Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA4 and LPA6 knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA2 knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2 S)-OMPT (LPA3 agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA4 and LPA6 knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr.

Different effects of lysophosphatidic acid receptor-2 (LPA2) and LPA5 on the regulation of chemoresistance in colon cancer cells.

Lysophosphatidic acid (LPA) is a simple physiological lipid and exhibits several biological functions by binding to G-protein-coupled LPA receptors (LPA receptor-1 (LPA1) to LPA6). The present study aimed to evaluate whether LPA signaling via LPA2 and LPA5 is involved in the chemoresistance to anticancer drugs in colon cancer DLD1 cells. In cell survival assay, cells were treated with fluorouracil (5-FU) every 24 h for 2 days. The cell survival rate to 5-FU of DLD1 cells was significantly decreased by LPA treatment. In the presence of LPA, the cell survival rate to 5-FU was significantly elevated by LPA5 knockdown. Before initiation of the cell survival assay, cells were pretreated with an LPA2 agonist, GRI-977143. The cell survival rate to 5-FU was markedly increased in DLD1 cells treated with GRI-977143. In the presence of GRI-977143, the elevated cell survival rate of DLD1 cells was reduced by LPA2 knockdown. To assess the effects of LPA2 and LPA5 on the enhancement of chemoresistance, long-term 5-FU treated (DLD-5FU) cells were generated from DLD1 cells. The cell survival rate to 5-FU of DLD-5FU cells were significantly elevated by LPA5 knockdown. GRI-977143 treatment increased the cell survival rate to 5-FU of DLD-5FU cells. These results suggest that LPA2 promotes and LPA5 suppresses the acquisition of chemoresistance in colon cancer cells treated with anticancer drugs.

Roles of endothelial cells in the regulation of cell motility via lysophosphatidic acid receptor-2 (LPA2) and LPA3 in osteosarcoma cells.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of biological responses. In tumor microenvironment, endothelial cells promote cancer cell functions. In this study, we investigated the roles of endothelial cells in the regulation of cell motile activity via LPA2 and LPA3 in human osteosarcoma MG-63 cells. In cell motility assay, the cell motile activity of MG-63 cells was markedly increased by the supernatants of endothelial F2 cells. MG-63 cell motility elevated by the supernatants was enhanced by GRI-977143 (LPA2 agonist) and reduced by (2S)-OMPT (LPA3 agonist). LPAR2 and LPAR3 expressions were increased in highly migratory MG63-CR7(F2) cells, which were generated from MG-63 cells by co-culture with F2 cell supernatants. MG63-CR7(F2) cell motility was stimulated by LPA treatment. In the presence of F2 cell supernatants, MG63-CR7(F2) cell motility was markedly enhanced by GRI-977143 and suppressed by (2S)-OMPT. Autotaxin (ATX) enzymatically converts lysophosphatidylcholine (LPC) to LPA. ATX expression was higher in MG63-CR(F2) cells than in MG-63 cells. MG63-CR7(F2) cell motility was markedly increased by LPC in comparison with MG-63 cells. In addition, MG63-CR(F2) cell motility was significantly stimulated by the supernatants of LPC treated F2 cells. The present results suggest that the activation of LPA signaling via LPA2 and LPA3 by endothelial cells is involved in the modulation of cell motile activity of MG-63 cells.

Cooperation of G12/13 and Gi proteins via lysophosphatidic acid receptor-2 (LPA2) signaling enhances cancer cell survival to cisplatin.

Lysophosphatidic acid (LPA) through six subtypes of G protein-coupled LPA receptors (LPA1 to LPA6) mediates a variety of cancer cell functions. The aim of this study was to evaluate the cooperative effects of G12/13 and Gi proteins through LPA2 on cancer cell survival to cisplatin (CDDP). In cell survival assay, cells were treated with CDDP every 24 h for 2 days. The long-term CDDP treated (HT-CDDP) cells established from fibrosarcoma HT1080 cells were pretreated with an LPA2 agonist, GRI-977143. The cell survival rate to CDDP of HT-CDDP cells was significantly increased by GRI-977143. The elevated cell survival to CDDP was suppressed by LPA2 knockdown. Since G12/13 protein stimulates Rho-mediated signaling, RhoA and RhoC knockdown cells were generated from HT1080 cells (HT1080-RhoA and HT1080-RhoC cells, respectively). In the presence of GRI-977143, HT1080-RhoA and HT1080-RhoC cells showed the low cell survival rates to CDDP. On the other hand, Gi protein inhibits adenylyl cyclase (AC) activity. Before cell survival assay, cells were treated with a Gi protein inhibitor, pertussis toxin (PTX) for 24 h. The cell survival rate to CDDP of HT1080 cells was significantly reduced by PTX. Furthermore, when HT1080-RhoA and HT1080-RhoC cells were pretreated with PTX, the cell survival rates to CDDP of both cells were markedly inhibited by PTX. The present results suggest that cooperation of G12/13 and Gi proteins activated by LPA2 enhances the cell survival of HT1080 cells treated with CDDP.

Lysophosphatidic acid receptor-2 (LPA2)-mediated signaling enhances chemoresistance in melanoma cells treated with anticancer drugs.

Lysophosphatidic acid (LPA) signaling through LPA receptors (LPA1 to LPA6) regulates a variety of malignant properties in cancer cells. Recently, we show that LPA2 expression is elevated by long-term cisplatin (CDDP) treatment in melanoma A375 cells. In the present study, we investigated whether LPA2-mediated signaling is involved in the modulation of chemoresistance in A375 cells. In cell survival assay, cells were treated with CDDP and dacarbazine (DTIC) every 24 h for 2 days. The cell survival rates to CDDP and DTIC were markedly increased by an LPA2 agonist, GRI-977143. To validate the effects of LPA2 on cell survival, LPA2 knockdown cells were generated from A375 cells. The cell survival rates elevated by GRI-977143 were suppressed by LPA2 knockdown. To evaluate the roles of LPA2-mediated signaling in cell survival, cells were pretreated with a Gi protein inhibitor, pertussis toxin (PTX). In the presence of GRI-977143, the cell survival rates to CDDP and DTIC were significantly lower in PTX-treated cells than in untreated cells. In addition, pretreatment of an adenylyl cyclase inhibitor, SQ22536, increased the cell survival of A375 cells treated with CDDP and DTIC. These results suggest that LPA2-mediated signaling plays an important role in the enhancement of chemoresistance of A375 cells treated with anticancer drugs.

Regulation of cell survival through free fatty acid receptor 1 (FFA1) and FFA4 induced by endothelial cells in osteosarcoma cells.

Free fatty acid receptor 1 (FFA1) and FFA4 belong to a family of free fatty acid (FFA) receptors. FFA1- and FFA4-mediated signaling regulates a variety of malignant properties in cancer cells. It is known that stromal cells in the tumor microenvironment promote tumor progression. In the present study, to assess the roles of FFA1 and FFA4 in cellular functions modulated by endothelial cells, highly migratory MG63-CR7(F2) cells were generated from osteosarcoma MG-63 cells, using endothelial F2 cell supernatants. Expression levels of FFAR1 and FFAR4 genes in MG63-CR7(F2) cells were significantly higher than those of MG-63 cells. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 2 days. The cell survival rate of MG-63 cells was significantly elevated by an FFA1 agonist TUG-770 as well as an FFA4 agonist TUG-891. Moreover, the cell survival rate of MG63-CR7(F2) cells was higher than that of MG-63 cells in the presence of TUG-770 or TUG-891, correlating with FFAR1 and FFAR4 expression levels. To validate the effects of FFA1 and FFA4 on cell survival to CDDP, FFA1 and FFA4 knockdown cell were generated from MG-63 cells. The cell survival rate of MG-63 cells was markedly inhibited by FFA1 or FFA4 knockdown. These results suggest that FFA1 and FFA4 may play an important role in the modulation of cellular functions by endothelial cells in osteosarcoma cells.

Effects of lysophosphatidic acid (LPA) receptor-2 (LPA2) and LPA3 on the regulation of chemoresistance to anticancer drug in lung cancer cells.

Lysophosphatidic acid (LPA) mediates a variety of biological functions via the binding of G protein-coupled LPA receptors (LPA receptor-1 (LPA1) to LPA6). This study aimed to investigate the roles of LPA2 and LPA3 in the modulation of chemoresistance to anticancer drug in lung cancer A549 cells. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 2 days. The cell survival rate to CDDP of A549 cells was significantly elevated by an LPA2 agonist, GRI-977143. To evaluate the roles of LPA2-mediated signaling in cell survival during tumor progression, highly migratory (A549-R10) cells were generated from A549 cells. In the presence of GRI-977143, the cell survival rate to CDDP of A549-R10 cells were markedly higher than that of A549 cells, correlating with LPAR2 expression level. Moreover, to assess the effects of long-term anticancer drug treatment on cell survival, the long-term CDDP treated (A549-CDDP) cells were established from A549 cells. The cell survival rate to CDDP of A549-CDDP cells was elevated by GRI-977143. Since LPAR3 expression level was significantly higher in A549-CDDP cells than in A549 cells, we investigated the roles of LPA3 in the cell survival to CDDP of A549 cells, using an LPA3 agonist, 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate ((2S)-OMPT). The cell survival rate to CDDP of A549 cells was significantly reduced by (2S)-OMPT treatment. In the presence of (2S)-OMPT, the cell survival rate to CDDP of A549 cells was elevated by LPA3 knockdown. These results suggest that LPA signaling via LPA2 and LPA3 is involved in the regulation of chemoresistance in A549 cells treated with CDDP.

LPA5-mediated signaling induced by endothelial cells and anticancer drug regulates cellular functions of osteosarcoma cells.

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.

Modulation of chemoresistance by lysophosphatidic acid (LPA) signaling through LPA5 in melanoma cells treated with anticancer drugs.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) contributes to the promotion of malignant potency in cancer cells. The cell motile activity are stimulated through the induction of LPA5 in melanoma cells treated with anticancer drugs. The present study aimed to investigate whether LPA signaling via LPA5 regulates chemoresistance in melanoma A375 cells. Cells were treated with cisplatin (CDDP) or dacarbazine (DTIC) every 24 h for 2 days. CDDP and DTIC treatment increased LPAR5 expressions. The cell survival rates of A375 cells treated with CDDP and DTIC were significantly decreased by LPA. In addition, LPAR5 expression was markedly elevated in long-term CDDP treated (A375-CDDP) cells. LPA decreased the cell survival rate of A375-CDDP cells treated with CDDP. To evaluate the roles of LPA5 in chemoresistance during tumor progression, highly migratory (A375-R11) cells were established from A375 cells. LPAR5 expression level was significantly lower in A375-R11 cells than in A375 cells. The cell survival rates of A375-R11 cells treated with CDDP and DTIC were increased, compared with A375 cells. Moreover, we generated LPA5 knockdown cells from A375 cells. The cell survival rates of A375 cells treated with CDDP and DTIC were significantly elevated by LPA5 knockdown. These results suggest that LPA signaling via LPA5 is involved in the modulation of chemoresistance in melanoma A375 cells.