Lysophosphatidic Acid (LPA) in Malignant Ascites StimulatesMotility of Human Pancreatic Cancer Cells through LPA1*

Received for publication, July 25, 2003, and in revised form, December 1, 2003Published, JBC Papers in Press, December 3, 2003, DOI 10.1074/jbc.M308133200

Takayuki Yamada‡§, Koichi Sato‡, Mayumi Komachi‡, Enkhzol Malchinkhuu‡, Masayuki Tobo‡,Takao Kimura¶, Atsushi Kuwabara¶, Yasuhiro Yanagita‡, Toshiro Ikeya�, Yoshifumi Tanahashi§,Tetsushi Ogawa§, Susumu Ohwada§, Yasuo Morishita§, Hideo Ohta**, Doon-Soon Im‡‡,Koichi Tamoto§§, Hideaki Tomura‡, and Fumikazu Okajima‡¶¶

From the ‡Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University,Maebashi 371-8512, Japan, the §Second Department of Surgery and the ¶Department of Laboratory Medicine,Graduate School of Medicine, Gunma University, Maebashi 371-8511, Japan, the �Maebashi Red Cross Hospital,3-21-36, Asahi-cho, Maebashi 371-0014, Japan, the **Research Laboratory, Kirin Brewery Co., LTD., 3 Miyahara,Takasaki 370-1295, Japan, the ‡‡Laboratory of Pharmacology, College of Pharmacy, Pusan National University,Busan, Republic of Korea 609-735, and the §§Department of Microbiology, Faculty of Pharmaceutical Sciences,Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-02, Japan

Cytokines and growth factors in malignant ascites arethought to modulate a variety of cellular activities ofcancer cells and normal host cells. The motility of can-cer cells is an especially important activity for invasionand metastasis. Here, we examined the components inascites, which are responsible for cell motility, from pa-tients and cancer cell-injected mice. Ascites remarkablystimulated the migration of pancreatic cancer cells. Thisresponse was inhibited or abolished by pertussis toxin,monoglyceride lipase, an enzyme hydrolyzing lysophos-phatidic acid (LPA), and Ki16425 and VPC12249, antag-onists for LPA receptors (LPA1 and LPA3), but not by anLPA3-selective antagonist. These agents also inhibitedthe response to LPA but not to the epidermal growthfactor. In malignant ascites, LPA is present at a highlevel, which can explain the migration activity, and thefractionation study of ascites by lipid extraction andsubsequent thin-layer chromatography indicated LPAas an active component. A significant level of LPA1 re-ceptor mRNA is expressed in pancreatic cancer cellswith high migration activity to ascites but not in cellswith low migration activity. Small interfering RNAagainst LPA1 receptors specifically inhibited the recep-tor mRNA expression and abolished the migration re-sponse to ascites. These results suggest that LPA is acritical component of ascites for the motility of pancre-atic cancer cells and LPA1 receptors may mediate thisactivity. LPA receptor antagonists including Ki16425are potential therapeutic drugs against the migrationand invasion of cancer cells.

Pancreatic cancer is a highly metastatic cancer characterizedby widespread intraperitoneal dissemination and ascites for-

mation, which frequently occur even after curative resectionand constitute the major cause of death in pancreatic cancerpatients (1, 2). Therefore, the suppression of dissemination isan important issue in the treatment of pancreatic cancer. Peri-toneal dissemination is thought to be composed of several pro-cesses, including cell adhesion, migration, invasion, and prolif-eration (3, 4). In the ascites and pleural effusions of patients, avariety of cytokines and growth factors are present; these cy-tokines were produced and secreted from cancer cells, as wellas from normal host cells, and have been suggested to affectthese processes of peritoneal dissemination (5–7).

Lysophosphatidic acid (LPA)1 has been shown to participatein diverse biological actions, including a change in cell shape,motility, and proliferation in a variety of cell types in associa-tion with the stimulation of early signaling events, such asCa2� mobilization, change in cAMP accumulation, and activa-tion of several protein kinases (8–11). Extracellular LPA hasalso been shown to be involved in certain diseases, such asatherosclerosis (12, 13) and cancer (11, 14–18). In fact, LPA hasbeen identified as a growth-promoting factor that promotes theproliferation of ovarian cancer cells in malignant ascites fromovarian cancer patients (14–16). LPA has also been reported tobe present in malignant effusions (19). Most of these LPAactions are mediated through the lipid-specific EDG family Gprotein-coupled receptors, i.e. LPA1/EDG-2, LPA2/EDG-4, andLPA3/EDG-7 (8–10, 20), although recent studies suggestedthat LPA actions are potentially mediated through LPA4/GPR23, another type of G protein-coupled receptor for LPA (21)and peroxisome proliferator-activated receptor �, a transcrip-tional factor identified as an intracellular LPA receptor (22).Although there are many reports suggesting the potential roleof LPA in cell growth, migration, and invasion of several cancercells (14, 18, 23–31), it was difficult to determine the extent towhich LPA, among a variety of potent cytokines and growthfactors present in ascites, serves as a mediator of these re-sponses. LPA receptor antagonists, LPA-neutralizing antibod-

* This work was supported by grants-in-aid for scientific researchfrom the Japan Society for the Promotion of Science, by research grantsfrom Pusan National University, the Mitsubishi Foundation, ONOMedical Research Foundation, and partly by the Japan-Korea BasicScientific Cooperation Program. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

¶¶ To whom correspondence should be addressed: Laboratory of Sig-nal Transduction, Institute for Molecular and Cellular Regulation,Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan.Tel.: 81-27-220-8850; Fax: 81-27-220-8895; E-mail.: fokajima@showa.gunma-u.ac.jp.

1 The abbreviations used are: LPA, lysophosphatidic acid; S1P, sphin-gosine 1-phosphate; LPC, lysophosphatidylcholine; PTX, pertussis tox-in; EDG, endothelial cell differentiation gene; G-protein, GTP-bindingregulatory protein; HPTLC, high performance thin layer chromatogra-phy; MG lipase, monoglyceride lipase; BuOH, n-butyl alcohol; DGPP8:0, dioctylglycerol pyrophosphate; BSA, bovine serum albumin; siRNA,small interfering RNA; EGF, epidermal growth factor; MOPS, 4-mor-pholinepropanesulfonic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 8, Issue of February 20, pp. 6595–6605, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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ies, or the LPA-specific degrading enzyme would be very usefulfor this purpose. Especially, receptor-selective antagonists arestrong tools for identifying the subtype of LPA receptors thatare responsible for the lipid action and would be a platform todevelop therapeutic agents.

We have recently developed a novel LPA receptor antagonist,Ki16425, which shows a preference for LPA1 and LPA3 overLPA2 (32). Ki16425 showed an extremely high specificity toLPA and its receptors. Thus, Ki16425 may be a useful tool forevaluating the role of LPA in biological samples, such as asci-tes. In the present paper, we showed that a considerably highamount of LPA is present in ascites from pancreatic cancerpatients and that the formation of LPA-rich ascites can beduplicated by an intraperitoneal injection of human pancreaticcancer cells in nude mice. Furthermore, our data indicated thatLPA in malignant ascites is an important component for themotility of pancreatic cancer cells through Ki16425-sensitiveLPA receptors, especially LPA1.

EXPERIMENTAL PROCEDURES

Materials—1-Oleoyl-sn-glycero-3-phosphate (LPA), L-�-lysophos-phatidylcholine palmitoyl (LPC, C16:0), and sphingosine 1-phosphate(S1P) were purchased from Cayman Chemical Co. (Ann Arbor, MI);fatty acid-free BSA was from Calbiochem-Novabiochem Co. (San Diego,CA); dioctylglycerol pyrophosphate (DGPP 8:0) was from Avanti PolarLipids, Inc. (Alabaster, AL); PTX was from List Biological Laboratories,Inc. (Campbell, CA); EGF was from Sigma; 3-(4,5-dimethythiazol-2-yl)-diphenyltetrazolium bromide was from Dojindo (Tokyo, Japan); MGlipase was from Asahi Kasei Corp. (Shizuoka, Japan); [3H]LPA (48Ci/mmol) was from PerkinElmer Life Sciences; and [�-32P]dATP (3000Ci/mmol) was from Amersham Biosciences. Ki16425 (3-(4-[4-([1-(2-chlo-rophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfo-nyl)propanoic acid) was synthesized by Kirin Brewery Co. (Takasaki,Japan), and VPC12249 was a generous gift from Prof. Kevin R. Lynch(University of Virginia School of Medicine).

Mice—Female BALB/c nude mice (5 weeks old) were obtained fromCharles River Japan, Inc. (Tokyo, Japan). Sterile food and water werefed to the mice ad libitum. The mice were maintained in sterile cageson sterile bedding and housed in rooms at a constant temperatureand humidity. All experiments using mice were performed accordingto procedures approved by the Gunma University Animal CareCommittee.

Ascites from Cancer Patients—Eight Japanese patients with pancre-atic cancer were available for this study at the Second Department ofSurgery, Gunma University Faculty of Medicine from 2001 through2003. Table I summarized the characteristics of the patients. A cyto-logical examination demonstrated that the ascites contains cancer cells.Plasma or ascites were collected in the presence of EDTA (at a finalconcentration of 2–3 mM) and centrifuged at 1,000 � g for 20 min toremove cells. The cell-free fluid was stored at �80 °C until use. In-formed consent was obtained from each patient for the use of samples.

Establishment of a Highly Peritoneal Metastatic Pancreatic CancerCell Line, YAPC-PD—A human pancreatic cancer cell line, YAPC-PD,was established from YAPC cells, which had been previously estab-lished in our laboratory (33). The YAPC is not a highly peritonealmetastatic cell line, but, in one nude mouse of 30, which were injectedin the peritoneal cavity with YAPC cells (1 � 107 per mouse), peritonealdissemination with bloody ascites was detected 12 weeks after theinjection. Cells in the bloody ascites were harvested and cultured inRPMI 1640 containing 10% fetal bovine serum. Intermingled mousefibroblasts gradually decreased in number and finally disappearedwithin a 4-week culture. The rest of the cells attached on dishes wereharvested, and 1 � 107 cells were then injected in the peritoneal cavityof nude mice. The same procedure was repeated in a fifth cycle, and weobtained the YAPC-PD cell line, which induces peritoneal dissemina-tion with high frequency. More than 90% of nude mice injected intra-peritoneally with this cell line developed peritoneal dissemination withbloody ascites within 4 to 6 weeks.

Ascites from Mice Injected with the Human Pancreatic Cancer CellLine, YAPC-PD—Ten million cells of YAPC-PD were injected into theperitoneal cavity of nude mice. Four to 6 weeks later, mice that devel-oped abdominal distension were killed, and bloody ascites was collectedin the presence of EDTA (at a final concentration of 2–3 mM). The bloodyascites was rendered cell-free by centrifugation as described above.

Cell Culture—Human pancreatic cancer cell lines, PK-1, PK-9, and

Panc-1, were kindly provided by the Cancer Cell Repository, TohokuUniversity (Sendai, Japan), and MIA PaCa-2, BxPC-3, CFPAC-1, andHPAC were purchased from the American Type Culture Collection(Rockville, MD). The YAPC cells were transfected with a pEFneo emptyvector alone or a pEFneo vector containing human LPA1 receptor (34)by electroporation, and the neomycin (G418 sulfate at 1 mg/ml)-resist-ant cells were selected. All pancreatic cancer cell lines and the receptor-transfected cells were cultured in RPMI 1640 containing 10% fetalbovine serum. Twenty-four hours before the experiments, the mediumwas changed to a fresh medium (without serum) containing 0.1% (w/v)BSA (fraction V) unless otherwise specified. Where indicated, PTX (100ng/ml) was added to the culture medium 24 h before the experiments.

Transfection of siRNA—Pancreatic cancer cell lines, YAPC-PD andPanc-1, were plated on 12 multiwell plates at �2.0 � 105 cells/well.Sixteen h later, siRNAs (total 30 nM) were introduced into cells using anRNAiFect reagent (Qiagen K.K., Tokyo, Japan) according to the man-ufacturer’s instructions and the cells were further cultured for 24 h. TheLPA receptor mRNA level was measured using real-time TaqMan tech-nology, and a cell migration assay was performed 24 h after serumstarvation as described later. The following 21-mer oligonucleotidepairs were used as siRNAs against human LPA1: LPA1-102, 5�-r(CCG-AAGUGGAAAGCAUCUU)d(TT)-3� and 5�-r(AAGAUGCUUUCCACU-UCGG)d(TT)-3�; LPA1-228, 5�-r(CCGCCGCUUCCAUUUUCCU)d(TT)-3�and 5�-r(AGGAAAAUGGAAGCGGCGG)d(TT)-3�; and LPA1-945, 5�-r(AGAAAUGAGCGCCACCUUU)d(TT)-3� and 5�-r(AAAGGUGGCGC-UCAUUUCU)d(TT)-3�. The numbers 102, 228, and 945 represent theposition in the nucleotide sequence of the coding region. The following21-mer RNA was used as a negative control: 5�-r(UUCUCCGAACGU-GUCACGU)d(TT)-3� and 5�-r(ACGUGACACGUUCGGAGAA)d(TT)-3�.These annealed oligonucleotides were obtained from Qiagen K.K. ashigh performance purity grade and used according to the manufactur-er’s instructions.

Evaluation of LPA-like Activity—LPA in malignant ascites andplasma (0.5 ml, unless otherwise stated) was selectively extracted asalkaline-soluble lipids as described previously (35). By this procedure,major lipid components, such as phosphatidylcholine, sphingomyelin,and other neutral lipids, can be removed. To evaluate the content ofLPA in this extract, a sensitive and specific bioassay based on theability of LPA to inhibit cAMP accumulation in LPA1-expressingRH7777 cells was used because vector-transfected RH7777 cells do notrespond to LPA (20). The LPA1-expressing RH7777 cells (a generousgift from Prof. Kevin R. Lynch, University of Virginia School of Medi-cine) were cultured in minimal essential medium containing 10% fetalbovine serum. Three days before the experiments, cells were seeded on12-well plates that were coated with rat-tail collagen (400 �g/ml).Twenty-four hours before the experiments, the medium was changed tofresh minimal essential medium containing 0.1% BSA. The cells werewashed twice with a HEPES-buffered medium (36) and incubated withthe extract or LPA (C18:1) standard in a HEPES-buffered mediumcontaining 10 �M forskolin and 0.5 mM isobutylmethylxanthine at afinal concentration of 500 �l. After a 10-min incubation, the reactionwas terminated by adding 100 �l of 1 N HCl. Cyclic AMP in the acidextracts was measured (36). The cAMP-inhibiting activity of LPA ortest samples was completely lost by MG lipase, an enzyme hydrolyzingmonoglycerides, such as LPA, or Ki16425, an LPA antagonist. Further-more, the activity was unchanged even when the LPA-like activity wasmeasured after further purification with silica gel high performancethin-layer chromatography (HPTLC) (Merck) using a solvent systemconsisting of BuOH/acetic acid/water (3:1:1). These results support thespecificity of this bioassay. By this method, we detected LPA C18:1(1-oleoyl-sn-glycero-3-phosphate) equivalent activity that was as low as1 pmol/assay well. The evaluated LPA-like activity in the test samplewas presented as an LPA C18:1 equivalent level.

MG Lipase Treatment—LPA (1 �M), EGF (100 ng/ml), or ascites(10%) were treated with MG lipase at 10 units/ml for 30 min at 37 °C inRPMI 1640 containing 0.1% BSA. In separate experiments, we added[3H]LPA to the assay medium and confirmed that more than 98% ofLPA in the test samples was degraded under these conditions. TheseMG lipase-treated samples were finally used by 10 times dilution withthe assay medium.

Extraction of an Active Component from Ascites—The ascites weretreated with 2 volumes of BuOH and separated into two phases. TheBuOH-extracted components were further separated by HPTLC using asolvent system consisting of BuOH/acetic acid/water (3:1:1) (37). Thesilica gel with the resolved lipids (about 1-cm length each) was scrapedoff to obtain lipids covering the entire area of migration. The lipids wereeluted and dried by evaporation. All fractions thus separated weredissolved in an assay medium containing 0.1% BSA and used at the

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final concentration corresponding to 1% ascites.Cell Migration Assay—The migration experiment was performed

using a Boyden chamber apparatus, as previously described (38). Inbrief, YAPC-PD cells and other cancer cells were harvested with 0.05%trypsin containing 0.02% EDTA, washed once, and resuspended withRPMI 1640 containing 0.1% BSA. The cells were loaded into the upperchamber, and test agents were placed in the lower chamber, unlessotherwise specified. A membrane filter with 8-mm pores was precoatedovernight at 4 °C with 100 �g/ml rat-tail collagen. When the effects ofLPA antagonists were examined, the cells were preincubated for 10 minwith antagonists before loading. The number of cells that had migratedfor 4 h to the lower surface were determined by counting the cells in fourplaces under microscopy at �400 magnification. Unless otherwisestated, this Boyden chamber method was used for the evaluation of themigratory activity of the cells. In some experiments, Transwell chemo-taxis chambers (6.5 mm diameter, 8 �m pore size) (Costar, Inc.) wereused. Chemotaxis filters were soaked in 100 �g/ml rat-tail collagenovernight, similarly to the Boyden chamber method. The cells were firstattached on the filters; unattached cells were then removed 1 h afterseeding, and test agents were placed in the lower chamber. The numberof the cells that migrated to the lower surface during a 4-h incubationwas determined as described above.

Matrigel Invasion Assay—Cell invasion activity was assessed byusing a Matrigel invasion chamber (BD Biosciences, San Jose, CA)according to the instructions provided by the manufacturer. The proce-dures are essentially the same as those for the migration assay usingTranswell chemotaxis chambers, except that the incubation time was24 h.

Cell Adhesion Assay—Cells were harvested by trypsin, seeded on48-well plates at a density of 5 � 104 cells per well in RPMI 1640containing 0.1% BSA and test agents, and then incubated for 1 or 4 h.At each time, floating cells were aspirated, plates were rinsed withphosphate-buffered saline, and the attached cells were then evaluatedby an 3-(4,5-dimethythiazol-2-yl)-diphenyltetrazolium bromide assayas described previously (33).

Cell Proliferation—Cells were seeded on 48-well plates at 2 � 104

cells in 0.4 ml. Twenty-four hours before the experiments, the mediumwas changed to RPMI 1640 containing 0.1% BSA, and cells were ex-posed to the test agents for an additional 24 h. Cell proliferation wasevaluated by an 3-(4,5-dimethythiazol-2-yl)-diphenyltetrazolium bro-mide assay (33).

Quantitative Reverse Transcriptase-PCR Using Real-time TaqManTechnology—Total RNA was isolated using Tri-Reagent (Sigma) accord-ing to the instructions from the manufacturer. After DNase I (Promega,Madison, WI) treatment to remove possible traces of genomic DNAcontaminating the RNA preparations, 5 �g of the total RNA was reversetranscribed using random priming and Multiscribe reverse tran-scriptase according to the instructions from the manufacturer (Applied

Biosystems, Foster City, CA). To evaluate the expression levels of theLPA1, LPA2, LPA3, and LPA4/GPR23 mRNAs, quantitative reversetranscriptase-PCR was performed using real-time TaqMan technologywith a sequence detection system model 7700 (Applied Biosystems,Foster City, CA). The human LPA1-, LPA2-, LPA3-, LPA4/GPR23-, andglyceraldehyde-3-phosphate dehydrogenase-specific probes were ob-tained from Assay-on-Demand products (Applied Biosystems, FosterCity, CA). The ID numbers of the products are Hs00173500 for LPA1,Hs00173704 for LPA2, Hs00173857 for LPA3, Hs00271072 for LPA4/GPR23, and Hs99999905 for glyceraldehyde-3-phosphate dehydrogen-ase. Amplification reaction was performed using the TaqMan universalPCR master mixture following the instructions from the manufacturer(Applied Biosystems). The thermal cycling conditions were as follows: 2min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 1 min at60 °C. The procedure for the calculation of their expression was essen-tially the same as that described previously (39). The expression level ofthe target mRNA was normalized to the relative ratio of the expressionof glyceraldehyde-3-phosphate dehydrogenase mRNA. Each reversetranscriptase-PCR assay was performed at least three times, and theresults are expressed as mean � S.E.

Northern Blot Analysis—Total RNA was prepared as describedabove. Twenty �g of total RNA was electrophoresed in a 1% agarose gelcontaining 3.7% formaldehyde in a 20 mM MOPS buffer and blotted ontoa nylon membrane (Hybond-N) with 20� standard saline citrate (SSC).The cDNA probe of LPA1 (20 ng) was labeled with [�-32P]dATP byrandom oligonucleotide priming and added to the blots at a concentra-tion of about 5 � 106 disintegrations/min in 5 ml of a hybridizationbuffer as described previously (34). The hybridization was carried out at60 °C. Following hybridization, the blots were washed at 60 °C with0.2� SSC and 0.1% SDS as described previously (34).

Data Presentation—All experiments were performed in duplicate ortriplicate. The results of multiple observations are presented as themean � S.E. or as representative results from more than three different

TABLE ILPA-equivalent level in malignant ascites and plasma

Malignant ascites and plasma were collected from pancreatic cancerpatients (8 cases) and healthy volunteers. Malignant ascitic fluid wasalso collected from mice injected with YAPC-PD cells as described under“Experimental Procedures.” Their LPA levels were evaluated as LPA-equivalent levels by a bioassay based on the ability to inhibit cAMPaccumulation in LPA1-expressing RH7777 cells, as shown under “Ex-perimental Procedures.” The number of observations is shown inparentheses.

Patient Gender AgeLPA-equivalent level

Ascites Plasma

nM

Case 1 Male 75 4560 404Case 2 Male 40 534 73Case 3 Male 72 2318 158Case 4 Male 73 6637 189Case 5 Male 51 5469 200Case 6 Male 79 1208 100Case 7 Male 45 557 160Case 8 Female 68 537 107

2728 � 876 (8) 174 � 36 (8)

Normalhuman

241 � 22 (14)

Ascites frommouse

624 � 213 (6)

FIG. 1. LPA and ascites stimulate the migration of YAPC-PDcells but not their adhesion. A, the migration activity of YAPC-PDcells was examined using a Boyden chamber in the presence or absenceof LPA (100 nM) or Case 2 ascites (1%) in the lower and upper chambers,as indicated. B, the migration activity of the cells was examined usinga Transwell chamber in the presence or absence of EGF (10 ng/ml), LPA(100 nM), or ascites (1%) from two pancreatic cancer patients (Case 1and Case 2) in the lower chamber. C, the adhesion of YAPC-PD cells tocollagen-coated dishes was examined in the presence or absence of theindicated agents. Concentrations were the same as those for B.

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FIG. 2. Inhibition by Ki16425 and PTX of the migration response to malignant ascites and LPA but not to EGF in YAPC-PD cells.A, dose-dependent increase in the migration response to ascites and its inhibition by Ki16425. B, dose-dependent inhibition by Ki16425 of themigration response to ascites (1%). C, migration response to the increasing dose of ascites from a mouse (PD-1) that was injected with YAPC-PDcells and its inhibition by Ki16425. D, inhibition by Ki16425 of the migration response to ascites (1%) obtained from YAPC-PD cell-injected mice(PD-2–PD-6). E, dose-dependent inhibition by Ki16425 of the migration response to LPA but not to EGF (10 ng/ml). F, inhibition by PTX of themigration response to LPA (100 nM) and ascites (1%) but not to EGF (10 ng/ml).

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batches of cells unless otherwise stated. Statistical significance wasassessed by the Student’s t test; values were considered significant atp � 0.05.

RESULTS

Presence of a High Level of LPA in Malignant Ascites fromPancreatic Cancer Patients and Cancer Cell-injected Mice—Because there was no previous information concerning thepresence of LPA in ascites of patients with pancreatic cancer,we first measured the LPA content in ascites of pancreaticcancer patients. Many forms of LPA have been identified on thebasis of differences in the species of fatty acid, the position ofthe fatty acid substituent (1- or 2-position of the glycerol back-bone), and the attachment to the backbone (acy, alkyl, or alke-nyl) (11). In the present study, we evaluated the amount of LPAon the basis of its ability to stimulate the LPA1 receptor after apartial purification as alkaline-soluble lipids from other lipidand protein components. The LPA levels in ascites and plasmaare summarized in Table I. There was no significant differencein the plasma LPA levels between cancer patients and normalhealthy volunteers (Table I). In all the cases of ascites (8patients), however, we detected a significantly high amount ofLPA, and its level was always higher than that in the plasmaof individuals.

The formation of peritoneal dissemination and ascites with asignificant level of LPA was reproduced in mice by an intrap-eritoneal injection of YAPC-PD, a highly invasive pancreaticcancer cell line (Table I). The mean LPA equivalent level was2728 nM in ascites from cancer patients and 624 nM in themalignant ascites from mice; these values are high enough tobind to and activate the known LPA receptor subtypes; theapparent dissociation constant of LPA receptors is around 100nM (8–10, 20, 21). These results raised the possibility that LPAin malignant ascites may participate in the regulation of thecellular activities of cancer cells as well as host normal cells.

LPA and Malignant Ascites Stimulate the Motility ofYAPC-PD Cells—In Fig. 1, we examined whether LPA andascites can affect the cellular activities of the pancreatic cancercell line. When LPA or ascites were loaded into the lowerchamber of the Boyden apparatus, YAPC-PD cells migratedtoward the lower chamber. The migration activity was stilleffective even when the same concentration of LPA or asciteswas present in both chambers, suggesting that LPA and ascitessimulate random migration (chemokinetics) as well as direc-tional migration (chemotaxis) of pancreatic cancer cells (Fig.1A). We also used Transwell chambers, in which the cells werefirst attached on the filter and the migration activity in re-sponse to test agents was then determined. LPA, ascites fromtwo patients, and EGF significantly stimulated the migration(Fig. 1B). In separate experiments, we observed no significanteffect by these test agents on the adhesion of cells on the dishes(Fig. 1C). These results support the idea that LPA and ascitesenhance the migratory activity of the cells but do not stimulatetheir adhesion activity.

LPA Is a Component Responsible for the Stimulation of Mo-tility in Malignant Ascites—To assess the role of LPA in ma-lignant ascites in cell migration activity, we examined theeffects of Ki16425, a novel LPA receptor antagonist with highspecificity to LPA (32). As shown in Fig. 2, Ki16425 at 1 to 10�M markedly inhibited the migration of YAPC-PD cells by theascites from a pancreatic cancer patient (Fig. 2A). The inhibi-tion by Ki16425 was also observed in a dose-dependent mannerfor malignant ascites from other patients (Fig. 2B). The migra-tion of YAPC-PD cells was also induced by ascites obtainedfrom mice injected with the same cells and the response to theascites was again markedly inhibited by Ki16425 (Fig. 2, C andD). Ki16425 was also an effective inhibitor of the migration

response to LPA but not to EGF, supporting the specificity ofKi16425 (Fig. 2E). Because Ki16425-sensitive LPA receptorsseem to be coupled to PTX-sensitive G-proteins (32), the effectof PTX on the migration of YAPC-PD cells was examined in Fig.2F. As expected, PTX treatment almost completely inhibitedthe migration response to LPA and ascites but not to EGF.These results suggest that the ascites-induced migration re-sponse is mediated by Ki16425-sensitive LPA receptors thatare coupled to PTX-sensitive G-proteins.

To further confirm the involvement of LPA in the inductionof migration responses to malignant fluids, we performed sev-eral experiments (Fig. 3). The ascites-induced action was in-hibited by a prior treatment of ascites with MG lipase, anenzyme hydrolyzing monoglycerides (Fig. 3A). Although it isuncertain whether LPA is a substrate for MG lipase underphysiological conditions, we confirmed that this enzyme de-graded more than 98% of LPA in the test samples. In fact, theLPA-induced migration was markedly inhibited by the enzymetreatment. These results suggest that LPA or related mono-glycerides in ascites may participate in the induction of migra-tion. In Fig. 3B, we extracted the active components fromascites that induce migration. About 70% of the active compo-nent(s) sensitive to MG lipase was extracted in the BuOHfraction. The recovery rate was almost identical to the recoveryrate of [3H]LPA in the same lipid extraction (data not shown).The BuOH fraction was further processed by HPTLC separa-tion of the active components. The highest cell migration ac-

FIG. 3. LPA is an important component of ascites for the mi-gration response. YAPC-PD cells were used throughout the experi-ments. A, inhibition by MG lipase (MGLP) of the migration response toLPA (100 nM) and ascites (1%) but not to EGF (10 ng/ml). B, the activecomponent(s) of ascites from a patient (Case 2) was purified by lipidextraction with BuOH and subsequent HPTLC separation. The activityin the HPTLC fraction is shown. [3H]LPA included in the samplerecovered to about 60% in fraction 3 (marked at arrow) and 20% infraction 4. C, dose-dependent effect of LPA (E), S1P (●), and LPC (Œ) onthe migration of cells.

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tivity was detected in fraction 3, which is the LPA-richestfraction (Fig. 3B). Because S1P has a similar property to LPAwith respect to its solubility to BuOH and migration onHPTLC, it is not easy to clearly separate LPA and S1P inbiological samples. However, compared with LPA, S1P exertedonly a small effect on the migration of YAPC-PD cells (Fig. 3C).In this figure, we also examined the effect of LPC, which maybe present in ascites at a high level, but its effect on migrationwas marginal (Fig. 3C). These results support the idea thatLPA is an important component for the migration responseto ascites.

Involvement of the LPA1 Receptor in the Migration of Pan-creatic Cancer Cell Lines—In Fig. 4 we examined the migrationactivity in pancreatic cancer cell lines other than YAPC-PD.Among the seven cell lines used, a significant migration re-sponse to EGF, LPA, and ascites was observed in PK-1, PK-9,Panc-1, BxPC-3, CFPAC-1, and HPAC. In MiaPaCa-2 cells,

however, no significant effect was observed by these test agents(Fig. 4). In all pancreatic cell lines responsive to ascites,Ki16425 (Fig. 4) and MG lipase (data not shown) specificallyinhibited the migration response to LPA and ascites, as was thecase for YAPC-PD cells.

Table II shows the expression of the mRNA of LPA receptorsubtypes by real-time PCR. In all cell lines responsive to LPAand ascites, a significant amount of LPA1 mRNA expressionwas detected, although there are variations in their expressionamong cell types. In MiaPaCa-2, an unresponsive cell line toLPA and ascites, however, LPA1 mRNA expression was notdetected by real-time PCR (Table II) or by Northern blotting(data not shown). On the other hand, LPA2 mRNAs were ex-pressed in all of the pancreatic cancer cell lines, includingMiaPaCa-2. In the case of LPA3, mRNA expression was verysmall compared with that of LPA1 and LPA2, except forBxPC-3, in which the expression of LPA3 mRNA was compa-

FIG. 4. Inhibition by Ki16425 of themigration response to LPA and as-cites in pancreatic cancer cell lines.The pancreatic cancer cell lines, PK-1,PK-9, Panc-1, BxPC-3, CFPAC-1, HPAC,and MiaPaCa-2, were used for the migra-tion responses to EGF (10 ng/ml), LPA(100 nM), and ascites (1%) in the presence(closed column) or absence (open column)of Ki16425 (10 �M). Other experimentalconditions were essentially the same asthose for Fig. 2.

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rable with the level of LPA2 mRNA. Thus, in PK-1 and HPAC,cell lines that are highly responsive to LPA and ascites, only amarginal or no significant expression of LPA3 mRNA was de-tected. As for LPA4/GPR23, its expression was marginal or notsignificant. Although the expression of receptor proteins wasnot successfully detected in the present study, LPA1 expressionseems to correlate with the migration activity by LPA andascites in pancreatic cancer cell lines. Thus, there was a sig-nificant correlation between LPA-induced migration responsein eight cell lines and their mRNA expression of LPA1 but notLPA2 or LPA3 mRNA (Table II).

The ability of LPA1 to stimulate the migration activity wasconfirmed by overexpressing LPA1 in YAPC, a parent cell lineof YAPC-PD cells (Fig. 5). YAPC cells showed less expression ofLPA1 mRNA and less responsiveness to LPA than YAPC-PDcells. Transfection of LPA1 in YAPC cells, however, resulted inan enhancement of the migration response to LPA to the levelof YAPC-PD cells (Fig. 5).

To further examine the LPA receptor subtypes responsiblefor the migration response, we performed two lines of experi-ments. In the first line of experiments, siRNAs against LPA1

receptors were transfected into YAPC-PD cells (Fig. 6A) andPanc-1 cells (Fig. 6C) to decrease the expression of the receptormRNA. The siRNA transfection resulted in a marked reductionof the expression of LPA1 mRNA without a significant changein the expression of LPA2 and LPA3 mRNAs, which was accom-panied by a remarkable inhibition of migration response toLPA and ascites but not to EGF (Fig. 6, B and D). Thus,LPA1-specific siRNA inhibited LPA- and ascites-induced mi-gration responses. These results are consistent with the ideathat LPA1 is a critical receptor for the migration response toLPA and ascites in pancreatic cancer cell lines, at least inYAPC-PD and Panc-1.

In the second line of experiments, we compared the effects ofseveral LPA receptor antagonists. Ki16425 has preference forLPA1 and LPA3 over LPA2 (32), whereas VPC12249 showedpreference for LPA1 and LPA3 but not for LPA2 (40), and DGPP8:0, only for LPA3 (41) (Table III). In the case of YAPC-PD cells,the migration response to LPA and ascites was significantlyinhibited by VPC12249 as well as Ki16425, antagonists forLPA1 and LPA3, but not by DGPP 8:0, an LPA3-specific antag-onist. None of these LPA receptor antagonists affected theEGF-induced migration, suggesting the specificity of the an-tagonists. In other cell lines as well, the migration response toascites was sensitive to both Ki16425 and VPC12249 but not toDGPP 8:0 (Table III). These pharmacological studies also sup-port a critical role of LPA1 in the migration response to ascitesin pancreatic cancer cell lines.

Ascites and LPA Induce Matrigel Invasion in a Manner Sen-sitive to Ki16425—Proteolysis of extracellular matrix proteins

is an important step for the invasion and metastasis of cancercells. The possibility that LPA and ascites stimulate the inva-sion of pancreatic cancer cells was studied using a Matrigelinvasion assay. YAPC-PD cells were loaded on Matrigel-coatedpore filters for 24 h in the absence or presence of EGF, LPA, orascites in the lower chamber, and the cells that had migrated to

FIG. 5. Overexpression of LPA1 receptors enhances the migra-tion response to LPA. Northern blot of LPA1 mRNA expression (up-per panel) and migration response to 100 nM LPA (lower panel) in YAPCcells (PC), YAPC-PD cells (PD), YAPC cells transfected with a vector(PC/V), and YAPC cells transfected with human LPA1 cDNA (PC/LPA1).Exogenous LPA1 mRNA (exo LPA1) was detected at a size similar tothat of 18 S ribosomal RNA.

TABLE IIExpression of mRNAs for LPA receptor subtypes in various types of pancreatic cancer cell lines

The mRNA expression of LPA receptor subtypes was assessed by real-time PCR in the indicated pancreatic cancer cell lines. Results areexpressed as the relative ratios to glyceraldehyde-3-phosphate dehydrogenase mRNA expression (�1000). A ratio of less than 0.00001 (describedas �0.01 in the table) is not significant by this method. A correlation coefficient between the expression of each LPA receptor subtype mRNA andthe LPA-induced migration activity in eight cell lines was calculated as 0.761 (p � 0.05) for LPA1, 0.045 (not significant) for LPA2, and 0.647 (notsignificant) for LPA3, where the migration activities shown in Fig. 3C were used for YAPC-PD and those in Fig. 4 were for other cell lines.

Cell lines LPA1 LPA2 LPA3 LPA4

YAPC-PD 4.68 � 1.29 4.55 � 0.84 0.25 � 0.10 �0.01PK-1 9.82 � 4.30 3.58 � 0.90 0.02 � 0.00 �0.01PK-9 16.1 � 6.91 1.56 � 0.40 0.48 � 0.08 �0.01Panc-1 2.98 � 0.72 2.96 � 0.38 0.55 � 0.11 �0.01BxPC-3 19.5 � 1.60 2.02 � 0.14 2.76 � 0.62 NDa

CFPAC-1 28.7 � 4.60 3.89 � 0.30 0.30 � 0.03 NDHPAC 20.2 � 2.50 0.45 � 0.11 �0.01 �0.01Mia PaCa-2 �0.01 2.23 � 0.67 0.35 � 0.13 �0.01

a ND, not determined.

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the lower surface of the filters were counted. As shown in Fig.7, ascites and other agents stimulated the invasion, and theresponse to ascites and LPA, but not to EGF, was markedlyinhibited by Ki16425, suggesting that LPA is also an importantcomponent for the invasive activity of ascites.

Role of LPA in Pancreatic Cancer Cell Proliferation—To clar-ify the role of LPA in malignant fluids in cell growth, theproliferation activity was evaluated by a 3-(4,5-dimethythiazol-2-yl)-diphenyltetrazolium bromide assay. LPA stimulated theproliferation about 150% of the level of the control in YAPC-PD

FIG. 6. siRNA specific to LPA1 receptors inhibits the migration response to ascites. The pancreatic cancer cell lines, YAPC-PD (A andB) and Panc-1 (C and D), were transfected with nonsense RNA (control; open column), LPA1-228 (hatched column), or a mixture of siRNA (10 nM

each of LPA1-102, LPA1-228, and LPA1-945; closed column). The respective LPA receptor mRNA level and cell migration activity in response to theindicated agents were measured. *, the effect of siRNA was significant.

TABLE IIIEffects of LPA antagonists on the migration response to EGF, LPA, and ascites

The indicated cell lines were used for the evaluation of the ability of LPA antagonists to inhibit the migration response to EGF (10 ng/ml), LPA(100 nM), and malignant ascites (Case 2 or Case 3 at 1%) from patients with pancreatic cancer. The results are expressed as percentages of thecontrol migration activity (increment from basal value) induced by the respective agent without any antagonist. LPA antagonists hardly affectedthe basal activity without test agents. The control activity (cell numbers per 4 macroscopic fields) was 57 � 2 for EGF, 87 � 12 for LPA, 85 � 13for ascites from Case 2, and 94 � 14 for ascites from Case 3 in YAPC-PD cells; and these values for ascites in other cell lines were 100–200, asdescribed in Fig. 4.

Cell line Test agent

Ki16425 VPC12249 DGPP 8:0

LPA1, LPA3 � LPA2 LPA1, LPA3 LPA3

0.1 10 0.1 10 0.1 10

�M �M �M

YAPC-PD EGF 92 � 10 89 � 5 89 � 7 88 � 7 87 � 8 85 � 4LPA 59 � 7a 16 � 4a 50 � 5a 30 � 3a 85 � 8 87 � 4Case 2 52 � 2a 18 � 6a 59 � 6a 34 � 4a 90 � 2 88 � 5Case 3 49 � 4a 15 � 5a 46 � 6a 34 � 3a 89 � 3 85 � 7

PK-1 Case 2 30 � 2a 27 � 2a 36 � 4a 29 � 2a NDb 93 � 2PK-9 Case 2 33 � 2a 14 � 7a 55 � 5a 27 � 6a NDb 97 � 2Panc-1 Case 2 40 � 10a 11 � 3a 56 � 6a 27 � 4a 96 � 3 98 � 5BxPC-3 Case 2 ND 28 � 2a ND 30 � 2a ND 100 � 8CFPAC-1 Case 2 ND 16 � 3a ND 29 � 1a ND 91 � 4HPAC Case 2 ND 25 � 2a ND 28 � 2a ND 100 � 1

a Significantly different from the control activity.b ND, not determined.

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cells at 1–10 �M and in Panc-1 cells at 0.1–1 �M, but weobserved only inhibitory effects by ascites at up to 10% (datanot shown). In PK-9 cells, these malignant fluids significantlypromoted their proliferation (Fig. 8). The cell proliferation,however, was insensitive to Ki16425 (Fig. 8A) and MG lipase(Fig. 8B); both compounds are potent inhibitors of the migra-tion of pancreatic cancer cell lines, including PK-9. Further-more, LPA had no stimulatory effect at 100 nM or less (Fig. 8),and the effect was inhibitory at more than 1 �M (data notshown). Thus, the LPA component in these fluids does not seemto be an important regulator for the proliferation in this cellline. Thus, it remains unclear whether LPA participates in theregulation of the proliferation of pancreatic cancer cells. In anyevent, components in ascites other than LPA may be involvedin the regulation of the proliferation of the cells.

DISCUSSION

In the present study, we showed that ascites from patientswith pancreatic cancer contain a considerably high LPA-likeactivity, which was assessed by the ability of LPA to stimulateLPA1 receptors. This bioassay excludes LPA species that can-not stimulate LPA1, although all the LPA species usually pres-ent in biological samples seem to stimulate LPA1 (20). More-over, LPA C18:1 (1-oleoyl-sn-glycero-3-phosphate), used as astandard, is usually the most potent species for LPA receptors.Therefore, the true LPA content may be higher than thatestimated in the present study. Thus, the bioassay appears tobe unsuitable for a quantitative measurement of LPA but maybe superior to other methods, such as the combination of chro-matography and mass spectrometry (42, 43), for the evaluationof the active LPA that stimulates LPA receptors.

Pancreatic cancer is a highly metastatic disease character-ized by widespread intraperitoneal dissemination and ascitesformation. Indeed, we showed that intraperitoneal injection ofa highly invasive pancreatic cancer cell line, YAPC-PD, intomice induced the formation of LPA-rich ascites. However, themolecular mechanism by which LPA accumulates in malignantascites remains to be elucidated. One possible mechanism isthat LPA might be produced from extracellular LPC by lyso-phospholipase D or autotaxin (44, 45). In our preliminary ex-periments, we detected significant lysophospholipase D activ-ity, which was assessed by choline formation from exogenousLPC, in malignant ascites from pancreatic cancer patients andYAPC-PD cell-injected mice. In any event, the pancreatic can-cer cell-injected mice may provide a useful in vivo model systemfor characterization of the mechanism of the formation of LPAin malignant ascites.

Intraperitoneal dissemination is composed of several pro-cesses, including adhesion, migration, invasion, and prolifera-tion (3, 4). We demonstrated in the present study that LPA inmalignant ascites is an important factor for the stimulation ofmotility, e.g. migration and invasion in vitro. This is supportedby the following observations. First, LPA receptor-selectiveantagonists, including Ki16425 and VPC12249, inhibited themigration response to ascites as well as LPA. Second, treat-ment of ascites with MG lipase, an enzyme that hydrolyzesmonoglyceride, including LPA, markedly inhibited the migra-tion response. Third, a fractionation study of ascites by lipidextraction and subsequent HPTLC indicated that the activecomponent was recovered in the LPA fraction. Finally, the LPAequivalent level in ascites was high enough to stimulate themigration and invasion of pancreatic cancer cells.

As for proliferation, however, we failed to demonstrate apositive role of LPA in the proliferative activity of malignantascites. In PK-9 cells, ascites significantly stimulated prolifer-ation, but their effect was insensitive to Ki16425, an LPAreceptor antagonist, and MG lipase, an LPA-hydrolyzing en-zyme. Furthermore, LPA was ineffective for the induction ofthe stimulation of proliferation in PK-9 cells (Fig. 8). Theseresults suggest that cytokines and growth factors other thanLPA may be responsible for the proliferative activity of ascitesin pancreatic cancer cells. It should be noted, however, thatLPA stimulated the proliferation of YAPC-PD cells, eventhough a stimulatory effect was not detected by ascites andeffusions (data not shown). Thus, the regulatory mechanism ofthe proliferation appears to differ in different pancreatic cancercell lines.

On the other hand, the motility of pancreatic cancer cellsseems to be regulated by similar mechanisms involving a Gprotein-coupled receptor LPA1. First, the migration response toascites and LPA was almost completely suppressed by PTX

FIG. 7. Effect of Ki16425 on the invasion of YAPC-PD cells.Matrigel invasion activity in response to EGF (10 ng/ml), LPA (1 �M),and ascites (1 and 10%) was measured in the presence or absenceof Ki16425.

FIG. 8. Ascites stimulates the proliferation of PK-9 cells in amanner independent of LPA. The effects of Ki16425 (10 �M) (A) andMG lipase (MGLP) (B) on the proliferation of PK-9 cells by EGF (10ng/ml), LPA (100 nM), and ascites (1%) were examined.

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(Fig. 2F), a toxin that ADP-ribosylates and inactivates Gi/o

proteins, suggesting a mediation by Gi/o protein-coupled recep-tors. Among four LPA receptor subtypes, EDG family receptors(LPA1, LPA2, and LPA3), but not LPA4, have been shown to becoupled to the PTX-sensitive G proteins (9, 10, 20, 21). Second,the migration response appears to be correlated with the ex-pression of LPA1 mRNA (Fig. 4 and Table II). A significantamount of LPA1 mRNA is expressed in pancreatic cancer celllines with high migration activity for ascites and LPA, but notin MiaPaCa-2 cells with low migration activity. In all cells,however, LPA2 mRNA was similarly expressed, and the expres-sion of LPA3 and LPA4/GPR23 mRNAs was low or marginalexcept for LPA3 mRNA in BxPC-3. Third, transfection of LPA1

in YAPC cells, a parent cell line for YAPC-PD cells, resulted inan enhancement of the migration response to LPA to the levelof that in YAPC-PD cells (Fig. 5). Fourth, Ki16425, an antag-onist for all EDG family LPA receptors, albeit with less pref-erence for LPA2, and VPC12249, an LPA1- and LPA3-selectiveantagonist, but not DGPP 8:0, an LPA3-selective antagonist,inhibited the migration response (Table III). Ki16425 was alsoeffective for inhibiting the invasion response to LPA and asci-tes (Fig. 7). Finally, siRNA against LPA1 markedly inhibitedthe migration response to LPA and ascites in association witha specific inhibition of the receptor mRNA expression (Fig. 6).Taken together, these results suggest that LPA1 plays a criticalrole in the migration response to LPA and malignant ascites inpancreatic cancer cells. Although no intracellular signalingmechanism other than the involvement of Gi/o proteins hasbeen analyzed in the present study, recent studies have shownthat two small molecule G proteins, Rho and Rac, which regu-late actin cytoskeleton rearrangement, are involved in LPA-induced random and directional migration (chemokinetics andchemotaxis, respectively) and tumor cell invasion (24, 25,29–31).

Thus, the present study suggests the importance of LPA1 inthe migratory response in pancreatic cancer cells, but thisfinding can never rule out the possible involvement of LPAreceptor subtypes other than LPA1 receptor, especially LPA2,in LPA-induced actions, including the migration response. Inthe case of LPA3, the LPA3 receptor antagonist DGPP 8:0 wasineffective for the migration response, and LPA3 receptormRNA expression was marginal in the highly responsive pan-creatic cell lines including HPAC and PK-1, suggesting theminor role of the LPA3 receptor. However, LPA2 mRNA isexpressed at a high level in all pancreatic cancer cells andmight be involved in the signaling pathways leading to migra-tion in collaboration with LPA1. In such a situation, inhibitionof either receptor would lead to the loss of the response. Thestimulatory role of LPA2 in migration has been shown in T-lymphoma, whereas LPA1 has a rather inhibitory effect on themigration response in the same cells (27). With respect toproliferation, LPA2 has been suggested to be stimulatory forovarian cancer cells (26) and colon cancer cells (31), althoughthe role of LPA receptors in cell proliferation remains to beelucidated in pancreatic cancer cells. Thus, LPA and its recep-tors are involved in the migration, invasion, and proliferationof various types of cancer cells and may be potential targets fortherapy. Indeed, the acceleration of LPA degradation by theintroduction of lipid phosphate phosphohydrolase-3 appears tobe effective for the control of tumor growth in ovarian cancer(46).

In conclusion, LPA in malignant ascites is an importantcomponent for the migration of pancreatic cancer cells througha G protein-coupled LPA receptor LPA1. Malignant ascites andLPA also induced the in vitro invasion of pancreatic cancer

cells, which was inhibited by Ki16425, an LPA receptor antag-onist. Thus, LPA receptors, especially LPA1, may be therapeu-tic targets for the control of the migration and invasion ofpancreatic cancer cells. Ki16425 is a potential drug for thispurpose.

Acknowledgements—We are grateful to Prof. Kevin R. Lynch for hisgenerous gifts of LPA1-expressing RH7777 cells and VPC12249 andMasayo Yanagita for technical assistance.

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LPA As a Potent Motility Factor in Ascites 6605

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Im, Koichi Tamoto, Hideaki Tomura and Fumikazu OkajimaTanahashi, Tetsushi Ogawa, Susumu Ohwada, Yasuo Morishita, Hideo Ohta, Doon-SoonTobo, Takao Kimura, Atsushi Kuwabara, Yasuhiro Yanagita, Toshiro Ikeya, Yoshifumi Takayuki Yamada, Koichi Sato, Mayumi Komachi, Enkhzol Malchinkhuu, Masayuki

1Pancreatic Cancer Cells through LPALysophosphatidic Acid (LPA) in Malignant Ascites Stimulates Motility of Human

doi: 10.1074/jbc.M308133200 originally published online December 3, 20032004, 279:6595-6605.J. Biol. Chem. 

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