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LPA5-mediated signaling induced by endothelial cells and anticancer drug regulates cellular functions of osteosarcoma cells
Journal Pre-proof LPA5-mediated signaling induced by endothelial cells and anticancer drug regulates cellular functions of osteosarcoma cells Kanako Minami, Nanami Ueda, Kaichi Ishimoto, Toshifumi Tsujiuchi PII:
S0014-4827(20)30001-X
DOI:
https://doi.org/10.1016/j.yexcr.2020.111813
Reference:
YEXCR 111813
To appear in:
Experimental Cell Research
Received Date: 28 October 2019 Revised Date:
27 December 2019
Accepted Date: 1 January 2020
Please cite this article as: K. Minami, N. Ueda, K. Ishimoto, T. Tsujiuchi, LPA5-mediated signaling induced by endothelial cells and anticancer drug regulates cellular functions of osteosarcoma cells, Experimental Cell Research (2020), doi: https://doi.org/10.1016/j.yexcr.2020.111813. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Credit Author Statement All authors have seen and approved the final version of the manuscript being submitted. They warrant that the article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere.
LPA5-mediated signaling induced by endothelial cells and anticancer drug regulates cellular functions of osteosarcoma cells
Kanako Minami, Nanami Ueda, Kaichi Ishimoto, Toshifumi Tsujiuchi *
Division of Molecular Oncology, Department of Life Science, Faculty of Science and Engineering, Kindai University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502, Japan
* Corresponding author. Fax: +81 6 6721 2721. E-mail: [email protected] (T. Tsujiuchi)
Keywords: lysophosphatidic acid; LPA5; osteosarcoma cells; endothelial cells; cisplatin.
Abbreviations: LPA, lysophosphatidic acid; LPA5, LPA receptor-5; CDDP, cisplatin; RT-PCR, reverse transcription-polymerase chain reaction.
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Abstract Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) regulates a variety of malignant properties of cancer cells. It is known that endothelial cells promote tumor progression and chemoresistance. The present study aimed to investigate the roles of LPA5 in cellular functions modulated by endothelial cells and anticancer drug in osteosarcoma cells. Human osteosarcoma MG-63 cells were maintained in endothelial F2 cell supernatants. After culturing for 3 months, MG63-F2 cells were established. LPAR5 expression level in MG63-F2 cells was significantly elevated, compared with MG-63 cells. The cell motile activity of MG63-F2 cells was markedly higher than that of MG-63 cells. To validate the effects of LPA5 on cell motile activity, LPA5 knockdown cells were generated from MG-63 cells. The cell motile activity of MG-63 cells was inhibited by LPA5 knockdown. The cell survival to cisplatin (CDDP) was reduced in MG-63 cells treated with LPA. In the presence of LPA, the cell survival rate was significantly lower in MG63-F2 cells than MG-63 cells, correlating with LPAR5 expression. LPA5 knockdown cells indicated the high cell survival rate to CDDP. Moreover, LPAR5 expression level was increased in the long-term CDDP treated MG63-C cells. The cell survival to CDDP of MG63-C cells was enhanced by LPA5 knockdown. These results suggest that cellular functions are regulated through LPA5-mediatd signaling induced by endothelial cells and CDDP in MG-63 cells.
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Introduction Lysophosphatidic acid (LPA) is a simple physiological lipid that consists of a glycerol, a phosphate and a fatty acid [1-4]. LPA regulates diverse cellular functions through binding to G protein-coupled seven-transmembrane LPA receptors. Although six subtypes of LPA receptors (LPA receptor-1 (LPA1) to LPA6) have been identified, LPA4, LPA5 and LPA6 are structurally distinct from other LPA receptors. The individual LPA receptors exhibit the different responses to cellular functions [1-4]. It is considered that LPA signaling via LPA receptors is involved in the pathogenesis of several diseases, including cardiovascular disease, fibrosis, neuropathic pain and cancer [5,6]. In cancer cells, LPA receptor-mediated signaling contributes to the acquisition of malignant properties [5,6]. Our recent studies indicate that cellular functions are regulated through LPA receptors induced by anticancer drug treatment in cancer cells. For instance, LPA2 stimulates the cell motile and invasive activities of the long-term CDDP treated fibrosarcoma cells [7]. The motility, invasion and tumorigenicity are enhanced by LPA1 and LPA3 in pancreatic cancer cells treated with cisplatin (CDDP) [8]. In addition, LPA5 enhances the cell motile activity of melanoma cells treated with CDDP and dacarbazine [9]. It is known that stromal cells in the tumor microenvironment promote the malignant process of cancer cells [10]. Endothelial cells contribute to the acquisition of tumor cell properties, such as cell growth, invasion, metastasis and
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chemoresistance [10,11]. In the present investigation, we assessed the involvement of LPA receptors in cellular functions modulated by endothelial cells and anticancer drug in osteosarcoma MG-63 cells. Our recent study shows that the cell survival to CDDP is reduced by LPA5 in melanoma cells [12]. Therefore, we focused on LPA5 to investigate the roles of LPA signaling in cellular functions, using MG-63 cells cultured in F2 cell supernatants for 3 months. Moreover, the roles of LPA5-mediated signaling in cell survival to CDDP of the long-term CDDP treated cells were examined.
Materials and methods Cell culture and treatment Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wako Pure Chemical Industries Ltd., Osaka, Japan) containing 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere at 37°C. To obtain F2 cell supernatants, F2 cells were seeded at 5 × 105 cells in a 6-cm diameter dish and maintained in DMEM containing 10% FBS for 48 h. The conditioned mediums were collected and purified by centrifugation at 3000 rpm for 10 min. MG-63 cells were cultured in the cell-free supernatants of F2 cells. The conditioned mediums were changed twice or triplicate per week. After culturing for 3 months, MG63-F2 cells were established.
Cell proliferation and survival assays
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Cells were seeded at 3000 cells/well in 96-well plates and maintained in 100 µl of DMEM containing 10% FBS. Cell proliferation rate was measured by the Cell Counting Kit-8 (CCK-8) (Dojin Chemistry, Kumamoto, Japan). CCK-8 working solution was added to each well on days 0, 1 or 2. The absorbance of the conditioned medium at 450 nm was measured. For cell survival assay, cells were plated at 5000 cells/well in 96-well plates and cultured in DMEM containing 5% charcoal stripped FBS (Sigma, St. Louis, MO). Cells were treated with CDDP (Sigma) every 24 h for 2 days. After culturing, the CCK-8 working solution was added to each well. These assays were performed in triplicate [12].
Cell motility assay Cells were seeded at 1x105 cells on the Cell Culture Insert with 8 µm pore size (BD Falcon, Franklin Lakes, NJ) in 200 µl of serum-free DMEM (upper chamber). The filters were placed in a 24-well plate containing 800 µl of DMEM supplemented with 5% charcoal stripped FBS with or without LPA (Avanti Polar Lipids, Inc., AL) at a concentration of 10 µM (lower chamber), and cultured for 16 h. Cells remaining on the upper side of the filters were carefully removed with cotton swabs. After Giemsa staining, the numbers of cells that had moved to the lower side of the filters were counted. Before initiation of the cell motility assay, some cells were pretreated with TC LPA5 4 (Tocris Bioscience, Bristol, UK) for 30 min [13].
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Quantitative real-time reverse transcription (RT) - polymerase chain reaction (PCR) analysis To measure LPA receptor expressions, template cDNA was synthesized from total RNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics Co. Ltd., Mannheim, Germany). Real time quantitative RT-PCR analysis using a Smart Cycler II System (TaKaRa Bio, Inc., Shiga, Japan) and SYBR Premix Ex Taq (Tli RNaseH Plus) (TaKaRa) was performed according to the manufacturer’s protocol. Expression levels of the target genes were normalized to those of GAPDH gene. Each assay was repeated at least twice for confirmation [12,13].
Establishment of LPA5 knockdown cells Briefly, a HuSH short hairpin RNA plasmid (29-mer) against human LPAR5 (Origene, Rockville, MD) was transiently transfected into MG-63 cells, using X-tremeGENE HP Transfection Reagent (Roche Diagnostics). To generate control (MG63-GFP) cells, a control (vector) plasmid without the target sequence was also transfected into MG-63 cells [12].
Statistical analysis Analysis of variance (ANOVA) was performed to determine statistical significance. The data were recognized to differ significantly for values of p<0.01. The results are given as means + SD.
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Results Characteristics of osteosarcoma cells cultured in endothelial cell supernatants MG63-F2 cells were generated from MG-63 cells by culturing in F2 cell supernatants for 3 months (Fig.1A). In cell proliferation assay, MG63-F2 cells had a higher cell growth rate compared with MG-63 cells (Fig.1B). The intrinsic cell motile activity of MG63-F2 cells was approximately 11.1 times higher than that of MG-63 cells (Fig.1C). When F2 cell supernatants at 0, 5 and 10% were added in lower chamber, MG-63 cells indicated the high cell motile activity by 10% supernatants. On the other hand, the cell motile activity of MG63-F2 cells was markedly stimulated by 5 and 10% supernatants (Fig.1D). The cell growth activity of MG-63 cells was decreased by 5 and 10% supernatants in a dose-dependent manner. In contrast, F2 cell supernatants did not reduce MG63-F2 cell growth (Fig.1E).
LPA receptor expressions and cell motility of osteosarcoma cells cultured in endothelial cell supernatants Expression levels of LPAR4, LPAR5 and LPAR6 genes were not changed in MG-63 cells cultured in F2 cell supernatants for 3 days (Fig.2A). After culturing for 2 months, LPAR5 expression was significantly higher in MG63-F2 cells than in MG-63 cells, while LPAR4 and LPAR6 expression levels remained unchanged (Fig.2B). The elevated cell motile activity of MG63-F2 cells was markedly
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stimulated by LPA treatment, compared with MG-63 cells (Fig.2C).
Effects of LPA5 on cell motility of osteosarcoma cells Before initiation of the cell motility assay, MG-63 cells were pretreated with TC LPA5 4, an antagonist of LPA5 [14]. The cell motile activity of MG-63 cells was significantly suppressed by TC LPA5 4 in the presence of LPA (Fig.3A). To confirm the effects of LPA5 on cell motile activity, LPA5 knockdown (MG63-L5) cells were generated from MG-63 cells (Fig.3B). The cell motile activity of MG63-L5 cells was significantly lower than that of control (MG63-GFP) cells. LPA reduced the cell motile activity of MG63-L5 cells, while MG63-GFP cell motility was stimulated by LPA (Fig.3C).
Roles of LPA5 signaling in cell survival of osteosarcoma cells cultured in endothelial cell supernatants To assess whether LPA5 was involved in the modulation of cell survival to anticancer drug, MG-63 cells were treated with CDDP every 24 h for 2 days. The cell survival rate of MG-63 cells treated with CDDP was reduced by LPA treatment (p<0.01) (Fig. 4A). In the presence of LPA, the cell survival rate of MG63-F2 cells was markedly lower than that of MG-63 cells (p<0.01) (Fig.4B). When MG63-F2 cells were cultured in F2 cell supernatants, the cell survival of MG63-F2 cells was significantly reduced (p<0.01) (Fig.4C). To validate the effects of LPA5 on cell survival, MG63-L5 cells was treated with CDDP in the 8
presence of LPA. The cell survival rate of MG63-L5 cells was significantly higher than that of MG63-GFP cells (p<0.01) (Fig.4D).
Effects of LPA5 on cell survival in the long-term CDDP treated cells In our previous report, we established the long-term CDDP treated (MG63-C) cells from MG-63 cells by the stepwise treatment of CDDP for approximately 6 months [15]. The growth rate of MG63-C cells was higher than that of MG-63 cells. Additionally, MG63-C cells indicated the high cell motile activity, compared with MG-63 cells [15]. LPAR5 expression level was significantly higher in MG63-C cells than in MG-63 cells, but not LPAR4 and LPAR6 expressions (Fig.5A). In MG63-C cells, the cell survival rate to CDDP was markedly inhibited by LPA treatment (p<0.01) (Fig.5B). The cell survival of MG63-C-L5 cells was significantly higher than that of control MG63-C-G cells, similar as observed with MG63-L5 cells (p<0.01) (Fig.5C).
Effects of LPA5 on cell survival in the long-term anticancer drug treated fibrosarcoma cells HT-CDDP and HT-MTX cells were established from fibrosarcoma HT1080 cells by the stepwise treatment of CDDP and methotrexate (MTX) for 6 months, respectively [7,13]. We investigated whether LPA signaling via LPA5 reduced cell survival in HT-CDDP and HT-MTX cells. LPAR5 expression level was significantly higher in HT-CDDP cells than in HT1080 cells. The high LPAR5
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expression in HT-MTX cells was confirmed [13] (Fig.6A). LPA5 knockdown HTCDDP-L5 and HTMTX-L5 cells were generated from HT-CDDP and HT-MTX cells, respectively. In the presence of LPA, HTCDDP-L5 cells indicated the high cell survival to CDDP, compared with control HTCDDP-GFP cells (p<0.01) (Fig.6B). The cell survival to MTX of HTMTX-L5 cells was significantly higher than that of HTMTX-GFP cells (p<0.01) (Fig.6C).
Discussion It is considered that endothelial cells play an important role in the regulation of cancer cell growth, invasion, and metastasis [10,11]. Moreover, cell growth activity is promoted by culturing of endothelial cell supernatants [16]. In the present study, we generated MG63-F2 cells by culturing of F2 cell supernatants for 3 months. Whereas LPAR4, LPAR5 and LPAR6 expressions were not affected by culturing of F2 cell supernatants for 2 days, LPAR5 expression was elevated in MG63-F2 cells. Based on these findings, the effects of LPA5 on cellular functions of MG63-F2 cells were investigated. The cell motile and growth activities of MG63-F2 cells were stimulated by F2 cell supernatants, compared with MG-63 cells. To validate the effects of LPA5 on cell motile activity, MG-63 cells were treated with the LPA5 antagonist TC LPA5 4 [14]. Treatment of TC LPA5 4 suppressed MG-63 cell motility. Moreover, the cell motile activity of MG-63 cells was inhibited by LPA5 knockdown. These results suggest that the high cell motile activity of MG63-F2 cells is due to the induction of LPA5 by 10
culturing of F2 cell supernatants. Since endothelial cells is also involved in the modulation of chemoresistance in cancer cells [10,11], the effects of LPA5 on cell survival to CDDP were evaluated. LPA decreased the cell survival to CDDP of MG-63 cells. The cell survival of MG63-F2 cells was lower than that of MG-63 cells, correlating with LPAR5 expression. In the presence of LPA, the cell survival to CDDP of MG63-F2 cells was reduced by culturing of F2 cell supernatants. These findings suggest that LPA5-mediated signaling is activated by endothelial cells in MG-63 cells, resulting in decreased cell survival to CDDP. The long-term anticancer drug treatment causes an increase in LPA receptor expressions [7-9]. Recently, we indicate that LPAR2 and LPAR3 expressions are elevated in MG-63 cells treated with CDDP [15]. In this study, LPAR5 expression was markedly enhanced in MG63-C cells, similar to the case for MG63-F2 cells. Thus, we assessed whether LPA5-mediated signaling induced by the long-term CDDP treatment regulated cell survival to CDDP. LPA treatment reduced the cell survival of MG63-C cells. The cell survival of MG63-C cells was increased by LPA5 knockdown. Additionally, to evaluate the involvement of LPA5 on cell survival to anticancer drugs of other sarcoma cells, the long-term CDDP and MTX treated cells were used. Since LPAR5 expressions were elevated in HT-CDDP and HT-MTX cells [13], the cell survival rates of HT-CDDP and HT-MTX cells were enhanced by LPA5 knockdown. LPA signaling via LPA5 facilitates intracellular cAMP accumulation [17,18]. 11
Treatment
of
cAMP
upregulates
glutathione-S-transferase
(GST)
and
ATP-binding cassette (ABC) transporter in colon cancer cells [19]. GST catalytic activity contributes to the detoxification for a variety of anticancer drugs [20]. ABC transporters regulate the efflux of anticancer drugs in cancer cells [21,22]. Conversely, LPA5-medicated signaling induced by F2 cell supernatants and CDDP treatment reduced cell survival to CDDP in MG-63 cells. LPA5 is couples with Gq and G12/13 [1,2]. G12/13 activates Rho-mediated signaling [1,2]. Whereas antiapoptotic effects by RhoA and RhoC induce chemoresistance to CDDP, a proapoptotic effect of RhoB enhances chemosensitivity [23]. Taken together, it is possible that LPA signaling via LPA5 may modulate Rho-mediated signaling, resulting in the reduced cell survival to CDDP in MG-63 cells. While LPA signaling via LPA5 reduced the cell survival to CDDP of MG-63 cells, the cell motile activity was elevated by LPA5. In melanoma cells, LPA5 decreases the cell survival to anticancer drugs and stimulates the cell motile activity [12,24]. It is well known that Rho-mediated signaling is involved in the regulation of cell motile activity [25]. The association between Rho- and LPA5-mediated signaling pathways in cellular functions of MG-63 cells remains to be clarified. In conclusion, our results show that cellular functions are regulated through the induction of LPA5-mediated signaling by endothelial cells and anticancer drug in MG-63 cells. Since the long-term CDDP treatment increases LPAR2 and LPAR3 expressions in MG-63 cells [15], the roles of LPA2 and LPA3 in cell survival to anticancer drugs of MG-63 cells should be examined.
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Acknowledgements This work was supported by JSPS KAKENHI Grant Number JP18K07249, and by research grants from the Faculty of Science and Engineering, Kindai University.
Conflict of interest statement The authors declare that they have no conflict of interest.
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Figure legends: Fig.1: Characteristics of osteosarcoma cells cultured in endothelial cell supernatants. (A) Morphology of MG63-F2 cells in DMEM containing 10% FBS. MG63-F2, MG-63 cells cultured in F2 cell supernatants. (B) Cell proliferation assay. Cells were cultured in 10% FBS-DMEM for 2 days. The cell growth rate was measured using the CCK-8. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG-63 cells. (C) Intrinsic cell motile activity. Cells were seeded at 1x105 cells on the Cell Culture Insert in serum-free DMEM (upper chamber). The filters were then placed in 24-well plates containing 10% FBS-DMEM (lower chamber) and incubated for 16 h. Columns indicate the mean of three studies; bars indicate SD. *; p<0.01 vs. MG-63 cells. (D) Effects of F2 cell supernatants on cell motile activity. Cells were incubated in DMEM supplemented with 5% charcoal-stripped FBS containing F2 cell supernatants at 0, 5 and 10% (lower chamber). Columns indicate the mean of three studies. Bars indicate SD. * ; p<0.01 vs. MG-63 cells cultured in DMEM supplemented with 5% charcoal-stripped FBS. (E) Effects of F2 cell supernatants on cell growth. Cells were cultured in 10% FBS-DMEM containing F2 cell supernatants at 0, 5 and 10% for 2 days. The cell growth rate was measured using the CCK-8. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG-63 cells cultured in 10% FBS-DMEM.
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Fig.2: LPA receptor expressions and cell motility of osteosarcoma cells cultured in endothelial cell supernatants. Expression levels of LPA receptor genes by quantitative real-time RT-PCR analysis. MG-63 cells were cultured in F2 cell supernatants for 3 days (A) and 3 months (B). Columns indicate the mean of three studies; bars indicate SD. *; p<0.01 vs. MG-63 cells. (C) Cell motility assay. Cells were incubated in DMEM supplemented with 5% charcoal-stripped FBS with or without LPA (10 µM). Columns indicate the mean of three studies. Bars indicate SD. * ; p<0.01 vs. untreated MG-63 cells. #; p<0.01 vs. LPA treated MG-63 cells. Fig.3: Roles of LPA5 signaling in cell motility of osteosarcoma cells. (A) Cell motility assay. Before initiating the cell motility assay, cells were pretreated with TC LPA5 4 (0.5 µM). Cells were incubated in DMEM supplemented with 5% charcoal-stripped FBS with LPA (10 µM) (lower chamber). Columns indicate the mean of three studies. Bars indicate SD. * ; p<0.01 vs. TC LPA5 4 untreated MG-63 cells. (B) LPAR5 expression levels by quantitative real-time RT-PCR analysis. Columns indicate the mean of three studies; bars indicate SD. *; p<0.01 vs. MG63-GFP cells. MG63-GFP, control (vector) cells generated from MG-63 cells. MG63-L5, LPA5 knockdown cells generated from MG-63 cells. (C) Cell motility assay. Cells were incubated in DMEM supplemented with 5% charcoal-stripped FBS with or without LPA (10 µM) (lower chamber). Columns indicate the mean of three studies. Bars indicate SD. * ; p<0.01 vs. LPA untreated
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MG63-GFP cells. #; p<0.01 vs. LPA treated MG63-GFP cells. Fig.4: Effects of LPA5 on cell survival of osteosarcoma cells cultured in endothelial cell supernatants. (A) Cell survival assay. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with or without LPA (10 µM). Cells were treated with CDDP for 2 days. Cell survival rate was measured using the CCK-8. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. control cells. (B) Cell survival of osteosarcoma cells cultured in F2 cell supernatants. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with LPA (10 µM). Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG-63 cells. MG63-F2, MG-63 cells cultured in F2 cell supernatants. (C) Effects of endothelial cell supernatants on cell survival. In the presence of LPA (10 µM), MG63-F2 cells were incubated in serum-free DMEM containing F2 cell supernatants at 50%. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG63-F2 cells treated without the supernatants. (D) Effects of LPA5 knockdown on cell survival of MG-63 cells. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with LPA (10 µM). Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG63-GFP cells. MG63-GFP, control (vector) cells generated from MG-63 cells. MG63-L5, LPA5 knockdown cells generated from MG-63 cells. Fig.5: Effects of LPA5 on cell survival of long-term CDDP-treated osteosarcoma 20
cells. (A) Expression levels of LPA receptor genes by quantitative real-time RT-PCR analysis. Columns indicate the mean of three studies; bars indicate SD. *; p<0.01 vs. MG-63 cells. MG63-C, long-term CDDP-treated MG-63 cells. (B) Cell survival assay. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with or without LPA (10 µM). Cells were treated with CDDP for 2 days. Cell survival rate was measured using the CCK-8. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. control cells. (C) Effects of LPA5 knockdown on cell survival of the long-term CDDP-treated cells.. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with LPA (10 µM). Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG63-C-G cells. MG63-C-G, control (vector) cells generated from MG6-C cells. MG63-C-L5, LPA5 knockdown cells generated from MG63-C cells. Fig.6: Effects of LPA5 on cell survival of long-term anticancer drug treated fibrosarcoma cells. (A) Expression levels of LPA receptor genes by quantitative real-time RT-PCR analysis. Columns indicate the mean of three studies; bars indicate SD. *; p<0.01 vs. HT1080 cells. HT-CDDP, long-term CDDP treated HT1080 cells. HT-MTX, long-term MTX treated HT1080 cells. (B) Effects of LPA5 on cell survival to CDDP. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with LPA (10 µM). Cells were treated with CDDP for 2 days. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. HTCDDP-GFP cells.
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HTCDDP-GFP, control (vector) cells generated from HT-CDDP cells. HTCDDP-L5, LPA5 knockdown cells generated from HT-CDDP cells. (C) Effects of LPA5 on cell survival to MTX. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with LPA (10 µM). Cells were treated with MTX for 2 days. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. HTMTX-GFP cells. HTMTX-GFP cells. HTMTX-GFP, control (vector) cells generated from HT-MTX cells. HTMTX-L5, LPA5 knockdown cells generated from HT-MTX cells.
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Highlights 1. High LPAR5 expression was induced by culturing of endothelial cell supernatants in MG-63 cells. 2. MG-63 cell motility was markedly stimulated by endothelial cell supernatants. 3. LPAR5 expression was elevated in the long-term CDDP treated MG63-C cells. 4. Cell survival rates to CDDP of MG-63 and MG63-C cells were increased by LPA5 knockdown. 5. Cellular functions are regulated via LPA5 induced by endothelial cells and CDDP in MG-63 cells.
Conflict of interest The authors declare that they have no conflict of interest.
1
S0014-4827(20)30001-X
DOI:
https://doi.org/10.1016/j.yexcr.2020.111813
Reference:
YEXCR 111813
To appear in:
Experimental Cell Research
Received Date: 28 October 2019 Revised Date:
27 December 2019
Accepted Date: 1 January 2020
Please cite this article as: K. Minami, N. Ueda, K. Ishimoto, T. Tsujiuchi, LPA5-mediated signaling induced by endothelial cells and anticancer drug regulates cellular functions of osteosarcoma cells, Experimental Cell Research (2020), doi: https://doi.org/10.1016/j.yexcr.2020.111813. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Credit Author Statement All authors have seen and approved the final version of the manuscript being submitted. They warrant that the article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere.
LPA5-mediated signaling induced by endothelial cells and anticancer drug regulates cellular functions of osteosarcoma cells
Kanako Minami, Nanami Ueda, Kaichi Ishimoto, Toshifumi Tsujiuchi *
Division of Molecular Oncology, Department of Life Science, Faculty of Science and Engineering, Kindai University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502, Japan
* Corresponding author. Fax: +81 6 6721 2721. E-mail: [email protected] (T. Tsujiuchi)
Keywords: lysophosphatidic acid; LPA5; osteosarcoma cells; endothelial cells; cisplatin.
Abbreviations: LPA, lysophosphatidic acid; LPA5, LPA receptor-5; CDDP, cisplatin; RT-PCR, reverse transcription-polymerase chain reaction.
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Abstract Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) regulates a variety of malignant properties of cancer cells. It is known that endothelial cells promote tumor progression and chemoresistance. The present study aimed to investigate the roles of LPA5 in cellular functions modulated by endothelial cells and anticancer drug in osteosarcoma cells. Human osteosarcoma MG-63 cells were maintained in endothelial F2 cell supernatants. After culturing for 3 months, MG63-F2 cells were established. LPAR5 expression level in MG63-F2 cells was significantly elevated, compared with MG-63 cells. The cell motile activity of MG63-F2 cells was markedly higher than that of MG-63 cells. To validate the effects of LPA5 on cell motile activity, LPA5 knockdown cells were generated from MG-63 cells. The cell motile activity of MG-63 cells was inhibited by LPA5 knockdown. The cell survival to cisplatin (CDDP) was reduced in MG-63 cells treated with LPA. In the presence of LPA, the cell survival rate was significantly lower in MG63-F2 cells than MG-63 cells, correlating with LPAR5 expression. LPA5 knockdown cells indicated the high cell survival rate to CDDP. Moreover, LPAR5 expression level was increased in the long-term CDDP treated MG63-C cells. The cell survival to CDDP of MG63-C cells was enhanced by LPA5 knockdown. These results suggest that cellular functions are regulated through LPA5-mediatd signaling induced by endothelial cells and CDDP in MG-63 cells.
2
Introduction Lysophosphatidic acid (LPA) is a simple physiological lipid that consists of a glycerol, a phosphate and a fatty acid [1-4]. LPA regulates diverse cellular functions through binding to G protein-coupled seven-transmembrane LPA receptors. Although six subtypes of LPA receptors (LPA receptor-1 (LPA1) to LPA6) have been identified, LPA4, LPA5 and LPA6 are structurally distinct from other LPA receptors. The individual LPA receptors exhibit the different responses to cellular functions [1-4]. It is considered that LPA signaling via LPA receptors is involved in the pathogenesis of several diseases, including cardiovascular disease, fibrosis, neuropathic pain and cancer [5,6]. In cancer cells, LPA receptor-mediated signaling contributes to the acquisition of malignant properties [5,6]. Our recent studies indicate that cellular functions are regulated through LPA receptors induced by anticancer drug treatment in cancer cells. For instance, LPA2 stimulates the cell motile and invasive activities of the long-term CDDP treated fibrosarcoma cells [7]. The motility, invasion and tumorigenicity are enhanced by LPA1 and LPA3 in pancreatic cancer cells treated with cisplatin (CDDP) [8]. In addition, LPA5 enhances the cell motile activity of melanoma cells treated with CDDP and dacarbazine [9]. It is known that stromal cells in the tumor microenvironment promote the malignant process of cancer cells [10]. Endothelial cells contribute to the acquisition of tumor cell properties, such as cell growth, invasion, metastasis and
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chemoresistance [10,11]. In the present investigation, we assessed the involvement of LPA receptors in cellular functions modulated by endothelial cells and anticancer drug in osteosarcoma MG-63 cells. Our recent study shows that the cell survival to CDDP is reduced by LPA5 in melanoma cells [12]. Therefore, we focused on LPA5 to investigate the roles of LPA signaling in cellular functions, using MG-63 cells cultured in F2 cell supernatants for 3 months. Moreover, the roles of LPA5-mediated signaling in cell survival to CDDP of the long-term CDDP treated cells were examined.
Materials and methods Cell culture and treatment Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wako Pure Chemical Industries Ltd., Osaka, Japan) containing 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere at 37°C. To obtain F2 cell supernatants, F2 cells were seeded at 5 × 105 cells in a 6-cm diameter dish and maintained in DMEM containing 10% FBS for 48 h. The conditioned mediums were collected and purified by centrifugation at 3000 rpm for 10 min. MG-63 cells were cultured in the cell-free supernatants of F2 cells. The conditioned mediums were changed twice or triplicate per week. After culturing for 3 months, MG63-F2 cells were established.
Cell proliferation and survival assays
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Cells were seeded at 3000 cells/well in 96-well plates and maintained in 100 µl of DMEM containing 10% FBS. Cell proliferation rate was measured by the Cell Counting Kit-8 (CCK-8) (Dojin Chemistry, Kumamoto, Japan). CCK-8 working solution was added to each well on days 0, 1 or 2. The absorbance of the conditioned medium at 450 nm was measured. For cell survival assay, cells were plated at 5000 cells/well in 96-well plates and cultured in DMEM containing 5% charcoal stripped FBS (Sigma, St. Louis, MO). Cells were treated with CDDP (Sigma) every 24 h for 2 days. After culturing, the CCK-8 working solution was added to each well. These assays were performed in triplicate [12].
Cell motility assay Cells were seeded at 1x105 cells on the Cell Culture Insert with 8 µm pore size (BD Falcon, Franklin Lakes, NJ) in 200 µl of serum-free DMEM (upper chamber). The filters were placed in a 24-well plate containing 800 µl of DMEM supplemented with 5% charcoal stripped FBS with or without LPA (Avanti Polar Lipids, Inc., AL) at a concentration of 10 µM (lower chamber), and cultured for 16 h. Cells remaining on the upper side of the filters were carefully removed with cotton swabs. After Giemsa staining, the numbers of cells that had moved to the lower side of the filters were counted. Before initiation of the cell motility assay, some cells were pretreated with TC LPA5 4 (Tocris Bioscience, Bristol, UK) for 30 min [13].
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Quantitative real-time reverse transcription (RT) - polymerase chain reaction (PCR) analysis To measure LPA receptor expressions, template cDNA was synthesized from total RNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics Co. Ltd., Mannheim, Germany). Real time quantitative RT-PCR analysis using a Smart Cycler II System (TaKaRa Bio, Inc., Shiga, Japan) and SYBR Premix Ex Taq (Tli RNaseH Plus) (TaKaRa) was performed according to the manufacturer’s protocol. Expression levels of the target genes were normalized to those of GAPDH gene. Each assay was repeated at least twice for confirmation [12,13].
Establishment of LPA5 knockdown cells Briefly, a HuSH short hairpin RNA plasmid (29-mer) against human LPAR5 (Origene, Rockville, MD) was transiently transfected into MG-63 cells, using X-tremeGENE HP Transfection Reagent (Roche Diagnostics). To generate control (MG63-GFP) cells, a control (vector) plasmid without the target sequence was also transfected into MG-63 cells [12].
Statistical analysis Analysis of variance (ANOVA) was performed to determine statistical significance. The data were recognized to differ significantly for values of p<0.01. The results are given as means + SD.
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Results Characteristics of osteosarcoma cells cultured in endothelial cell supernatants MG63-F2 cells were generated from MG-63 cells by culturing in F2 cell supernatants for 3 months (Fig.1A). In cell proliferation assay, MG63-F2 cells had a higher cell growth rate compared with MG-63 cells (Fig.1B). The intrinsic cell motile activity of MG63-F2 cells was approximately 11.1 times higher than that of MG-63 cells (Fig.1C). When F2 cell supernatants at 0, 5 and 10% were added in lower chamber, MG-63 cells indicated the high cell motile activity by 10% supernatants. On the other hand, the cell motile activity of MG63-F2 cells was markedly stimulated by 5 and 10% supernatants (Fig.1D). The cell growth activity of MG-63 cells was decreased by 5 and 10% supernatants in a dose-dependent manner. In contrast, F2 cell supernatants did not reduce MG63-F2 cell growth (Fig.1E).
LPA receptor expressions and cell motility of osteosarcoma cells cultured in endothelial cell supernatants Expression levels of LPAR4, LPAR5 and LPAR6 genes were not changed in MG-63 cells cultured in F2 cell supernatants for 3 days (Fig.2A). After culturing for 2 months, LPAR5 expression was significantly higher in MG63-F2 cells than in MG-63 cells, while LPAR4 and LPAR6 expression levels remained unchanged (Fig.2B). The elevated cell motile activity of MG63-F2 cells was markedly
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stimulated by LPA treatment, compared with MG-63 cells (Fig.2C).
Effects of LPA5 on cell motility of osteosarcoma cells Before initiation of the cell motility assay, MG-63 cells were pretreated with TC LPA5 4, an antagonist of LPA5 [14]. The cell motile activity of MG-63 cells was significantly suppressed by TC LPA5 4 in the presence of LPA (Fig.3A). To confirm the effects of LPA5 on cell motile activity, LPA5 knockdown (MG63-L5) cells were generated from MG-63 cells (Fig.3B). The cell motile activity of MG63-L5 cells was significantly lower than that of control (MG63-GFP) cells. LPA reduced the cell motile activity of MG63-L5 cells, while MG63-GFP cell motility was stimulated by LPA (Fig.3C).
Roles of LPA5 signaling in cell survival of osteosarcoma cells cultured in endothelial cell supernatants To assess whether LPA5 was involved in the modulation of cell survival to anticancer drug, MG-63 cells were treated with CDDP every 24 h for 2 days. The cell survival rate of MG-63 cells treated with CDDP was reduced by LPA treatment (p<0.01) (Fig. 4A). In the presence of LPA, the cell survival rate of MG63-F2 cells was markedly lower than that of MG-63 cells (p<0.01) (Fig.4B). When MG63-F2 cells were cultured in F2 cell supernatants, the cell survival of MG63-F2 cells was significantly reduced (p<0.01) (Fig.4C). To validate the effects of LPA5 on cell survival, MG63-L5 cells was treated with CDDP in the 8
presence of LPA. The cell survival rate of MG63-L5 cells was significantly higher than that of MG63-GFP cells (p<0.01) (Fig.4D).
Effects of LPA5 on cell survival in the long-term CDDP treated cells In our previous report, we established the long-term CDDP treated (MG63-C) cells from MG-63 cells by the stepwise treatment of CDDP for approximately 6 months [15]. The growth rate of MG63-C cells was higher than that of MG-63 cells. Additionally, MG63-C cells indicated the high cell motile activity, compared with MG-63 cells [15]. LPAR5 expression level was significantly higher in MG63-C cells than in MG-63 cells, but not LPAR4 and LPAR6 expressions (Fig.5A). In MG63-C cells, the cell survival rate to CDDP was markedly inhibited by LPA treatment (p<0.01) (Fig.5B). The cell survival of MG63-C-L5 cells was significantly higher than that of control MG63-C-G cells, similar as observed with MG63-L5 cells (p<0.01) (Fig.5C).
Effects of LPA5 on cell survival in the long-term anticancer drug treated fibrosarcoma cells HT-CDDP and HT-MTX cells were established from fibrosarcoma HT1080 cells by the stepwise treatment of CDDP and methotrexate (MTX) for 6 months, respectively [7,13]. We investigated whether LPA signaling via LPA5 reduced cell survival in HT-CDDP and HT-MTX cells. LPAR5 expression level was significantly higher in HT-CDDP cells than in HT1080 cells. The high LPAR5
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expression in HT-MTX cells was confirmed [13] (Fig.6A). LPA5 knockdown HTCDDP-L5 and HTMTX-L5 cells were generated from HT-CDDP and HT-MTX cells, respectively. In the presence of LPA, HTCDDP-L5 cells indicated the high cell survival to CDDP, compared with control HTCDDP-GFP cells (p<0.01) (Fig.6B). The cell survival to MTX of HTMTX-L5 cells was significantly higher than that of HTMTX-GFP cells (p<0.01) (Fig.6C).
Discussion It is considered that endothelial cells play an important role in the regulation of cancer cell growth, invasion, and metastasis [10,11]. Moreover, cell growth activity is promoted by culturing of endothelial cell supernatants [16]. In the present study, we generated MG63-F2 cells by culturing of F2 cell supernatants for 3 months. Whereas LPAR4, LPAR5 and LPAR6 expressions were not affected by culturing of F2 cell supernatants for 2 days, LPAR5 expression was elevated in MG63-F2 cells. Based on these findings, the effects of LPA5 on cellular functions of MG63-F2 cells were investigated. The cell motile and growth activities of MG63-F2 cells were stimulated by F2 cell supernatants, compared with MG-63 cells. To validate the effects of LPA5 on cell motile activity, MG-63 cells were treated with the LPA5 antagonist TC LPA5 4 [14]. Treatment of TC LPA5 4 suppressed MG-63 cell motility. Moreover, the cell motile activity of MG-63 cells was inhibited by LPA5 knockdown. These results suggest that the high cell motile activity of MG63-F2 cells is due to the induction of LPA5 by 10
culturing of F2 cell supernatants. Since endothelial cells is also involved in the modulation of chemoresistance in cancer cells [10,11], the effects of LPA5 on cell survival to CDDP were evaluated. LPA decreased the cell survival to CDDP of MG-63 cells. The cell survival of MG63-F2 cells was lower than that of MG-63 cells, correlating with LPAR5 expression. In the presence of LPA, the cell survival to CDDP of MG63-F2 cells was reduced by culturing of F2 cell supernatants. These findings suggest that LPA5-mediated signaling is activated by endothelial cells in MG-63 cells, resulting in decreased cell survival to CDDP. The long-term anticancer drug treatment causes an increase in LPA receptor expressions [7-9]. Recently, we indicate that LPAR2 and LPAR3 expressions are elevated in MG-63 cells treated with CDDP [15]. In this study, LPAR5 expression was markedly enhanced in MG63-C cells, similar to the case for MG63-F2 cells. Thus, we assessed whether LPA5-mediated signaling induced by the long-term CDDP treatment regulated cell survival to CDDP. LPA treatment reduced the cell survival of MG63-C cells. The cell survival of MG63-C cells was increased by LPA5 knockdown. Additionally, to evaluate the involvement of LPA5 on cell survival to anticancer drugs of other sarcoma cells, the long-term CDDP and MTX treated cells were used. Since LPAR5 expressions were elevated in HT-CDDP and HT-MTX cells [13], the cell survival rates of HT-CDDP and HT-MTX cells were enhanced by LPA5 knockdown. LPA signaling via LPA5 facilitates intracellular cAMP accumulation [17,18]. 11
Treatment
of
cAMP
upregulates
glutathione-S-transferase
(GST)
and
ATP-binding cassette (ABC) transporter in colon cancer cells [19]. GST catalytic activity contributes to the detoxification for a variety of anticancer drugs [20]. ABC transporters regulate the efflux of anticancer drugs in cancer cells [21,22]. Conversely, LPA5-medicated signaling induced by F2 cell supernatants and CDDP treatment reduced cell survival to CDDP in MG-63 cells. LPA5 is couples with Gq and G12/13 [1,2]. G12/13 activates Rho-mediated signaling [1,2]. Whereas antiapoptotic effects by RhoA and RhoC induce chemoresistance to CDDP, a proapoptotic effect of RhoB enhances chemosensitivity [23]. Taken together, it is possible that LPA signaling via LPA5 may modulate Rho-mediated signaling, resulting in the reduced cell survival to CDDP in MG-63 cells. While LPA signaling via LPA5 reduced the cell survival to CDDP of MG-63 cells, the cell motile activity was elevated by LPA5. In melanoma cells, LPA5 decreases the cell survival to anticancer drugs and stimulates the cell motile activity [12,24]. It is well known that Rho-mediated signaling is involved in the regulation of cell motile activity [25]. The association between Rho- and LPA5-mediated signaling pathways in cellular functions of MG-63 cells remains to be clarified. In conclusion, our results show that cellular functions are regulated through the induction of LPA5-mediated signaling by endothelial cells and anticancer drug in MG-63 cells. Since the long-term CDDP treatment increases LPAR2 and LPAR3 expressions in MG-63 cells [15], the roles of LPA2 and LPA3 in cell survival to anticancer drugs of MG-63 cells should be examined.
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Acknowledgements This work was supported by JSPS KAKENHI Grant Number JP18K07249, and by research grants from the Faculty of Science and Engineering, Kindai University.
Conflict of interest statement The authors declare that they have no conflict of interest.
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Figure legends: Fig.1: Characteristics of osteosarcoma cells cultured in endothelial cell supernatants. (A) Morphology of MG63-F2 cells in DMEM containing 10% FBS. MG63-F2, MG-63 cells cultured in F2 cell supernatants. (B) Cell proliferation assay. Cells were cultured in 10% FBS-DMEM for 2 days. The cell growth rate was measured using the CCK-8. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG-63 cells. (C) Intrinsic cell motile activity. Cells were seeded at 1x105 cells on the Cell Culture Insert in serum-free DMEM (upper chamber). The filters were then placed in 24-well plates containing 10% FBS-DMEM (lower chamber) and incubated for 16 h. Columns indicate the mean of three studies; bars indicate SD. *; p<0.01 vs. MG-63 cells. (D) Effects of F2 cell supernatants on cell motile activity. Cells were incubated in DMEM supplemented with 5% charcoal-stripped FBS containing F2 cell supernatants at 0, 5 and 10% (lower chamber). Columns indicate the mean of three studies. Bars indicate SD. * ; p<0.01 vs. MG-63 cells cultured in DMEM supplemented with 5% charcoal-stripped FBS. (E) Effects of F2 cell supernatants on cell growth. Cells were cultured in 10% FBS-DMEM containing F2 cell supernatants at 0, 5 and 10% for 2 days. The cell growth rate was measured using the CCK-8. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG-63 cells cultured in 10% FBS-DMEM.
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Fig.2: LPA receptor expressions and cell motility of osteosarcoma cells cultured in endothelial cell supernatants. Expression levels of LPA receptor genes by quantitative real-time RT-PCR analysis. MG-63 cells were cultured in F2 cell supernatants for 3 days (A) and 3 months (B). Columns indicate the mean of three studies; bars indicate SD. *; p<0.01 vs. MG-63 cells. (C) Cell motility assay. Cells were incubated in DMEM supplemented with 5% charcoal-stripped FBS with or without LPA (10 µM). Columns indicate the mean of three studies. Bars indicate SD. * ; p<0.01 vs. untreated MG-63 cells. #; p<0.01 vs. LPA treated MG-63 cells. Fig.3: Roles of LPA5 signaling in cell motility of osteosarcoma cells. (A) Cell motility assay. Before initiating the cell motility assay, cells were pretreated with TC LPA5 4 (0.5 µM). Cells were incubated in DMEM supplemented with 5% charcoal-stripped FBS with LPA (10 µM) (lower chamber). Columns indicate the mean of three studies. Bars indicate SD. * ; p<0.01 vs. TC LPA5 4 untreated MG-63 cells. (B) LPAR5 expression levels by quantitative real-time RT-PCR analysis. Columns indicate the mean of three studies; bars indicate SD. *; p<0.01 vs. MG63-GFP cells. MG63-GFP, control (vector) cells generated from MG-63 cells. MG63-L5, LPA5 knockdown cells generated from MG-63 cells. (C) Cell motility assay. Cells were incubated in DMEM supplemented with 5% charcoal-stripped FBS with or without LPA (10 µM) (lower chamber). Columns indicate the mean of three studies. Bars indicate SD. * ; p<0.01 vs. LPA untreated
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MG63-GFP cells. #; p<0.01 vs. LPA treated MG63-GFP cells. Fig.4: Effects of LPA5 on cell survival of osteosarcoma cells cultured in endothelial cell supernatants. (A) Cell survival assay. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with or without LPA (10 µM). Cells were treated with CDDP for 2 days. Cell survival rate was measured using the CCK-8. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. control cells. (B) Cell survival of osteosarcoma cells cultured in F2 cell supernatants. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with LPA (10 µM). Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG-63 cells. MG63-F2, MG-63 cells cultured in F2 cell supernatants. (C) Effects of endothelial cell supernatants on cell survival. In the presence of LPA (10 µM), MG63-F2 cells were incubated in serum-free DMEM containing F2 cell supernatants at 50%. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG63-F2 cells treated without the supernatants. (D) Effects of LPA5 knockdown on cell survival of MG-63 cells. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with LPA (10 µM). Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG63-GFP cells. MG63-GFP, control (vector) cells generated from MG-63 cells. MG63-L5, LPA5 knockdown cells generated from MG-63 cells. Fig.5: Effects of LPA5 on cell survival of long-term CDDP-treated osteosarcoma 20
cells. (A) Expression levels of LPA receptor genes by quantitative real-time RT-PCR analysis. Columns indicate the mean of three studies; bars indicate SD. *; p<0.01 vs. MG-63 cells. MG63-C, long-term CDDP-treated MG-63 cells. (B) Cell survival assay. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with or without LPA (10 µM). Cells were treated with CDDP for 2 days. Cell survival rate was measured using the CCK-8. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. control cells. (C) Effects of LPA5 knockdown on cell survival of the long-term CDDP-treated cells.. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with LPA (10 µM). Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. MG63-C-G cells. MG63-C-G, control (vector) cells generated from MG6-C cells. MG63-C-L5, LPA5 knockdown cells generated from MG63-C cells. Fig.6: Effects of LPA5 on cell survival of long-term anticancer drug treated fibrosarcoma cells. (A) Expression levels of LPA receptor genes by quantitative real-time RT-PCR analysis. Columns indicate the mean of three studies; bars indicate SD. *; p<0.01 vs. HT1080 cells. HT-CDDP, long-term CDDP treated HT1080 cells. HT-MTX, long-term MTX treated HT1080 cells. (B) Effects of LPA5 on cell survival to CDDP. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with LPA (10 µM). Cells were treated with CDDP for 2 days. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. HTCDDP-GFP cells.
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HTCDDP-GFP, control (vector) cells generated from HT-CDDP cells. HTCDDP-L5, LPA5 knockdown cells generated from HT-CDDP cells. (C) Effects of LPA5 on cell survival to MTX. Cells were cultured in DMEM supplemented with 5% charcoal stripped FBS with LPA (10 µM). Cells were treated with MTX for 2 days. Data are expressed as the percentage of cell number on day 0. *; p<0.01 vs. HTMTX-GFP cells. HTMTX-GFP cells. HTMTX-GFP, control (vector) cells generated from HT-MTX cells. HTMTX-L5, LPA5 knockdown cells generated from HT-MTX cells.
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Highlights 1. High LPAR5 expression was induced by culturing of endothelial cell supernatants in MG-63 cells. 2. MG-63 cell motility was markedly stimulated by endothelial cell supernatants. 3. LPAR5 expression was elevated in the long-term CDDP treated MG63-C cells. 4. Cell survival rates to CDDP of MG-63 and MG63-C cells were increased by LPA5 knockdown. 5. Cellular functions are regulated via LPA5 induced by endothelial cells and CDDP in MG-63 cells.
Conflict of interest The authors declare that they have no conflict of interest.
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