects on ngiogenesis in previous studies (Norry, 1993; Pieper et l., ).

8 3 H. Tkhshi et l Fctor X inhiition nd solid tumours 8 8 VEGF in serum (pg ml -1 ) 6 VEGF in tumor (x1-9 ) 6 Dl Dn Dl Dn c 5 d VEGF in vitro (pg ml -1 ) 3 Rtio to β-ctin mrna (u ml -1 ) (u ml -1 ) Control Dl Dn.1 1 Control Dl Figure 5 VEGF protein levels in serum () nd tumours () in mice with LLC tumours were quntified y ELISA. The vlues represent the men7s.e. (n ¼ 5 7 per group, Po.5, Po.1 compred to other groups using one-wy ANOVA with Fisher s lest-significnt-difference test). (c) The VEGF protein levels in LLCs in culture medium. The vlues represent the men7s.e. of triplicte wells (Po.5, Po.1 vs control using Student s unpired t-test). (d) VEGF mrna of LLCs treted with the PSAs. Results of quntittive RT PCR re expressed s the rtio to -ctin mrna. The vlues represent the men7s.e. of triplicte wells. The dt re representtive results of three independent experiments. Dn It ws interesting tht, in the sence of cncer cells, ll three PSAs induced ngiogenesis in vivo when they were included in the mtrigel plug (Figure 3). In prticulr, the mtrigel plug contining dnproid hd higher hemogloin content thn the others. Angiogenesis in mtrigel plugs hs lso een found to e mrkedly enhnced y the ddition of heprin (Pssniti et l., 199). However, when PSAs were not included in the mtrigel, VEGF-induced vessel formtion in the mtrigel ws similrly inhiited y sucutneous injections of ech of the PSAs (Figure 3). This suggests tht the PSAs re le to modulte ngiogenic growth fctors. Although vrious studies hve evluted the effects of nd LMWH on the prolifertion of vsculr endothelil cells in vitro (Smorenurg & vn Noorden, 1), few hve investigted the effect of exogenously dded heprn sulphte. In vitro, ll three PSAs significntly inhiited the prolifertion nd migrtion of vsculr endothelil cells (Figure 3 nd ). However, the PSAs hd no significnt effect on endothelil cell morphogenesis in vitro (Figure 3c). The results depicted in Figures 3 nd suggest tht, in the sence of cncer cells, the effects of the PSAs on ngiogenesis re similr oth in vitro nd in vivo. In contrst, the effect of dnproid on tumour ngiogenesis ws different in LLC nd KLN5 tumours. Since the induction nd mintennce of tumour ngiogenesis requires ngiogenic growth fctors produced y cncer cells (Crmeliet, ), the diverse effects of dnproid on ngiogenesis in LLC nd KLN5 tumours could e due to differences in the effects of dnproid on the production of these ngiogenic growth fctors. Of ll the ngiogenic fctors produced y tumours, VEGF is one of the most potent (Ferrr et l., 3). The interction of VEGF with its receptor is elieved to ply mjor role in ngiogenesis in humn tumours (Ferrr, ). We found tht, in LLC tumours, nd dlteprin suppressed VEGF production in vivo, wheres dnproid did not. This result is consistent with those from the in vitro experiments in which VEGF relese from LLCs ws determined. In KLN5 tumours, ll the PSAs significntly inhiited VEGF production in tumours, in vivo, nd from KLN5 cells in vitro. Thus, the effect of dnproid on VEGF production in cncer cells depends on their type. Although VEGF is ound to heprins (El-Sheikh et l., ; Lever & Pge, ), our study showed tht oth nd dlteprin hd

9 H. Tkhshi et l Fctor X inhiition nd solid tumours 31 VEGF in serum (pg ml -1 ) VEGF in tumor (x1-9 ) c 1 Dl Dn Dl Dn d 5. VEGF in vitro (pg ml -1 ) 8 6 Rtio to β-ctin mrna (u ml -1 ) Control Dl Dn (u ml -1 ) 1 Control Dl Figure 6 VEGF protein levels in serum () nd tumours () in mice with KLN5 tumours were quntified y ELISA. The vlues represent the men7s.e. (n ¼ 5 7 per group, Po.5 compred to other groups using one-wy ANOVA with Fisher s lestsignificnt-difference test). (c) The VEGF protein levels in KLN5 cell culture medium. The vlues represent the men7s.e. of triplicte wells (Po.5, Po.1 vs control using Student s unpired t-test). (d) VEGF mrna in KLN5 cells treted y PSAs. Results of quntittive RT PCR re expressed s the rtio to -ctin mrna. The vlues represent the men7s.e. of triplicte wells. The dt re representtive results from three independent experiments. Dn the ility to suppress VEGF production in cncer cells t the mrna level. Owing to structurl similrities with heprn sulphte, nd LMWH inhiited the enzyme heprnse, which hydrolyses the internl glycosidic linkge of heprn sulphte in sement memrnes nd ECM (Prish et l., 1987; Voldvsky & Friedmnn, 1). In cncer progression, heprnse hs role not only in invsion nd metstsis ut lso in the induction of tumour ngiogenesis (Elkin et l., 1; Goldshmidt et l., ). nd LMWH oth inhiited heprnse ctivity in LLC tumours, wheres dnproid did not (Figure 7), which is consistent with the inhiitory effects of nd dlteprin on heprnse mrna expression in LLCs in vivo. On the other hnd, heprnse ctivity in KLN5 tumours ws significntly inhiited only y dlteprin, suggesting the lck of reltionship etween heprnse ctivity nd tumour ngiogenesis in KLN5 cells (Figure 7). Tken together, our dt from LLC nd KLN5 tumours suggest tht PSAs inhiit tumour ngiogenesis, t lest in prt, y suppressing VEGF production rther thn heprnse production in cncer cells. The effect of dnproid on VEGF production in cncer cells might e cell type specific, resulting in different effects on tumour ngiogenesis. Since there re severl other ngiogenic growth fctors such s sic firolst growth fctor, plcent-like growth fctor, pltelet-derived growth fctors nd interleukin-8, further studies re needed to clrify the ntingiogenic mechnism of PSAs. Sucutneous injections of dnproid did not inhiit heprnse in either LLC or KLN5 tumours. Heprnse is known to hve n importnt role in cncer metstsis s it degrdes the extrcellulr mtrix (Prish et l., 1987) nd is involved in the progression of cncer (Tkhshi et l., ). Our results suggest tht, in contrst to heprins, dnproid does not inhiit cncer metstsis. In our experiments, lthough oth nd dlteprin inhiited ngiogenesis in vivo nd in vitro, the effect of dlteprin ws generlly seen efore tht of. Moreover, t doses tht re similrly potent t inhiiting FX, dlteprin is less likely to cuse leeding thn (Figure 1). Therefore, our results suggest tht LMWH my hve dvntges compred to other PSAs s tretment for DIC in ptients with solid tumours. Recently, dnproid hs ecome widely used s n nticogulnt in ptients with ctive or pst heprin-induced thromocytopeni (Trdy-Poncet et l., 1999). However, the precise mechnism of its selective

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