LPA5-mediated signaling induced by endothelial cells and anticancer drug regulates cellular functions of osteosarcoma cells
Introduction
Lysophosphatidic acid (LPA) is a simple physiological lipid that consists of a glycerol, a phosphate and a fatty acid [[1], [2], [3], [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], [2], [3], [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 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.
Section snippets
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.
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
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
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number JP18K07249, and by research grants from the Faculty of Science and Engineering, Kindai University.
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