Lysophosphatidic Acid (LPA) in Malignant Ascites Stimulates Motility of Human Pancreatic Cancer Cells through LPA1
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m308133200
ISSN1083-351X
AutoresTakayuki Yamada, Kōichi 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, Dong‐Soon Im, Koichi Tamoto, Hideaki Tomura, Fumikazu Okajima,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoCytokines and growth factors in malignant ascites are thought to modulate a variety of cellular activities of cancer cells and normal host cells. The motility of cancer cells is an especially important activity for invasion and metastasis. Here, we examined the components in ascites, which are responsible for cell motility, from patients and cancer cell-injected mice. Ascites remarkably stimulated the migration of pancreatic cancer cells. This response was inhibited or abolished by pertussis toxin, monoglyceride lipase, an enzyme hydrolyzing lysophosphatidic acid (LPA), and Ki16425 and VPC12249, antagonists for LPA receptors (LPA1 and LPA3), but not by an LPA3-selective antagonist. These agents also inhibited the response to LPA but not to the epidermal growth factor. In malignant ascites, LPA is present at a high level, which can explain the migration activity, and the fractionation study of ascites by lipid extraction and subsequent thin-layer chromatography indicated LPA as an active component. A significant level of LPA1 receptor mRNA is expressed in pancreatic cancer cells with high migration activity to ascites but not in cells with low migration activity. Small interfering RNA against LPA1 receptors specifically inhibited the receptor mRNA expression and abolished the migration response to ascites. These results suggest that LPA is a critical component of ascites for the motility of pancreatic cancer cells and LPA1 receptors may mediate this activity. LPA receptor antagonists including Ki16425 are potential therapeutic drugs against the migration and invasion of cancer cells. Cytokines and growth factors in malignant ascites are thought to modulate a variety of cellular activities of cancer cells and normal host cells. The motility of cancer cells is an especially important activity for invasion and metastasis. Here, we examined the components in ascites, which are responsible for cell motility, from patients and cancer cell-injected mice. Ascites remarkably stimulated the migration of pancreatic cancer cells. This response was inhibited or abolished by pertussis toxin, monoglyceride lipase, an enzyme hydrolyzing lysophosphatidic acid (LPA), and Ki16425 and VPC12249, antagonists for LPA receptors (LPA1 and LPA3), but not by an LPA3-selective antagonist. These agents also inhibited the response to LPA but not to the epidermal growth factor. In malignant ascites, LPA is present at a high level, which can explain the migration activity, and the fractionation study of ascites by lipid extraction and subsequent thin-layer chromatography indicated LPA as an active component. A significant level of LPA1 receptor mRNA is expressed in pancreatic cancer cells with high migration activity to ascites but not in cells with low migration activity. Small interfering RNA against LPA1 receptors specifically inhibited the receptor mRNA expression and abolished the migration response to ascites. These results suggest that LPA is a critical component of ascites for the motility of pancreatic cancer cells and LPA1 receptors may mediate this activity. LPA receptor antagonists including Ki16425 are potential therapeutic drugs against the migration and invasion of cancer cells. Pancreatic cancer is a highly metastatic cancer characterized by widespread intraperitoneal dissemination and ascites formation, which frequently occur even after curative resection and constitute the major cause of death in pancreatic cancer patients (1Niederhuber J.E. Brennan M.F. Menck H.R. Cancer. 1995; 76: 1671-1677Crossref PubMed Scopus (405) Google Scholar, 2Kayahara M. Nagakawa T. 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Cancer Res. 2003; 63: 1706-1711PubMed Google Scholar), it was difficult to determine the extent to which LPA, among a variety of potent cytokines and growth factors present in ascites, serves as a mediator of these responses. LPA receptor antagonists, LPA-neutralizing antibodies, or the LPA-specific degrading enzyme would be very useful for this purpose. Especially, receptor-selective antagonists are strong tools for identifying the subtype of LPA receptors that are responsible for the lipid action and would be a platform to develop therapeutic agents. We have recently developed a novel LPA receptor antagonist, Ki16425, which shows a preference for LPA1 and LPA3 over LPA2 (32Ohta K. Sato K. Murata N. Damirin A. Malchinkhuu E. Kon J. Kimura T. Tobo M. Yamazaki Y. Watanabe T. Yagi M. Sato M. Suzuki R. Murooka H. Sakai T. Nishitoba T. Im D.-S. Nochi H. Tamoto K. Tomura H. Okajima F. Mol. Pharmacol. 2003; 64: 994-1005Crossref PubMed Scopus (336) Google Scholar). Ki16425 showed an extremely high specificity to LPA and its receptors. Thus, Ki16425 may be a useful tool for evaluating the role of LPA in biological samples, such as ascites. In the present paper, we showed that a considerably high amount of LPA is present in ascites from pancreatic cancer patients and that the formation of LPA-rich ascites can be duplicated by an intraperitoneal injection of human pancreatic cancer cells in nude mice. Furthermore, our data indicated that LPA in malignant ascites is an important component for the motility of pancreatic cancer cells through Ki16425-sensitive LPA receptors, especially LPA1. Materials—1-Oleoyl-sn-glycero-3-phosphate (LPA), l-α-lysophosphatidylcholine 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 Polar Lipids, 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); MG lipase was from Asahi Kasei Corp. (Shizuoka, Japan); [3H]LPA (48 Ci/mmol) was from PerkinElmer Life Sciences; and [α-32P]dATP (3000 Ci/mmol) was from Amersham Biosciences. Ki16425 (3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfonyl)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 from Charles River Japan, Inc. (Tokyo, Japan). Sterile food and water were fed to the mice ad libitum. The mice were maintained in sterile cages on sterile bedding and housed in rooms at a constant temperature and humidity. All experiments using mice were performed according to procedures approved by the Gunma University Animal Care Committee. Ascites from Cancer Patients—Eight Japanese patients with pancreatic cancer were available for this study at the Second Department of Surgery, Gunma University Faculty of Medicine from 2001 through 2003. Table I summarized the characteristics of the patients. A cytological examination demonstrated that the ascites contains cancer cells. Plasma or ascites were collected in the presence of EDTA (at a final concentration of 2–3 mm) and centrifuged at 1,000 × g for 20 min to remove cells. The cell-free fluid was stored at -80 °C until use. Informed consent was obtained from each patient for the use of samples.Table ILPA-equivalent level in malignant ascites and plasma Malignant ascites and plasma were collected from pancreatic cancer patients (8 cases) and healthy volunteers. Malignant ascitic fluid was also 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 cAMP accumulation in LPA1-expressing RH7777 cells, as shown under "Experimental Procedures." The number of observations is shown in parentheses.PatientGenderAgeLPA-equivalent levelAscitesPlasmanmCase 1Male754560404Case 2Male4053473Case 3Male722318158Case 4Male736637189Case 5Male515469200Case 6Male791208100Case 7Male45557160Case 8Female685371072728 ± 876 (8)174 ± 36 (8)Normal human241 ± 22 (14)Ascites from mouse624 ± 213 (6) Open table in a new tab Establishment of a Highly Peritoneal Metastatic Pancreatic Cancer Cell Line, YAPC-PD—A human pancreatic cancer cell line, YAPC-PD, was established from YAPC cells, which had been previously established in our laboratory (33Yamada T. Okajima F. Adachi M. Ohwada S. Kondo Y. Int. J. Cancer. 1998; 76: 141-147Crossref PubMed Scopus (14) Google Scholar). The YAPC is not a highly peritoneal metastatic cell line, but, in one nude mouse of 30, which were injected in the peritoneal cavity with YAPC cells (1 × 107 per mouse), peritoneal dissemination with bloody ascites was detected 12 weeks after the injection. Cells in the bloody ascites were harvested and cultured in RPMI 1640 containing 10% fetal bovine serum. Intermingled mouse fibroblasts gradually decreased in number and finally disappeared within a 4-week culture. The rest of the cells attached on dishes were harvested, and 1 × 107 cells were then injected in the peritoneal cavity of nude mice. The same procedure was repeated in a fifth cycle, and we obtained the YAPC-PD cell line, which induces peritoneal dissemination with high frequency. More than 90% of nude mice injected intraperitoneally with this cell line developed peritoneal dissemination with bloody ascites within 4 to 6 weeks. Ascites from Mice Injected with the Human Pancreatic Cancer Cell Line, YAPC-PD—Ten million cells of YAPC-PD were injected into the peritoneal cavity of nude mice. Four to 6 weeks later, mice that developed abdominal distension were killed, and bloody ascites was collected in the presence of EDTA (at a final concentration of 2–3mm). The bloody ascites 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, Tohoku University (Sendai, Japan), and MIA PaCa-2, BxPC-3, CFPAC-1, and HPAC were purchased from the American Type Culture Collection (Rockville, MD). The YAPC cells were transfected with a pEFneo empty vector alone or a pEFneo vector containing human LPA1 receptor (34Sato K. Ui M. Okajima F. Brain Res. Mol. Brain Res. 2000; 85: 151-160Crossref PubMed Scopus (44) Google Scholar) by electroporation, and the neomycin (G418 sulfate at 1 mg/ml)-resistant cells were selected. All pancreatic cancer cell lines and the receptor-transfected cells were cultured in RPMI 1640 containing 10% fetal bovine serum. Twenty-four hours before the experiments, the medium was changed to a fresh medium (without serum) containing 0.1% (w/v) BSA (fraction V) unless otherwise specified. Where indicated, PTX (100 ng/ml) was added to the culture medium 24 h before the experiments. Transfection of siRNA—Pancreatic cancer cell lines, YAPC-PD and Panc-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 an RNAiFect reagent (Qiagen K.K., Tokyo, Japan) according to the manufacturer's instructions and the cells were further cultured for 24 h. The LPA receptor mRNA level was measured using real-time TaqMan technology, and a cell migration assay was performed 24 h after serum starvation as described later. The following 21-mer oligonucleotide pairs were used as siRNAs against human LPA1: LPA1-102, 5′-r(CCGAAGUGGAAAGCAUCUU)d(TT)-3′ and 5′-r(AAGAUGCUUUCCACUUCGG)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(AAAGGUGGCGCUCAUUUCU)d(TT)-3′. The numbers 102, 228, and 945 represent the position in the nucleotide sequence of the coding region. The following 21-mer RNA was used as a negative control: 5′-r(UUCUCCGAACGUGUCACGU)d(TT)-3′ and 5′-r(ACGUGACACGUUCGGAGAA)d(TT)-3′. These annealed oligonucleotides were obtained from Qiagen K.K. as high performance purity grade and used according to the manufacturer's instructions. Evaluation of LPA-like Activity—LPA in malignant ascites and plasma (0.5 ml, unless otherwise stated) was selectively extracted as alkaline-soluble lipids as described previously (35Murata N. Sato K. Kon J. Tomura H. Okajima F. Anal. Biochem. 2000; 282: 115-120Crossref PubMed Scopus (91) Google Scholar). By this procedure, major lipid components, such as phosphatidylcholine, sphingomyelin, and other neutral lipids, can be removed. To evaluate the content of LPA in this extract, a sensitive and specific bioassay based on the ability of LPA to inhibit cAMP accumulation in LPA1-expressing RH7777 cells was used because vector-transfected RH7777 cells do not respond to LPA (20Im D.S. Heise C.E. Harding M.A. George S.R. O'Dowd B.F. Theodorescu D. Lynch K.R. Mol. Pharmacol. 2000; 57: 753-759Crossref PubMed Scopus (180) Google Scholar). The LPA1-expressing RH7777 cells (a generous gift from Prof. Kevin R. Lynch, University of Virginia School of Medicine) were cultured in minimal essential medium containing 10% fetal bovine serum. Three days before the experiments, cells were seeded on 12-well plates that were coated with rat-tail collagen (400 μg/ml). Twenty-four hours before the experiments, the medium was changed to fresh minimal essential medium containing 0.1% BSA. The cells were washed twice with a HEPES-buffered medium (36Kon J. Sato K. Watanabe T. Tomura H. Kuwabara A. Kimura T. Tamama K. Ishizuka T. Murata N. Kanda T. Kobayashi I. Ohta H. Ui M. Okajima F. J. Biol. Chem. 1999; 274: 23940-23947Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) and incubated with the extract or LPA (C18:1) standard in a HEPES-buffered medium containing 10 μm forskolin and 0.5 mm isobutylmethylxanthine at a final concentration of 500 μl. After a 10-min incubation, the reaction was terminated by adding 100 μl of 1 n HCl. Cyclic AMP in the acid extracts was measured (36Kon J. Sato K. Watanabe T. Tomura H. Kuwabara A. Kimura T. Tamama K. Ishizuka T. Murata N. Kanda T. Kobayashi I. Ohta H. Ui M. Okajima F. J. Biol. Chem. 1999; 274: 23940-23947Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The cAMP-inhibiting activity of LPA or test samples was completely lost by MG lipase, an enzyme hydrolyzing monoglycerides, such as LPA, or Ki16425, an LPA antagonist. Furthermore, the activity was unchanged even when the LPA-like activity was measured after further purification with silica gel high performance thin-layer chromatography (HPTLC) (Merck) using a solvent system consisting of BuOH/acetic acid/water (3:1:1). These results support the specificity of this bioassay. By this method, we detected LPA C18:1 (1-oleoyl-sn-glycero-3-phosphate) equivalent activity that was as low as 1 pmol/assay well. The evaluated LPA-like activity in the test sample was presented as an LPA C18:1 equivalent level. MG Lipase Treatment—LPA (1 μm), EGF (100 ng/ml), or ascites (10%) were treated with MG lipase at 10 units/ml for 30 min at 37 °C in RPMI 1640 containing 0.1% BSA. In separate experiments, we added [3H]LPA to the assay medium and confirmed that more than 98% of LPA in the test samples was degraded under these conditions. These MG lipase-treated samples were finally used by 10 times dilution with the assay medium. Extraction of an Active Component from Ascites—The ascites were treated with 2 volumes of BuOH and separated into two phases. The BuOH-extracted components were further separated by HPTLC using a solvent system consisting of BuOH/acetic acid/water (3:1:1) (37Kimura T. Sato K. Kuwabara A. Tomura H. Ishiwara M. Kobayashi I. Ui M. Okajima F. J. Biol. Chem. 2001; 276: 31780-31785Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The silica gel with the resolved lipids (about 1-cm length each) was scraped off to obtain lipids covering the entire area of migration. The lipids were eluted and dried by evaporation. All fractions thus separated were dissolved in an assay medium containing 0.1% BSA and used at the final concentration corresponding to 1% ascites. Cell Migration Assay—The migration experiment was performed using a Boyden chamber apparatus, as previously described (38Tamama K. Kon J. Sato K. Tomura H. Kuwabara A. Kimura T. Kanda T. Ohta H. Ui M. Kobayashi I. Okajima F. Biochem. J. 2001; 353: 139-146Crossref PubMed Scopus (103) Google Scholar). In brief, YAPC-PD cells and other cancer cells were harvested with 0.05% trypsin containing 0.02% EDTA, washed once, and resuspended with RPMI 1640 containing 0.1% BSA. The cells were loaded into the upper chamber, and test agents were placed in the lower chamber, unless otherwise specified. A membrane filter with 8-mm pores was precoated overnight at 4 °C with 100 μg/ml rat-tail collagen. When the effects of LPA antagonists were examined, the cells were preincubated for 10 min with antagonists before loading. The number of cells that had migrated for4htothe lower surface were determined by counting the cells in four places under microscopy at ×400 magnification. Unless otherwise stated, this Boyden chamber method was used for the evaluation of the migratory activity of the cells. In some experiments, Transwell chemotaxis chambers (6.5 mm diameter, 8 μm pore size) (Costar, Inc.) were used. Chemotaxis filters were soaked in 100 μg/ml rat-tail collagen overnight, similarly to the Boyden chamber method. The cells were first attached on the filters; unattached cells were then removed 1 h after seeding, and test agents were placed in the lower chamber. The number of the cells that migrated to the lower surface during a 4-h incubation was determined as described above. Matrigel Invasion Assay—Cell invasion activity was assessed by using a Matrigel invasion chamber (BD Biosciences, San Jose, CA) according to the instructions provided by the manufacturer. The procedures are essentially the same as those for the migration assay using Transwell chemotaxis chambers, except that the incubation time was 24 h. Cell Adhesion Assay—Cells were harvested by trypsin, seeded on 48-well plates at a density of 5 × 104 cells per well in RPMI 1640 containing 0.1% BSA and test agents, and then incubated for 1 or 4 h. At each time, floating cells were aspirated, plates were rinsed with phosphate-buffered saline, and the attached cells were then evaluated by an 3-(4,5-dimethythiazol-2-yl)-diphenyltetrazolium bromide assay as described previously (33Yamada T. Okajima F. Adachi M. Ohwada S. Kondo Y. Int. J. Cancer. 1998; 76: 141-147Crossref PubMed Scopus (14) Google Scholar). Cell Proliferation—Cells were seeded on 48-well plates at 2 × 104 cells in 0.4 ml. Twenty-four hours before the experiments, the medium was changed to RPMI 1640 containing 0.1% BSA, and cells were exposed to the test agents for an additional 24 h. Cell proliferation was evaluated by an 3-(4,5-dimethythiazol-2-yl)-diphenyltetrazolium bromide assay (33Yamada T. Okajima F. Adachi M. Ohwada S. Kondo Y. Int. J. Cancer. 1998; 76: 141-147Crossref PubMed Scopus (14) Google Scholar). Quantitative Reverse Transcriptase-PCR Using Real-time TaqMan Technology—Total RNA was isolated using Tri-Reagent (Sigma) according to the instructions from the manufacturer. After DNase I (Promega, Madison, WI) treatment to remove possible traces of genomic DNA contaminating the RNA preparations, 5 μg of the total RNA was reverse transcribed using random priming and Multiscribe reverse transcriptase according to the instructions from the manufacturer (Applied Biosystems, Foster City, CA). To evaluate the expression levels of the LPA1, LPA2, LPA3, and LPA4/GPR23 mRNAs, quantitative reverse transcriptase-PCR was performed using real-time TaqMan technology with a sequence detection system model 7700 (Applied Biosystems, Foster City, CA). The human LPA1-, LPA2-, LPA3-, LPA4/GPR23-, and glyceraldehyde-3-phosphate dehydrogenase-specific probes were obtained from Assay-on-Demand products (Applied Biosystems, Foster City, 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 dehydrogenase. Amplification reaction was performed using the TaqMan universal PCR master mixture following the instructions from the manufacturer (Applied Biosystems). The thermal cycling conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. The procedure for the calculation of their expression was essentially the same as that described previously (39Aihara Y. Kasuya H. Onda H. Hori T. Takeda J. Stroke. 2001; 32: 212-217Crossref PubMed Scopus (125) Google Scholar). The expression level of the target mRNA was normalized to the relative ratio of the expression of glyceraldehyde-3-phosphate dehydrogenase mRNA. Each reverse transcriptase-PCR assay was performed at least three times, and the results are expressed as mean ± S.E. Northern Blot Analysis—Total RNA was prepared as described above. Twenty μg of total RNA was electrophoresed in a 1% agarose gel containing 3.7% formaldehyde in a 20 mm MOPS buffer and blotted onto a nylon membrane (Hybond-N) with 20× standard saline citrate (SSC). The cDNA probe of LPA1 (20 ng) was labeled with [α-32P]dATP by random oligonucleotide priming and added to the blots at a concentration of about 5 × 106 disintegrations/min in 5 ml of a hybridization buffer as described previously (34Sato K. Ui M. Okajima F. Brain Res. Mol. Brain Res. 2000; 85: 151-160Crossref PubMed Scopus (44) Google Scholar). The hybridization was carried out at 60 °C. Following hybridization, the blots were washed at 60 °C with 0.2× SSC and 0.1% SDS as described previously (34Sato K. Ui M. Okajima F. Brain Res. Mol. Brain Res. 2000; 85: 151-160Crossref PubMed Scopus (44) Google Scholar). Data Presentation—All experiments were performed in duplicate or triplicate. The results of multiple observations are presented as the mean ± S.E. or as representative results from more than three different
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