RESUMO
G protein coupled free fatty acid receptors (FFARs) are involved in the pathogenesis of several human diseases. FFAR2 and FFAR3 are activated by the binding of short-chain fatty acids (SCFAs). This study aimed to evaluate the roles of FFAR2 in the regulation of cellular functions in osteosarcoma HOS cells, using acetic acid and propanoic acid as FFAR2 and FFAR3 agonists. FFAR2 and FFAR3 genes were expressed in HOS cells. The cell motile activity of HOS cells was significantly stimulated by acetic acid and propanoic acid. In contrast, acetic acid and propanoic acid had no impact on the activation of matrix metalloproteinase-2 (MMP-2) and MMP-9. In cell survival assay, the cell survival rate to cisplatin (CDDP) of HOS cells was elevated by acetic acid and propanoic acid. To assess the effects of FFAR2 on cellular functions, FFAR2 knockdown (HOS-FFAR2) cells were generated from HOS cells. The cell motile activity of HOS-FFAR2 cells was enhanced by acetic acid and propanoic acid. In the presence of acetic acid and propanoic acid, MMP-2 and MMP-9 activities were reduced in HOS-FFAR2 cells, compared with control cells. When cells were treated with acetic acid and propanoic acid, the cell survival rate to CDDP of HOS-FFAR2 cells was significantly lower than that of control cells. These results suggest that activation of FFAR2-mediated signaling is involved in the modulation of cellular functions in HOS cells.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Humanos , Propionatos/farmacologia , Metaloproteinase 2 da Matriz/genética , Receptores Acoplados a Proteínas G/metabolismo , Ácidos Graxos não Esterificados , Metaloproteinase 9 da Matriz/genética , Ácidos Graxos Voláteis/farmacologia , Osteossarcoma/genética , Cisplatino/farmacologia , Ácido Acético , Neoplasias Ósseas/genéticaRESUMO
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.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Trifosfato de Adenosina/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Movimento Celular , Etídio/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Lisofosfolipídeos/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismoRESUMO
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.
Assuntos
Neoplasias Ósseas/patologia , Movimento Celular , Células Endoteliais/patologia , Regulação Neoplásica da Expressão Gênica , Osteossarcoma/patologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Apoptose , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Proliferação de Células , Células Endoteliais/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Transdução de Sinais , Células Tumorais CultivadasRESUMO
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.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Fibrossarcoma/tratamento farmacológico , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/genética , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína de Ligação a GTP rhoC/metabolismoRESUMO
PURPOSE: Lysophosphatidic acid (LPA) receptor-mediated signaling regulates various biological functions in cancer cells. This study aimed to evaluate the roles of LPA receptor-2 (LPA2) in cellular responses induced by X-ray irradiation in pancreatic cancer PANC-1 cells. Since X-ray irradiation generates reactive oxygen species (ROS), PANC-1 cells were treated with hydrogen peroxide (H2O2). H2O2 is a key member of ROS. METHODS: To investigate the cell survival rate to X-ray irradiation, PANC-1 cells were irradiated with X-rays (2.5-15 Gy). LPAR2 expression levels were measured by quantitative real-time RT-PCR analysis. The effects of LPA2 on the cell survival and motility were evaluated using LPA2 knockdown cells. To establish H2O2 treated cells, PANC-1 cells were cultured in 10% FBS-DMEM with H2O2 (30 µM) for 2 weeks. The cell motility and survival rate to cisplatin (CDDP) of H2O2 treated cells were examined. RESULTS: LPAR2 expression was significantly increased in PANC-1 cells irradiated with X-rays. PANC-1 cell motility was markedly decreased by X-ray irradiation. The reduced cell motility activity by X-ray irradiation was enhanced by LPA2 knockdown. The cell survival to X-ray irradiation was elevated in PANC-1 cells treated with GRI-977143 (LPA2 agonist) and suppressed by LPA2 knockdown. On the other hand, LPAR2 expression was markedly higher in H2O2 treated cells than in H2O2 untreated cells. H2O2 treated cells showed the high cell survival to CDDP in comparison with H2O2 untreated cells. GRI-977143 increased the cell survival to CDDP of H2O2 treated cells, while LPA2 knockdown suppressed. CONCLUSIONS: The present results suggest that the activation of LPA2-mediated signaling plays an important role in the modulation of cellular functions induced by X-ray irradiation and H2O2 in PANC-1 cells.