Regulation of cellular responses to X-ray irradiation through the activation of lysophosphatidic acid (LPA) receptor-3 (LPA3) and LPA2 in osteosarcoma cells.

Lysophosphatidic acid (LPA) binds to its specific G protein-coupled LPA receptors (LPA1 to LPA6), resulting in the activation of various cellular functions. LPA receptor-mediated signaling facilitates tumor progression in human malignancies. In the present study, we investigated whether LPA receptor-mediated signaling contributes to cellular responses to X-ray irradiation in osteosarcoma MG-63 cells. After X-ray irradiation (2, 4 and 8 Gy), LPAR2 and LPAR3 expression levels in MG-63 cells were significantly elevated in a dose-dependent manner, but no change of LPAR1 expression level was observed. The cell growth activities of MG-63 cells irradiated with X-rays (2, 4 and 8 Gy) were reduced by LPA. Conversely, LPA3 agonist (2 S)-OMPT enhanced the cell growth activities of X-ray irradiated MG-63 cells. The cell movement of MG-63 cells exposed to X-ray irradiation (8 Gy) was inhibited by (2 S)OMPT. In cell survival assay, (2 S)-OMPT suppressed the cell survival to cisplatin (CDDP) of MG-63 cells irradiated with X-rays (8 Gy). The cell survival to CDDP of X-ray irradiated cells was elevated by LPA3 knockdown. Moreover, we evaluated the effects of LPA2 on the cell survival to CDDP of MG-63 cells exposed to X-ray irradiation (8 Gy). The cell survival to CDDP of X-ray irradiated cells was increased by LPA2 agonist GRI-977143 and reduced by LPA2 knockdown. These results suggest that LPA receptor-signaling participates in the modulation of cellular functions induced by X-ray irradiation in osteosarcoma cells.

Roles of lysophosphatidic acid (LPA) receptor-mediated signaling in cellular functions modulated by endothelial cells in pancreatic cancer cells under hypoxic conditions.

BACKGROUND: In the tumor environment, malignant characteristics of cancer cells are promoted by stromal cells under hypoxia. It is unknown whether lysophosphatidic acid (LPA) receptor-mediated signaling is involved in the regulation of cellular functions by endothelial cells in pancreatic cancer cells under hypoxic conditions. METHODS: Pancreatic cancer (PANC-1) cells were co-cultured with endothelial (F2) cells and F2 cell supernatants at 21% and 1% O2. The Cell Culture Insert was used to assess the cell motile activity. The cell growth and viability to cisplatin (CDDP) were measured, using the Cell Counting Kit-8. RESULTS: LPA receptor expression levels were changed in PANC-1 cells co-cultured with F2 cells at 21% and 1% O2. The cell motile activities of PANC-1 cells co-cultured with F2 cells at 21% and 1% O2 were markedly elevated, compared with PANC-1 cells alone. The cell viabilities to CDDP of PANC-1 cells co-cultured with F2 cell supernatants at 21% and 1% O2 were regulated by the activation of LPA receptors. CONCLUSION: These results suggest that LPA receptor-mediated signaling plays an important role in the modulation of pancreatic cancer cell functions by endothelial cells under hypoxic conditions.

Effects of free fatty acid receptor (FFAR) signaling on the modulation of cancer cell functions under hypoxic conditions.

In the tumor environment, hypoxia promotes tumor progression, such as cancer cell growth, migration and chemoresistance. This study aimed to evaluate the roles of free fatty acid receptors (FFARs) in the regulation of cancer cell functions under hypoxic conditions, using fibrosarcoma HT1080 cells. HT1080 cells expressed FFAR1, FFAR2 and FFAR3 genes, but not FFAR4 gene. FFAR1, FFAR2 and FFAR3 expression levels in HT1080 cells cultured at 1 % O2 were elevated, compared with 21 % O2. The cell growth activities of HT1080 cells cultured at 21 % O2 were inhibited by acetic acid (AA) and propanoic acid (PA), but not 1 % O2. HT1080 cell motility was markedly reduced by culturing at 1 % O2. The cell growth and motility of HT1080 cells were enhanced by FFAR2 knockdown. The cell viability to cisplatin (CDDP) of HT1080 cells cultured at 1 % O2 was increased, compared with 21 % O2. FFAR2 knockdown suppressed the cell viability to CDDP of HT1080 cells. On the other hand, the cell motility and viability to CDDP of HT1080 cells cultured at 21 % O2 were suppressed by TUG-770. When HT1080 cells were cultured at 1 % O2, the cell motility and viability to CDDP were decreased, correlating with FFAR1 expression level. Moreover, FFAR1 knockdown increased the cell viability to CDDP of HT1080 cells cultured at 1 % O2. These results suggest that FFAR-mediated signaling plays an important role in the modulation of cellular functions of HT1080 cells under hypoxic conditions.

Induction of lysophosphatidic acid (LPA) receptor-mediated signaling regulates cell motility and survival to anticancer drugs in cancer cells treated with hydrogen peroxide.

Hydrogen peroxide (H2O2) is one of reactive oxygen species (ROS) and promotes malignant properties of cancer cells. Lysophosphatidic acid (LPA) signaling via LPA receptor (LPA1 to LPA6) regulates a variety of cellular functions, such as cell growth, migration and differentiation. This study aimed to evaluate the effects of LPA receptors on the cell motility and survival to anticancer drugs by H2O2 in colon cancer DLD-1 cells. To obtain H2O2 treated (DLD- H2O2) cells, cells were maintained in culture medium containing H2O2 (60 µM) for 2 months. LPAR2 and LPAR4 gene expressions were markedly elevated in DLD-H2O2 cells. The cell motility of DLD-H2O2 cells was significantly lower than that of DLD-1 cells. DLD-H2O2 cell motility was suppressed by LPA2 knockdown and stimulated by LPA4 knockdown. The cell survival rates to fluorouracil (5-FU), irinotecan (CPT-11) and oxaliplatin (L-OHP) of DLD-H2O2 cells were significantly higher than those of DLD-1 cells. The cell survival rate to 5-FU of DLD-H2O2 cells was decreased by LPA2 knockdown. Conversely, LPA4 knockdown enhanced the cell survival rate to 5-FU of DLD-H2O2 cells. In the tumor microenvironment, high levels of H2O2 production are observed under hypoxic conditions. The cell survival rate to 5-FU of DLD-H2O2 cells cultured at 1% O2 was significantly higher than that of DLD-1 cells cultured at 1% O2, correlating with LPAR2 gene expression. The present results suggest that the induction of LPA receptor-mediated signaling plays an important role in regulating cellular functions of DLD-1 cells treated with H2O2.

Effects of LPA receptor-mediated signaling on the modulation of cellular functions of pancreatic cancer cells cultured in fibroblast supernatants under hypoxic conditions.

The tumor microenvironment (TME) consists of various cell types, including fibroblasts. The TME plays a central role in the promotion of tumor progression. In the present study, we investigated whether lysophosphatidic acid (LPA) receptor-mediated signaling regulates cellular functions by the TME of pancreatic cancer PANC-1 cells. To obtain fibroblast 3T3 cell supernatants, 3T3 cells were cultured in 5% charcoal stripped FCS-DMEM for 48 h. LPAR2 and LPAR3 expression levels were elevated in PANC-1 cells cultured in 3T3 cell supernatants. While PANC-1 cell motility was decreased by 3T3 cell supernatants, the cell survival to cisplatin (CDDP) of PANC-1 cells was markedly enhanced. Moreover, the cell survival to CDDP of PANC-1 cells cultured in 3T3 cell supernatants was increased by GRI-977,143 (LPA2 agonist) and (2 S)-OMPT (LPA3 agonist). Since hypoxia is caused by the restriction of adequate vascular networks to deliver oxygen into solid tumors, PANC-1 cells were cultured in 3T3 cell supernatants at 1% O2 conditions. The cell survival to CDDP of PANC-1 cells cultured in 3T3 cell supernatants at 1% O2 was significantly elevated, correlating with LPAR2 and LPAR3 expressions. These results suggest that LPA signaling via LPA2 and LPA3 is involved in the promotion of malignant properties by the TME in PANC-1 cells.

Structure-Activity relationships of 2-substituted 5,7-Diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids as a novel class of endothelin receptor antagonists.

Synthesis and structure-activity relationships of 2-substituted-5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids, a novel class of endothelin receptor antagonists, were described. Derivatization of a lead structure 1 (IC(50)=2.4nM, 170-fold selectivity) by incorporating a substituent such as an alkyl, alkoxy, alkylthio, or alkylamino group into the 2-position of the cyclopenteno[1,2-b]pyridine skeleton was achieved via the key intermediate 8. Introduction of an alkyl group led to the identification of potent ET(A)/ET(B) mixed receptor antagonists, a butyl (2d: IC(50)=0.21nM, 52-fold selectivity) and an isobutyl (2f: IC(50)=0.32nM, 26-fold selectivity) analogue. In contrast, installment of a primary amino group resulted in ET(A) selective antagonists, a propylamino 2p (IC(50)=0.12nM, 520-fold selectivity) and an isopropylamino 2q (IC(50)=0.10nM, 420-fold selectivity) analogue. These results suggested that a substituent at the 2-position of the 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids played a key role in the binding affinity for both ET(A) and ET(B) receptors.