http://informahealthcare.com/rst ISSN: 1079-9893 (print), 1532-4281 (electronic) J Recept Signal Transduct Res, 2013; 33(5): 286–290 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10799893.2013.823998

RESEARCH ARTICLE

Eriko Tanabe1, Misaho Kitayoshi1, Miku Hirane1, Mutsumi Araki1, Yan Dong1, Nobuyuki Fukushima2, and Toshifumi Tsujiuchi1 1

Division of Cancer Biology and Bioinformatics and 2Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, Kowakae, Higashiosaka, Osaka, Japan Abstract

Keywords

Angiogenesis stimulates the invasive and metastatic process of cancer cells. It is also known that activated fibroblasts promote cancer cell growth and enhance invasive and metastatic potential. Lysophosphatidic acid (LPA) is a biological mediator and interacts with G proteincoupled transmembrane LPA receptors (LPA1 to LPA6). In this study, to assess an involvement of LPA3 on angiogenesis and fibroblast activation, the Lpar3-expressing cells were generated from mouse lung cancer LL/2 cells, which unexpressed LPA3. The Lpar3-expressing cells were maintained in serum-free Dulbecco’s modified Eagle’s medium for 48 h, and cell motility assay was performed with a cell culture Insert. When endothelial F-2 cells and 3T3 fibroblasts were cultured with conditioned medium from the Lpar3-expressing cells, their cell motile activities were significantly lower than the Lpar3-unexpressing (control) cells. Expression levels of vascular endothelial growth factor (Vegf) and fibroblast growth factor (Fgf) genes in the Lpar3expressing cells were measured by quantitative real time reverse transcription polymerase chain reaction analysis. The expressions of Vegf-A, Fgfa and Fgfb genes in the Lpar3-expressing cells were significantly lower than those in control cells, correlating with the effects on cell motile activities of F-2 and 3T3 cells. These results suggest that LPA signaling through LPA3 may inhibit angiogenesis and fibroblast activation in mouse lung cancer cells.

Angiogenesis, cell migration, fibroblast, LPA3, lung cancer

Introduction Lysophosphatidic acid (LPA) is biological signaling lipid that interacts with G protein-coupled transmembrane LPA receptors (1–3). Six LPA receptor subtypes (LPA1 to LPA6) have been identified to date (1). While LPA1 is ubiquitously expressed in normal tissues, the expression levels of other LPA receptor subtypes are relatively restricted (2,4). Moreover, the different expression patterns of LPA receptors were also found in cancer cells, correlating with DNA methylation status (5,6). LPA signaling throughout LPA receptors indicates a variety of intracellular responses, such as cell proliferation, migration, differentiation and morphogenesis (1–3). However, the function of each LPA receptor is not uniform. In fact, each LPA receptor acts a positive or negative regulator of biological effects in normal cells as well as cancer cells.

Address for correspondence: Toshifumi Tsujiuchi, Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1 Kowakae, Higashiosaka, Osaka 577-8502, Japan. Tel: +81 6 6721 2332. Fax: +81 6 6721 2721. E-mail: [email protected]

History Received 15 April 2013 Revised 4 July 2013 Accepted 8 July 2013 Published online 2 August 2013

For example, LPA1 inhibited and LPA3 stimulated cell motile activities induced by estrogens in rat liver epithelial cells (7). In ovarian cancer cells, exogenous LPA1, LPA2 and LPA3 increased cell migration activity in ovarian cancer cells (8). Recently, we indicated that LPA2 and LPA3 increased cell motility and invasion of neuroblastoma cells, whereas LPA1 suppressed those activities (9). LPA stimulated cell proliferation and migration activity in the Lpar5-expressing lung and liver cancer cells (10). It is well accepted that angiogenesis is the important process of developing new blood vessels from the existing vasculature and stimulates the invasive and metastatic potency of cancer cells (11,12). Angiogenesis is functionally regulated by several growth factors, such as vascular endothelial growth factors (VEGFs) (12). On the other hand, it is also considered that activated fibroblasts promote cancer cell growth, invasion, metastasis and angiogenesis, while normal conditioned fibroblasts are in an inactive quiescent state (13,14). In this study, to evaluate an involvement of LPA3 on angiogenesis and fibroblast activation in lung cancer cells, endothelial F-2 cells and 3T3 fibroblasts were cultured with conditioned medium from the Lpar3-expressing (LL/2-a3) cells and their growth rate and cell motile activities were

20 13

Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by University of Otago on 07/14/15 For personal use only.

Downregulation of activation factors of endothelia and fibroblasts via lysophosphatidic acid signaling in a mouse lung cancer LL/2 cell line

DOI: 10.3109/10799893.2013.823998

Downregulation of endothelia and fibroblasts via LPA signaling

measured (15,16). LL/2-a3 cells were generated from mouse lung cancer LL/2 cells, which unexpressed LPA3 (6,17). Recently, we demonstrated that exogenous LPA3 inhibited cell motile activity of LL/2 cells (17). Therefore, we investigated whether LPA3 acts as a positive or negative regulator of angiogenesis and fibroblast activation in LL/2 cells.

Materials and methods

Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by University of Otago on 07/14/15 For personal use only.

Cell culture Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) containing 10% fetal bovine serum in 5% CO2 atmosphere at 37  C. The Lpar3-expressing (LL/2-a3) cells were generated from LL/2 cells, using retroviruses co-expressing green fluorescent protein from an internal ribosomal entry site (17,18). LL/2-AB (control vector) cells were also used as a control clone. Cell growth of F-2 and 3T3 cells by the Lpar3-expressing cells F-2 and 3T3 were plated at 2000 cells/well in a 96-well plate and cultured with 100 ml of serum-free DMEM containing 30% supernatant from the Lpar3-expressing cells treated with LPA (10 mM) (Avanti Polar Lipid, Alabaster, AL) for 48 h. To assess cell proliferation rate for 3 d, solution from a Cell Counting Kit-8 (Dojin Chemistry, Kumamoto, Japan) was added to the plate at 1 or 3 d and cells were further incubated for 1 h. The absorbance of the culture medium at 450 nm was measured. The assay was always done in triplicate (15,16,18). Cell motile activity of F-2 and 3T3 cells by the Lpar3-expressing cells The effects of the Lpar3-expressing cells on cell motile activity of F-2 and 3T3 cells were measured using a Cell Culture Insert (BD, Falcon, NJ) with 8 mm pore size (15,16). F-2 and 3T3 cells were seeded in the filter at 1  105 cells in 200 ml serum-free DMEM (upper chamber) and placed in 24well plates (lower chamber) containing 800 ml of supernatants from the Lpar3-expressing cells treated with LPA (10 mM) (Avanti Polar Lipid) for 48 h. After 24 h, cells remaining in the upper side of the filter were removed with cotton swabs. The number of cells migrated to the lower side of the filter was counted after Giemsa staining (9,10,15–17). Quantitative real time reverse transcription polymerase chain reaction analysis Total RNA was extracted from each cell using ISOGEN (Nippon Gene, Inc., Toyama, Japan), and cDNA was then synthesized from 1.0 mg samples with Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics Co. Ltd., Mannheim, Germany). To measure the expression levels of Vegf-A, fibroblast growth factor (Fgf) a and Fgfb genes, quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis using a Smart Cycler II System (TaKaRa Bio, Inc., Shiga, Japan) and a SYBR Premix Ex Taq (Tli RNaseH Plus) (TaKaRa Bio, Inc.) was performed according to the manufacturer’s protocol

287

(10,15,16). Primer pairs used in this study were as follows: for Vegf-A (NCBI accession number NM_001025250), 50 -TGTGCAGGCTGCTGTAACGA-30 and 50 - TGTGCTGG CTTTGGTGAGGT-30 ; for Fgfa (NCBI accession number M30641), 50 -AACGGGGGCCACTTCTTGAG-30 and 50 -CG CACTTTCCGCACTGAGC-30 ; for Fgfb (NCBI accession number M30644), 50 -GCGAGAAGAGCGACCCACAC-30 and 50 -GGTTGGCACACACTCCCTTGA-30 . The data for these target genes were normalized to GAPDH (10,15,16). Effects of LPA signaling inhibitors on cell motile activity of F-2 and 3T3 cells LL/2-AB cells were pretreated with 100 ng/ml pertussis toxin (PTX) (Wako Pure Chemical Industries, Ltd.) for 24 h, 5 mM U-73122 (Cayman Chemical Co., Ann Arbor, MI) for 30 min, 1 mM Y-27632 (Mitsubishi Pharma Co., Osaka, Japan) for 30 min or 10 mM dioctylglycerol pyrophosphate (DGPP) (Avanti Polar Lipid) for 30 min in the condition of serumfree DMEM before the treatment of LPA (9,15,18). After 48 h, supernatants from LL/2-AB cells, treated with those inhibitors, were collected and used for the measurement of cell motile activity of F-2 and 3T3 cells. Cells were seeded into the filter at 1  105 and incubated for 24 h with LPA (10 mM) (15).

Results and discussion LPA3 indicated the opposite effects on cell motile activity of cancer cells. In neuroblastoma and hepatoma cells, LPA3 enhanced cell growth, motility, invasion and tumorigenicity (9,19). By contrast, LPA3 suppressed cell motile activity of LL/2 cells (17). In this study, we thus investigated the effects of LPA3 on angiogenesis and fibroblast activation in lung cancer cells. To measure cell growth and motile activities of F-2 and 3T3 cells, cells were cultured with conditioned medium from the Lpar3-expressing LL/2-a3 cells. No significant difference of cell growth rate of F-2 cells for 3 d was found (Figure 1A). For cell motile activity, F-2 cells were cultured with conditioned medium from LL/2-AB and LL/2-a3 cells treated with LPA (10 mM) for 48 h. A supernatant from LL/2-a3 cells indicated significantly lower cell motile activity of F-2 cells than LL/2-AB cells (Figure 1B,C). The 3T3 cells were also cultured with conditioned medium from LL/2-a3 and LL/2AB cells. The cell growth rate of 3T3 cells was at the same levels for 3 d (Figure 2A). The cell motile activity of 3T3 cells cultured with conditioned medium from LL/2-a3 cells treated with LPA was significantly lower than LL/2-AB cells (Figure 2B,C). Next, we measured the expression levels of Vegf-A, Fgfa and Fgfb genes in LL/2-AB and LL/2-a3 cells by quantitative real-time RT-PCR analysis. The expressions of Vegf-A, Fgfa and Fgfb genes in LL/2-a3 cells were significantly lower than those in LL/2-AB cells, correlating with the effects on cell motile activities of F-2 and 3T3 cells (Figure 3). To assess whether LPA receptor-coupled G proteins contribute to cell motile activities of F-2 and 3T3 cells, LL/ 2-AB cells were pretreated with PTX, U-73122 or Y-27632 before the treatment of LPA. LPA1 and LPA2 are coupled with Gi, Gq and G12/13, and LPA3 with Gi and Gq (9,18,20). PTX

E. Tanabe et al.

J Recept Signal Transduct Res, 2013; 33(5): 286–290

LL/2-AB LL/2-a3

120

p

2 cell line.

Angiogenesis stimulates the invasive and metastatic process of cancer cells. It is also known that activated fibroblasts promote cancer cell growth an...
455KB Sizes 0 Downloads 0 Views