Misaho Kitayoshi

Opposite roles of LPA1 and LPA3 on cell motile and invasive activities of pancreatic cancer cells

Lysophosphatidic acid (LPA) interacts with at least six G protein-coupled transmembrane LPA receptors. Recently, it has been demonstrated that each LPA receptor acts as a positive or negative regulator of cellular function. In the present study, to assess a biological role of LPA receptors on cell migration of pancreatic cancer cells, we generated LPA receptor-1 (LPA1) and LPA3 knockdown cells from hamster pancreatic cancer cells by transfection with short hairpin RNA plasmids and measured their cell motile and invasive abilities. In cell motility and invasion assay, a Cell Culture Insert, coated with or without a Matrigel, was used. While the cell motility and invasion of Lpar1 knockdown cells were markedly enhanced than those of control cells, Lpar3 knockdown cells showed significantly lower cell motility and invasion. Moreover, to investigate an involvement of LPA1 and LPA3 in the development of pancreatic cancers, we also measured the expression levels of Lpar1 and Lpar3 genes in hamster pancreatic duct adenocarcinomas (PDAs) induced by a nitroso compound. The expressions of Lpar1 gene in PDAs were significantly lower than those in normal pancreatic tissues. By contrast, the elevated expressions of Lpar3 gene were detected in PDAs. We thus demonstrate that LPA1 and LPA3 play the different roles on cell migration ability of pancreatic cancer cells, suggesting the opposite effects via LPA1 and LPA3 may contribute to the pathogenesis of pancreatic cancers in hamsters.

Constitutively active lysophosphatidic acid receptor-1 enhances the induction of matrix metalloproteinase-2

Lysophosphatidic acid (LPA) is a simple phospholipid which interacts with at least six G protein-coupled transmembrane LPA receptors (LPA(1)-LPA(6)). In rat neuroblastoma B103 cells, we have recently reported that each LPA receptor indicates the different cellular functions, including cell motility, invasion and tumorigenicity. Especially, mutated and constitutively active LPA(1) enhanced these cellular effects in B103 cells. In the present study, to better understand a role of mutated LPA(1) underlying progression of cancer cells, we measured the expression and activity levels of matrix metalloproteinases (MMPs) in constitutively active mutant Lpar1-expressing B103 cells (lpa1Δ-1), compared with each wild-type LPA receptor-expressing cells. LPA receptor-unexpressing cells were also used as control. In quantitative real time RT-PCR analysis, the expressions of Mmp-9 were detected at the same levels in all cells. By contrast, Mmp-2 expressions of lpa1Δ-1 were significantly higher than those of other cells. In gelatin zymography, proMmp-9 was observed at the same levels in all cells. Interestingly, markedly high levels of proMmp-2 and Mmp-2 were detected in lpa1Δ-1 cells, whereas no activation was in other cells. The increased expression and activity of Mmp-2 in lpa1Δ-1 cells were suppressed by the pretreatment with a Gq protein inhibitor. These results suggest that mutated LPA(1) may involve in the enhancement of Mmp-2 expression and activation in rat neuroblastoma cells.

AKT activation and telomerase reverse transcriptase expression are concurrently associated with prognosis of gastric cancer.

AKT is a protein in the phosphatidylinositol-3 kinase (PI3K) pathway and associated with diverse pro-tumoral responses. Activation of the human telomere reverse transcriptase (hTERT) is one of AKTs tumorigenic effects. In this study, the significance of AKT phosphorylation and hTERT on prognosis of gastric cancer were examined. AKT activation by epidermal growth factor increased hTERT expression and telomerase activity. In contrast, AKT inactivation by inhibitors and knockdown decreased hTERT expression and telomerase activity in MKN28 gastric cancer cells. In 40 gastric cancer tissues, significant correlations were found among the levels of phosphorylated AKT (pAKT), hTERT expression, and telomer length. The pAKT levels or the levels of pAKT/hTERT were not associated with clinicopathological parameters, including stage and nodal metastasis. However, survival rates of the pAKT-high patients or the pAKT-high and hTERT-high patients were significantly poorer than those in other patients. These findings suggest that AKT and hTERT are good molecular targets for the treatment of gastric cancer.

Hydrogen peroxide stimulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells

Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1 μM for 48 h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA3 on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA3 may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide.

Regulation of cell motile activity through the different induction of LPA receptors by estrogens in liver epithelial WB-F344 cells.

Lysophosphatidic acid (LPA) interacts with G protein-coupled transmembrane LPA receptors (LPA receptors; LPA(1)-LPA(6)). Recently, we demonstrated that each LPA receptor acts as a positive or negative regulator of cell migration ability. It is known that estrogens indicate a variety of biological functions, including cell motility. In the present study, to assess whether LPA signaling is involved in cell motile activity stimulated by estrogens, we measured cell motile activity and LPA receptor expressions of rat liver epithelial WB-F344 cells treated with 17β-estradiol (E(2)), ethinyl estradiol (EE) and diethylstilbestrol (DES) at concentrations of 0.1 and 1.0 μM for 48 h. The cell motility of E(2) and EE treated cells was significantly higher than that of untreated cells. By contrast, DES markedly inhibited cell motile activity. Using quantitative real time RT-PCR analysis, Lpar1 and Lpar3 expressions in E(2) treated cells were significantly higher than those in untreated cells. In EE treated cells, Lpar3 expression was markedly elevated, whereas Lpar1 expression was decreased. On the other hand, Lpar1 expression was significantly increased in DES treated cells. Interestingly, the effects of E(2), EE and DES on cell motility were suppressed by Lpar1 or Lpar3 knockdown. These results suggest that the different induction of LPA receptors by estrogens may regulate cell motile activity of WB-F344 cells.

Role of two types of angiotensin II receptors in colorectal carcinoma progression.

Angiotensin II (Ang-II) is a bioactive peptide associated closely with the progression and metastasis of colorectal cancer (CRC). We examined the expression and role of 2 Ang-II receptor types in 20 cases of CRC. Ang-II type 1 receptor (AT1R) protein was localized to the plasma membrane, whereas Ang-II type 2 receptor (AT2R) protein was localized to the nuclei. AT1R expression showed a direct correlation with tumor stage and liver metastasis, whereas AT2R expression showed an inverse correlation. A knockdown study of the AT1R or AT2R with Ang-II treatment was performed to reveal their individual roles in a mouse rectal cell line CMT93, which expresses both Ang-II receptor types. AT2R knockdown showed that the AT1R was associated with tumor growth, survival, invasion and VEGF-A secretion in CMT93 cells in a dose-dependent manner. In contrast, AT1R knockdown showed that the AT2R was associated with increased VEGF-A secretion at low Ang-II concentrations, whereas high concentrations of Ang-II inhibited tumor growth, survival, invasion and VEGF-A secretion. Thus, the AT1R showed a monophasic protumoral effect, while the AT2R showed a biphasic amphitumoral effect. Our findings suggest that a high angiotensinogen condition in the liver might evoke the antitumoral role of the AT2R in CRC cells.

Negative regulation of cell motile and invasive activities by lysophosphatidic acid receptor-3 in colon cancer HCT116 cells

Lysophosphatidic acid (LPA) mediates a wide range of biological responses with G protein-coupled transmembrane receptors (LPA receptors). So far, at least six types of LPA receptors (LPA receptor-1 (LPA1) to LPA6) have been identified. Recently, it has been reported that LPA3 indicates opposite effects on cellular functions of cancer cells. In the present study, to assess a biological role of LPA3 on cell migration ability of colon cancer cells, we generated LPA receptor-3 (LPAR3) knockdown (HCT-sh3-3) cells from HCT116 and measured cell motile and invasion activities. In motility assay with a cell culture insert, HCT-sh3-3 cells showed significantly high cell motile activity, compared with control cells. For invasion assay, the filter was coated with Matrigel. The invasive activity of HCT-sh3-3 cells was significantly higher than that of control cells. Furthermore, we also examined the effects of LPAR3 knockdown on the interaction between colon cancer cells and endothelial F-2 cells. When F-2 cells were cultured with serum-free DMEM containing a supernatant from HCT-sh3-3 cells, the cell growth rate and migration activity of F-2 cells were significantly stimulated, associating with the elevated expressions of vascular endothelial growth factor (VEGF)-A and VEGF-C genes in HCT-sh3-3 cells. These results suggest that LPA3 may act as a negative regulator on cell motile and invasive abilities of colon cancer HCT116 cells.

Loss of lysophosphatidic acid receptor-3 suppresses cell migration activity of human sarcoma cells

Lysophosphatidic acid (LPA) interacts with at least six G protein-coupled transmembrane LPA receptors (LPA1-LPA6). Recently, we have reported that LPA3 indicated opposite effects on cell migration, depending on the cell types. In the present study, to assess an involvement of LPA3 on cell migration of sarcoma cells, we generated LPA receptor-3 (LPAR3)-knockdown (HT1080-sh3 and HOS-sh3, respectively) cells from fibrosarcoma HT1080 and osteosarcoma HOS cells, and measured their cell migration abilities. In cell motility assay with a Cell Culture Insert, both LPAR3-knockdown cells showed significantly lower cell motile activities than control cells. Next, to investigate the effect of LPAR3-knockdown on invasion activity, which degraded the extracellular matrices, the Matrigel-coated filter was used. HT1080-sh3 cells showed significantly low invasive activity compared with control cells, while no invasive activity was found in HOS-sh3 cells. In gelatin zymography, no significant difference of matrix metalloproteinase (MMP)-2 and MMP-9 activities were detected in all cells. The results indicated that LPA3 acts as a positive regulator of cell motility and invasion in sarcoma cells, suggesting that LPA signaling pathway via LPA3 may be involved in the progression of sarcoma cells.

Enhancement of endothelial cell migration by constitutively active LPA(1)-expressing tumor cells.

Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA(1) to LPA(6)). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA(1) induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA(1) on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA(1) than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA(1). These results suggest that activation of LPA signaling via mutated LPA(1) may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

Different effects on cell proliferation and migration abilities of endothelial cells by LPA1 and LPA3 in mammary tumor FM3A cells

Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors and mediate a variety of cellular responses through the binding of LPA. So far, six types of LPA receptors (LPA receptor-1 (LPA1) to LPA6) have been identified. Recently, it has been demonstrated that each LPA receptor has opposite effects on malignant property of cancer cells. In this study, to evaluate an involvement of LPA receptors on angiogenic process in mammary tumor cells, we generated Lpar1- and Lpar3-expressing (FM3A-a1 and FM3A-a3A9, respectively) cells from FM3A cells, and investigated the effects on cell proliferation and migration abilities of endothelial F-2 cells by those cells. In Vegf-A and Vegf-C genes, FM3A-a1 cells indicated high expression and FM3A-a3A9 cells showed low expression, compared with control cells. When F-2 cells were cultured with a supernatant from FM3A-a1 cells, the cell growth rate and migration ability of F-2 cells was significantly higher than control cells. By contrast, a supernatant from FM3A-a3A9 cells significantly inhibited those abilities of F-2 cells. These results suggest that LPA1 and LPA3 may play opposite roles on the regulation of endothelial cells in mouse mammary tumor FM3A cells.