Expression of MAS1 in breast cancer.

MAS1 is a receptor for angiotensin 1-7 (A1-7), which is derived from angiotensin II (A-II) by the action of angiotensin converting enzyme (ACE) 2. MAS1 induces anti-A-II phenotypes, such as vessel dilation and depression of blood pressure. Using immunohistochemistry, we examined the role of MAS1 in 132 cases of invasive ductal carcinoma (IDC) of the breast. While benign mammary tissues expressed MAS1 at high levels, MAS1 expression was attenuated in all IDC, especially in scirrhous IDC. The decrease in MAS1 expression was associated with tumor growth, lymph node metastasis, and grade. MAS1 expression was inversely associated with the proliferation index and epidermal growth factor receptor and human epidermal growth factor receptor-2 expression. Of the 132 cases, 12 (9.1%) were triple-negative breast cancer (TNBC) cases. All TNBC cases (the 12 cases and the additional 36 cases using a tissue array) expressed MAS1. Using the TNBC cell lines 4T1 and MDA-MB-468, which expresses MAS1, we found that cell growth, anti-apoptotic survival and invasion were suppressed by MAS1 activation with A1-7 treatment and enhanced by MAS1 knockdown. In contrast, synergic effect was found between tamoxifen and A1-7 in a luminal A breast cancer cell line, MCF-7. Combination treatment with cisplatin, an ACE2 activator, and an A-II type 1 receptor blocker showed synergic effects on tumor growth inhibition of 4T1 tumors in a syngeneic mouse model. These findings suggest that MAS1 might act as an inhibitory regulator of breast cancer and may be a possible molecular target for this malignancy.

Inhibitory effects of lysophosphatidic acid receptor-5 on cellular functions of sarcoma cells.

Lysophosphatidic acid (LPA) is a bioactive lipid that interacts with G protein-coupled LPA receptors (LPA receptor-1 (LPA1) to LPA6). Here, we investigated the effects of LPA signaling via LPA5 on cellular functions of sarcoma cells by generating Lpar5 overexpressing and Lpar5 knockdown cells from rat osteosarcoma and malignant fibrous histiocytoma cells, respectively. The cell motility activity of Lpar5 overexpressing cells was significantly lower, while Lpar5 knockdown cells showed high cell motility, compared with respective controls. Gelatin zymography showed that LPA5 suppressed the activation of matrix metalloproteinase-2. LPA5 also inhibited the cell motility activity of endothelial cells, correlating with the expression levels of vascular endothelial growth factor genes. These results suggest that LPA signaling via LPA5 negatively regulates the cellular functions of rat sarcoma cells.

Serum CD10 is associated with liver metastasis in colorectal cancer.

INTRODUCTION: Colorectal cancer (CRC) is the third leading cause of cancer death in Japan. CD10 expression is closely associated with liver metastasis. In the present study, we explored the possibility of serum CD10 as a marker of liver metastasis in CRC. METHODOLOGY: BALB/c mouse with subcutaneous tumor of syngeneic CT26 CRC cells were examined serum CD10. In 84 CRC patients and patients undergoing hemodialysis, serum CD10 was examined. CD10 concentration was measured by enzyme-linked immunosorbent assay. RESULTS: In a mouse subcutaneous tumor model, serum CD10 correlated with the weight of the tumors. Serum CD10 was examined in 84 patients with CRC. The serum levels of CD10 were higher in patients with more advanced cancer stages. Patients with liver metastasis showed the highest levels of serum CD10 among all patients. Importantly, patients with high serum CD10 levels had metachronous liver metastasis. Healthy volunteers showed low levels of CD10; however, serum CD10 levels in patients undergoing hemodialysis showed levels as high as those with stage II and III CRC. A cutoff of serum CD10 set to >1000 pg/mL showed 70% sensitivity and 93% specificity for liver metastasis in CRC. This cutoff included all cases of metachronous liver metastasis. CONCLUSIONS: With the exclusion of mimicking factors, serum CD10 levels might serve as a useful marker of synchronous and metachronous liver metastasis in CRC.

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 AKT's 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.

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.

Involvement of oncogenic K-ras on cell migration stimulated by lysophosphatidic acid receptor-2 in pancreatic cancer cells.

Lysophosphatidic acid (LPA) mediates a variety of cellular responses with atleast six G protein-coupled transmembrane receptors (LPA receptor-1 (LPA(1)-LPA(6))). The interaction between LPA receptors and other cellular molecules on the biological function is not fully understood. Recently, we have reported that LPA(1) suppressed and LPA(3) stimulated cell migration of pancreatic cancer cells. In the present study, to evaluate the function of LPA(2) on motile and invasive activities of pancreatic cancer cells, we generated Lpar2 knockdown (HPD-sh2) cells from hamster pancreatic cancer cells and measured their cell migration ability. In cell motility and invasive assays with an uncoated Cell Culture Insert, HPD-sh2 cells showed significantly lower intrinsic activity than control (HPD-GFP) cells. Since K-ras mutations were frequently detected in pancreatic cancer, we next investigated whether oncogenic K-ras is involved in cell migration induced by LPA(2) using K-ras knockdown (HPD-K2) cells. The cell motile ability of HPD-K2 cells was significantly lower than that of control cells. To confirm LPA(2) increases cell migration activity, cells were pretreated with dioctylglycerol pyrophosphate (DGPP) which is the antagonist of LPA(1)/LPA(3). The cell motile and invasive abilities of DGPP -treated HPD-GFP cells were markedly higher than those of untreated cells, but DGPP did not stimulate cell migration of HPD-K2 cells. These results suggest that cell migration activity of pancreatic cancer cells stimulated by LPA(2) may be enhanced by oncogenic K-ras.

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.

Downregulation of activation factors of endothelia and fibroblasts via lysophosphatidic acid signaling in a mouse lung cancer LL/2 cell line.

Angiogenesis stimulates the invasive and metastatic process of cancer cells. It is also known that activated fibroblasts promote cancer cell growth and enhance invasive and metastatic potential. Lysophosphatidic acid (LPA) is a biological mediator and interacts with G protein-coupled transmembrane LPA receptors (LPA1 to LPA6). In this study, to assess an involvement of LPA3 on angiogenesis and fibroblast activation, the Lpar3-expressing cells were generated from mouse lung cancer LL/2 cells, which unexpressed LPA3. The Lpar3-expressing cells were maintained in serum-free Dulbecco's modified Eagle's medium for 48 h, and cell motility assay was performed with a cell culture Insert. When endothelial F-2 cells and 3T3 fibroblasts were cultured with conditioned medium from the Lpar3-expressing cells, their cell motile activities were significantly lower than the Lpar3-unexpressing (control) cells. Expression levels of vascular endothelial growth factor (Vegf) and fibroblast growth factor (Fgf) genes in the Lpar3-expressing cells were measured by quantitative real time reverse transcription polymerase chain reaction analysis. The expressions of Vegf-A. Fgfa and Fgfb genes in the Lpar3-expressing cells were significantly lower than those in control cells, correlating with the effects on cell motile activities of F-2 and 3T3 cells. These results suggest that LPA signaling through LPA3 may inhibit angiogenesis and fibroblast activation in mouse lung cancer cells.

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 17ß-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.

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.

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.

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 (LPA(1)-LPA(6)). Recently, we have reported that LPA(3) indicated opposite effects on cell migration, depending on the cell types. In the present study, to assess an involvement of LPA(3) 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 LPA(3) acts as a positive regulator of cell motility and invasion in sarcoma cells, suggesting that LPA signaling pathway via LPA(3) may be involved in the progression of sarcoma 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.

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 (LPA(1)) and LPA(3) 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 LPA(1) and LPA(3) 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 LPA(1) and LPA(3) play the different roles on cell migration ability of pancreatic cancer cells, suggesting the opposite effects via LPA(1) and LPA(3) may contribute to the pathogenesis of pancreatic cancers in hamsters.

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 (LPA(1)) to LPA(6)) have been identified. Recently, it has been reported that LPA(3) indicates opposite effects on cellular functions of cancer cells. In the present study, to assess a biological role of LPA(3) 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 LPA(3) may act as a negative regulator on cell motile and invasive abilities of colon cancer HCT116 cells.

Fatty acids inhibit anticancer effects of 5-fluorouracil in mouse cancer cell lines.

The present study investigated the effects of two major dietary fatty acid components, linoleic acid (LA) and elaidic acid (EA), on the antitumor effects of 5-fluorouracil (5-FU) in the LL2, CT26 and CMT93 mouse cancer cell lines. Concurrent treatment with LA and 5-FU elicited a decreased cell viability compared with treatment with 5-FU alone. In addition, increased inhibition of growth was observed following concurrent treatment with EA and 5-FU. Sequential treatment of LA followed by 5-FU abrogated the anticancer effects of 5-FU, and treatment with EA followed by 5-FU increased cancer cell growth in addition to abrogating the anticancer effects of 5-FU. The expression of the stem cell markers CD133 and nucleostemin (NS) increased in all three cell lines treated concurrently with 5-FU and either LA or EA when compared with cells treated with 5-FU alone. Aldehyde dehydrogenase activity in the cancer stem cells (CSCs), in response to concurrent treatment with 5-FU and either LA or EA, was increased compared with 5-FU treatment alone. 5-FU inhibited the growth of CT26 tumors, but co-treatment with either LA or EA abrogated this effect. NS-positive CSCs were more abundant in CT26 tumors treated with 5-FU and either LA or EA compared with those treated with 5-FU alone. The results of the present study suggested that, rather than altering the sensitivity of cancer cells to 5-FU, LA and EA may promote the survival of CSCs. The results indicated that dietary composition during chemotherapy is an important issue.

Increased phosphorylation of AKT in high-risk gastric mucosa.

AIM: To establish the role of oxidative stress and v-akt murine thymoma viral oncogene homolog (AKT) activation in gastric cancer development, we examined the levels of phosphorylated AKT (pAKT), inducible nitric oxide synthase (iNOS), nitrotyrosine (NT), and human telomerase reverse transcriptase (hTERT) by enzyme-linked immunosorbent assay in 73 non-cancerous gastric mucosa and 10 gastric carcinomas. We found that the levels of pAKT were associated with the levels of iNOS, NT, and hTERT. Gastric mucosa was classified into four categories: chronic gastritis without Helicobacter pylori (CG), chronic active gastritis with H. pylori (CAG), chronic metaplastic gastritis without H. pylori (CMG), and chronic gastritis with atypia without H. pylori (CGA). We found increasing levels of pAKT, iNOS, and NT in the order of CG, CAG, CMG, and CGA. hTERT was detected only in CGA. These findings suggest that oxidative stress might be associated with AKT activation and hTERT induction and that mucosa in CGA might confer a high-risk status for gastric carcinogenesis.

Enhancement of Drug Resistance by Lysophosphatidic Acid Receptor-3 in Mouse Mammary Tumor FM3A Cells.

Lysophosphatidic acid (LPA) acts as a simple phospholipid that interacts with G protein-coupled transmembrane LPA receptors. Recently, it has been reported that each LPA receptor plays different biological roles in acquisition of the malignant property of tumor cells. In this study, to assess the involvement of LPA receptor-3 (LPA(3)) in cell survival after treatment with anticancer drugs, we generated Lpar3-expressing FM3A-a3A9 cells from mouse mammary tumor FM3A cells and examined the cell survival rate after treatment with anticancer drugs compared with Lpar3-unexpressing cells. Cells were treated with 0.005 to 10 µM of cisplatin (CDDP) or doxorubicin (DOX) for 3 days. For the CDDP and DOX treatments, the cell survival rate of FM3A-a3A9 cells was significantly higher than that of Lpar3-unexpressing cells. The expression level of the Mdr1a gene in FM3A-a3A9 cells was higher than that of Lpar3-unexpressing cells, whereas no significant difference in multidrug resistance 1b (Mdr1b) and glutathione S-transferase mu1 (Gstm1) expressions was found. These results suggest that LPA(3) may enhance the cell survival rate after treatment with anticancer drugs in mouse mammary tumor cells, correlating with increased expression of the Mdr1 gene.