Endoglin Expression Research Articles

227: The Role of lactate and oxidative stress in hypoxia enhanced soluble endoglin expression in human umbilical vein endothelial cells

Preeclampsia, a hypertensive disorder of pregnancy, is thought to be caused by hypoxia. Lactate has been shown to be increased in hypoxic conditions. Certain antiangiogenic markers such as soluble endoglin (sEng) are elevated in response to hypoxia in microvascular endothelial cells but not in villous trophoblast cells. Proposed mechanisms of hypoxia mediated sEng increase involve both lactate and oxidative stress. We hypothesized that hypoxia generated oxidative stress increases lactate leading to increase in sEng expression.

Expression and functional analysis of endoglin in isolated liver cells and its involvement in fibrogenic Smad signalling

Endoglin is an accessory component of the TGF-β-binding receptor complex that differentially modulates TGF-β and BMP responses. The existence of two splice variants L- and S-endoglin which differ in their cytoplasmic domain has already been shown in human and mice. Endoglin is located on the cell surfaces of cultured hepatic stellate cells and transdifferentiated myofibroblasts suggesting that this receptor might be associated with the profibrogenic attributes of these liver cell subpopulations. We now show that endoglin expression is increased in transdifferentiating hepatic stellate cells and in two models of liver fibrosis (i.e. bile duct ligation and carbon tetrachloride model) and further detectable in cultured portal fibroblasts representing another important fibrogenic cell type but not in hepatocytes. In respect to TGF-β1-signalling, we demonstrate that endoglin interacts with and is phosphorylated by TβRII. In hepatic stellate cells, TGF-β1 upregulates endoglin expression most likely via the ALK5 pathway and requires the SP1 transcription factor. We further identified a novel rat splice variant that is structurally and functionally different from that identified in human and mouse. Transient overexpression of endoglin resulted in a strong increase of TGF-β1-driven Smad1/5 phosphorylation and α-smooth muscle actin expression in a hepatic stellate cell line. In supernatants of respective cultures, we could detect the ectodomain of endoglin suggesting that shedding is a further key process involved in the regulation of this surface receptor.

Expression of the TGF-beta1 system in human testicular pathologies.

BackgroundIn non-obstructive azoospermia, histological patterns of Sertoli cell-only Syndrome (SCO) and hypospermatogenesis (H) are commonly found. In these pathologies, Leydig cell hyperplasia (LCH) is detected in some patients. Since TGF-β1 is involved in cellular proliferation/development, the aim of this work was to analyze the expression of TGF-β1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5), and the co-receptor endoglin in human biopsies from patients with idiopathic infertility.MethodsSpecific immunostaining of TGF-β1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5), co-receptor endoglin and Smads proteins, were carried out in testicular biopsies from normal and infertile men with SCO or H. Gene expression of TGF-β1 system were made in biopsies from infertile patients with semi-quantitative and quantitative PCR.ResultsImmunohistochemical studies revealed that TGF-β1 and its specific receptors are present in Leydig cells in biopsies from normal tissue or patients with SCO or H with or without LCH. Smad proteins, which are involved in TGF-β1 signaling, are also detected in both their phosphorylated (activated) and dephosphorylated form in all samples TGF-β1, ALK-1 and endoglin gene expression are stronger in human biopsies with LCH than in those with SCO or H. Neither TGFBRII nor ALK-5 gene expression showed significant differences between pathologies. A significant correlation between ALK-1 and endoglin expression was observed.ConclusionsIn conclusion, the high levels of mRNA and protein expression of the TGF-β1 system in patients with LCH, particularly ALK1 and its correlation with endoglin, suggest that these proteins acting in concert might be, at least in part, committed actors in the Leydig cell hyperplasia.

Expression of midkine and endoglin in breast carcinomas with different immunohistochemical profiles

The aim of this study was to investigate the midkine and endoglin expression in breast carcinomas with five different immunohistochemical profiles and their relevance to histopathologic and clinicopathologic features. We analyzed 161 archival tissues immunohistologically. The level of midkine expression in breast cancer significantly correlated with lymph node metastasis (p = 0.001) and TNM staging (p = 0.003). High microvessel density (MVD) was associated with higher midkine reactivity group (p = 0.036). Although the basal-like subtype had higher midkine expression level and MVD, no significant difference with the other breast cancer subtypes was found. In conclusion, midkine was a promising target for tumor prognosis in clinical diagnosis and treatment. This study found no significant differences in tumor angiogenesis in different molecular subtypes of breast cancer.

Endoglin expression in breast tumor cells suppresses invasion and metastasis and correlates with improved clinical outcome

Tumor growth factor-β (TGF-β) signaling in cancer has been implicated in growth suppression of early lesions and enhancing tumor cell invasion and metastasis. However, the cellular mechanisms that determine this signaling output in individual tumors are still largely unknown. In endothelial cells, TGF-β signaling is modulated by the TGF-β co-receptor endoglin (CD105). Here we demonstrate that endoglin is expressed in a subset of invasive breast cancers and cell lines and is subject to epigenetic silencing by gene methylation. Endoglin downregulation in non-tumorigenic MCF10A breast cells leads to the formation of abnormal acini in 3D culture, but does not promote cell migration or transformation. In contrast, in the presence of activated ErbB2, endoglin downregulation in MCF10A cells leads to enhanced invasion into a 3D matrix. Consistent with these data, ectopic expression of endoglin in MDA-MB-231 cells blocks TGF-β-enhanced cell motility and invasion and reduces lung colonization in an in vivo metastasis model. Unlike endothelial cells, endoglin does not modulate Smad-mediated TGF-β signaling in breast cells but attenuates the cytoskeletal remodeling to impair cell migration and invasion. Importantly, in a large cohort of invasive breast cancers, lack of endoglin expression in the tumor cell compartment correlates with ENG gene methylation and poor clinical outcome.

Effect of Reishi polysaccharides on human stem/progenitor cells

The polysaccharide fraction of Ganoderma lucidum (F3) was found to benefit our health in many ways by influencing the activity of tissue stem/progenitor cells. In this study, F3 was found to promote the adipose tissue MSCs’ aggregation and chondrosphere formation, with the increase of CAM (N-CAM, I-CAM) expressions and autokine (BMP-2, IL-11, and aggrecan) secretions, in an in vitro chondrogenesis assay. In a stem cell expansion culture, it possesses the thrombopoietin (TPO) and GM-CSF like functions to enhance the survival/renewal abilities of primitive hematopoietic stem/progenitor cells (HSCs). F3 was found to promote the dendrite growth of blood mononuclear cells (MNCs) and the expression of cell adhesion molecules in the formation of immature dendritic cells (DC). On the other hand, F3 exhibited inhibitory effects on blood endothelial progenitor (EPC) colony formation, with concomitant reduction of cell surface endoglin (CD105) and vascular endothelial growth factor receptor-3 (VEGFR-3) marker expressions, in the presence of angiogenic factors. A further cytokine array analysis revealed that F3 indeed inhibited the angiogenin synthesis and enhanced IL-1, MCP-1, MIP-1, RANTES, and GRO productions in the blood EPC derivation culture. Collectively, we have demonstrated that the polysaccharide fraction of G. lucidum F3 exhibits cytokine and chemokine like functions which are beneficial to human tissue stem/progenitor cells by modulating their CAM expressions and biological activities. These findings provide us a better the observation that F3 glycopolysaccharides indeed possesses anti-angiogenic and immune-modulating functions and promotes hematopoietic stem/progenitor cell homing for better human tissue protection, reducing disease progression and health.

P114 Effect of preeclampsia sera on mRNA expression of soluble Endoglin and HO-1 from cultured choriocarcinoma cells

P114 Effect of preeclampsia sera on mRNA expression of soluble Endoglin and HO-1 from cultured choriocarcinoma cells

Spreading endothelial cell dysfunction in response to necrotic trophoblasts. Soluble factors released from endothelial cells that have phagocytosed necrotic shed trophoblasts reduce the proliferation of additional endothelial cells

The pathogenesis of preeclampsia is not clear but the disease is characterised by systemic endothelial cell dysfunction that is considered to be triggered by a placental factor. Necrotic trophoblastic debris that is deported in the maternal blood is one possible placental trigger for preeclampsia. Syncytial knots were first associated with preeclampsia over 100 years ago. However, syncytial knots are very large and most are trapped in the pulmonary capillaries making it difficult to envisage how they could lead to widespread systemic endothelial cell dysfunction. This study was undertaken to examine whether conditioned medium from endothelial cells that have phagocytosed necrotic trophoblastic debris could adversely affect the proliferation or survival of fresh endothelial cells. Trophoblastic cellular debris, harvested from placental explants was added to endothelial cell monolayers directly or after induction of necrosis by freeze–thawing. Conditioned medium from the endothelial cell cultures was exposed to fresh endothelial cells and their proliferation measured by Alamar Blue, and CyQUANT ®NF cell proliferation assays. Endothelial cell death was examined by a fluorogenic caspase-3 activity assay and LDH release. Conditioned medium from endothelial cells that had phagocytosed necrotic but not apoptotic trophoblastic debris significantly inhibited the proliferation of fresh endothelial cells but did not induce their death. The conditioned medium also reduced cell-surface endoglin expression by fresh endothelial cells. These results confirm that phagocytosis of necrotic trophoblastic debris by endothelial cells results in the secretion of soluble factors that might explain how necrotic trophoblastic debris trapped in the pulmonary capillaries could induce systemic endothelial cell dysfunction.

Abnormal expression of Endoglin and its receptor complex (TGF-β1 and TGF-β receptor II) as early angiogenic switch indicator in premalignant lesions of the colon mucosa

The precise timing of the angiogenic switch in colorectal cancer development is still unclear. The simultaneous expression of Endoglin (CD105), transforming growth factor (TGF)-β1 and TGF-β receptor (R) II were quantified in surgical specimens comprising normal human colon, pre-malignant dysplastic tissue, in situ, and invasive colon cancer specimens, at mRNA and protein levels, respectively by real-time PCR and immunohistochemistry. Serum concentrations of soluble Endoglin and TGF-β1 were evaluated. mRNA and CD105+-microvessel density (MVD) increased significantly in dysplastic colon and carcinoma versus normal tissues; values correlated respectively with dysplasia degree and Dukes' stages. TGF-β1 expression was significantly upregulated in most severe dysplastic adenoma specimens, while TGF-β1 transcript and protein signals were intense in carcinoma, positively-correlated with tumor progression. TGF-β1 RII was overexpressed in adenoma and carcinoma versus normal samples, but unrelated with dysplasia or Dukes' stage. Soluble Endoglin serum levels were equivalent in adenoma and normal tissues; in carcinoma the highest levels were in invasive tumor. Circulating TGF-β1 levels were increased in severe dysplasia and progressed with tumor progression. Correlations between adenoma dysplasia degree and TGF-β RII and CD105+-MVD, and between tumor Dukes' staging and TGF-β1 and CD105+-MVD, were significant. TGF-β1 and Endoglin and TGF-β1 serum levels, TGF-β1 staining and CD105+-MVD were significantly and inversely associated with disease-free survival. TGF-β1 levels were an independent and significant prognostic factor of disease-free survival. These findings suggest active angiogenesis occurs in many pre-malignant colon cases and supports more careful evaluation of different chemopreventive agents.

Critical role of endoglin in tumor cell plasticity of Ewing sarcoma and melanoma

Tumor cell plasticity enables certain types of highly malignant tumor cells to dedifferentiate and engage a plastic multipotent embryonic-like phenotype, which enables them to 'adapt' during tumor progression and escape conventional therapeutic strategies. This plastic phenotype of aggressive cancer cells enables them to express endothelial cell-specific markers and form tube-like structures, a phenotype that has been linked to aggressive behavior and poor prognosis. We demonstrate here that the transforming growth factor (TGF)-β co-receptor endoglin, an endothelial cell marker, is expressed by tumor cells and its expression correlates with tumor cell plasticity in two types of human cancer, Ewing sarcoma and melanoma. Moreover, endoglin expression was significantly associated with worse survival of Ewing sarcoma patients. Endoglin knockdown in tumor cells interferes with tumor cell plasticity and reduces invasiveness and anchorage-independent growth in vitro. Ewing sarcoma and melanoma cells with reduced endoglin levels showed reduced tumor growth in vivo. Mechanistically, we provide evidence that endoglin, while interfering with TGF-β signaling, is required for efficient bone morphogenetic protein, integrin, focal adhesion kinase and phosphoinositide-3-kinase signaling in order to maintain tumor cell plasticity. The present study delineates an important role of endoglin in tumor cell plasticity and progression of aggressive tumors.

Endoglin differentially regulates TGF-β-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes

Transforming growth factor-β (TGF-β) plays a critical role in cartilage homeostasis and deregulation of its signalling is implicated in osteoarthritis (OA). TGF-β isoforms signal through a pair of transmembrane serine/threonine kinases known as the type I and type II TGF-β receptors. Endoglin is a TGF-β co-receptor that binds TGF-β with high affinity in the presence of the type II TGF-β receptor. We have previously shown that endoglin is expressed in human chondrocytes and that it forms a complex with the TGF-β signalling receptors. However, the functional significance of endoglin expression in chondrocytes is unknown. Our objective was to determine whether endoglin regulates TGF-β/Smad signalling and extracellular matrix (ECM) production in human chondrocytes and whether its expression varies with chondrocyte differentiation state. Endoglin function was determined by overexpression or antisense morpholino/siRNA knockdown of endoglin in human chondrocytes and measuring TGF-β-induced Smad phosphorylation, transcriptional activity and ECM production. Alterations in endoglin expression levels were determined during subculture-induced dedifferentiation of human chondrocytes and in normal vs OA cartilage samples. Endoglin enhances TGF-β1-induced Smad1/5 phosphorylation and inhibits TGF-β1-induced Smad2 phosphorylation, Smad3-driven transcriptional activity and ECM production in human chondrocytes. In addition, the enhancing effect of endoglin siRNA knockdown on TGF-β1-induced Smad3-driven transcription is reversed by ALK1 overexpression. Furthermore, endoglin levels are increased in chondrocytes following subculture-induced dedifferentiation and in OA cartilage as compared to normal cartilage. Together, our results suggest that endoglin regulates the balance between TGF-β/ALK1/Smad1/5 and ALK5/Smad2/3 signalling and ECM production in human chondrocytes and that endoglin may represent a marker for chondrocyte phenotype.

Differential expression of endoglin in human melanoma cells expressing the V3 isoform of versican by microarray analysis

Versican is a large chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The large isoforms V0 and V1 promote melanoma cell proliferation. We previously described that overexpression of the short V3 isoform in MeWo human melanoma cells markedly reduced tumor cell growth in vitro and in vivo, but favored the appearance of secondary tumors. This study aimed to elucidate the mechanisms of V3 by identifying differentially expressed genes between parental and V3-expressing MeWo melanoma cells using microarray analysis. V3 expression significantly reduced the expression of endoglin, a transforming growth factor-β superfamily co-receptor. Other differentially expressed genes were VEGF and PPP1R14B. Changes in endoglin levels were validated by qRT-PCR and Western blotting.

Adhesion, proliferation, and gene expression profile of human umbilical vein endothelial cells cultured on bilayered polyelectrolyte coatings composed of glycosaminoglycans

This study characterized human umbilical vein endothelial cell (HUVEC) adhesion, proliferation, and gene expression on bilayered polyelectrolyte coatings composed of an outermost layer of glycosaminoglycans (hyaluronan, heparin, or chondroitin sulfate), with an underlying layer of poly-L-lysine or chitosan. The proportion of cells that adhered to the various polyelectrolyte coatings after 1 and 2 h incubations was quantified by the WST-8 assay. Interchanging poly-L-lysine with chitosan resulted in significant differences in cellular adhesion to the outermost glycosaminoglycan layer after 1 h, but these differences became insignificant after 2 h. The proliferation of HUVEC on the various bilayered polyelectrolyte coatings over 10 days was characterized using the WST-8 assay. Regardless of whether the underlying layer was poly-L-lysine or chitosan, HUVEC proliferation on the hyaluronan outermost layer was significantly less than on heparin or chondroitin sulfate. Additionally, it was observed that there was more proliferation with poly-L-lysine as the underlying layer, compared to chitosan. Subsequently, real-time polymerase chain reaction was used to analyze the expression of seven genes related to adhesion, migration, and endothelial function (VWF, VEGFR, VEGFA, endoglin, integrin-α5, ICAM1, and ICAM2) by HUVEC cultured on the various bilayered polyelectrolyte coatings for 3 days. With poly-L-lysine as the underlying layer, biologically significant differences (greater than twofold) in the expression of VWF, VEGFR, VEGFA, endoglin, and ICAM1 were observed among the three glycosaminoglycans. With chitosan as the underlying layer, all three glycosaminoglycans displayed biologically significant differences in the expression of VWF and VEGFR compared to the chitosan control. CT-HA displayed the highest level of expression of VWF, whereas expression levels of VEGFR were almost similar among the three glycosaminoglycans.

Endoglin (CD105) expression and angiogenesis status in small cell lung cancer

It is well established that angiogenesis is crucial for tumor development and progression. Among the angiogenesis immunomarkers defined to date, endoglin (CD105) has been shown to be a useful marker of angiogenesis. To investigate the degree of angiogenesis status in small cell lung cancer (SCLC) tissue, we assessed 35 cases of SCLC at autopsy using immunohistochemical staining of CD31 and CD105. The intratumoral area, peritumoral area, and background pulmonary alveoli were then observed under low magnification, and the microvessel density (MVD) for each area was determined. The MVD-CD31 was the highest in the background alveoli, followed by the intratumoral and peritumoral areas. The MVD-CD105 was highest in the intratumoral area, followed by the peritumoral area and the background lung. The ratio of CD105/CD31 revealed that almost 78% of the intratumoral area, 63% of the peritumoral area, and 4.6% of the background lung alveoli were newly formed and expressed CD105. This result indicated that SCLC is predominantly supported by newly formed vessels that are generated by CD105-mediated angiogenesis. These findings suggest that anti-angiogenic therapy, especially CD105-targeting, may prove an effective form of SCLC treatment.

CD105 (Endoglin) expression in breast carcinoma effusions is a marker of poor survival

We analyzed the expression and clinical role of endoglin (CD105) in breast carcinoma effusions. Endoglin levels were measured in 36 effusion supernatants by ELISA and studied for association with the cancer-associated markers calprotectin, VEGF, and the VEGF receptor sFlt1. Endoglin expression was further studied in 46 effusions and 22 primary carcinomas using immunohistochemistry. The four secreted molecules were detected in all specimens and their levels significantly correlated ( p < 0.001). In effusions, endoglin was localized to carcinoma cells and reactive mesothelium using immunohistochemistry. Tumor cell expression was higher in effusions compared to primary carcinomas ( p = 0.025), and in post-chemotherapy compared to pre-chemotherapy effusions ( p = 0.017). Higher tumor endoglin expression was associated with poor overall ( p = 0.021) and disease-free ( p = 0.032) survival in univariate analysis, and was an independent predictor in Cox multivariate analysis ( p = 0.001 and p = 0.038, respectively). Our data suggest that endoglin may be an important therapeutic target in metastatic breast cancer.

Spheroid‐forming subpopulation of breast cancer cells demonstrates vasculogenic mimicry via hsa‐miR‐299–5p regulated de novo expression of osteopontin

The growth of cancer cells as multicellular spheroids has frequently been reported to mimic the in vivo tumour architecture and physiology and has been utilized to study antitumour drugs. In order to determine the distinctive characteristics of the spheroid-derived cells compared to the corresponding monolayer-derived cells, we enriched multicellular spheroid-forming subpopulations of cells from three human breast cancer cell lines (MCF7, MCF10AT and MCF10DCIS.com). These spheroid-derived cells were injected into female athymic nude mice to assess their tumorigenic potential and were profiled for their characteristic miRNA signature. We discovered that the spheroid-derived cells expressed increased levels of osteopontin (OPN), an oncogenic protein that has been clinically correlated with increased tumour burden and adverse prognosis in patients with breast cancer metastasis. Our studies further show that increased OPN levels are brought about in part, by decreased levels of hsa-mir-299–5p in the spheroid-forming population from all three cell lines. Moreover, the spheroid-forming cells can organize into vascular structures in response to nutritional limitation; these structures recapitulate a vascular phenotype by the expression of endothelial markers CD31, Angiopoeitin-1 and Endoglin. In this study, we have validated that hsa-mir-299–5p targets OPN; de novo expression of OPN in turn plays a critical role in enhancing proliferation, tumorigenicity and the ability to display vasculogenic mimicry of the spheroid-forming cells.

Transforming growth factor-beta co-receptor endoglin suppresses breast cancer invasion and metastasis

Transforming growth factor-beta (TGFβ) signaling in cancer has been implicated in both growth suppression in early lesions as well as enhancing tumor cell invasion and metastasis. However, the cellular mechanisms that determine the signaling output in individual tumors are still largely unknown. In endothelial cells, TGFβ signaling is modulated by the TGFβ co-receptor endoglin (CD105). Here we demonstrate that endoglin is differentially expressed in invasive breast cancers and breast cancer cell lines, and is subject to epigenetic silencing by gene methylation. Downregulation of endoglin expression in nontumorigenic MCF10A cells leads to the formation of abnormal acini in 3D culture but does not promote cell migration or result in cell transformation. In contrast, in the presence of an activated oncogene, loss of endoglin in MCF10A cells leads to enhanced migration and invasion into a 3D matrix. Consistent with these data, ectopic expression of endoglin in the endoglin-negative MDA-MB-231 cell line blocks TGFβ-enhanced cell motility and invasion and reduces the ability of cells to successfully colonize the lung parenchyma in an in vivo metastasis model. Unlike endothelial cells, endoglin does not does not modulate canonical TGFβ signaling in breast cells but attenuates the cytoskeletal remodeling to impair cell migration and invasion. Importantly, lack of endoglin expression in clinical samples significantly correlates with ENG gene methylation and poor clinical outcome. Together these data identify endoglin as a key component suppressing the invasive activities of breast cancer cells.

Endoglin negatively regulates transforming growth factor β1-induced profibrotic responses in intestinal fibroblasts

Fibroblasts isolated from strictures in Crohn's disease (CD) exhibit reduced responsiveness to stimulation with transforming growth factor (TGF) beta1. TGF-beta1, acting through the smad pathway, is critical to fibroblast-mediated intestinal fibrosis. The membrane glycoprotein, endoglin, is a negative regulator of TGF-beta1. Intestinal fibroblasts were cultured from seromuscular biopsies of patients undergoing intestinal resection for CD strictures or from control patients. Endoglin expression was assessed using confocal microscopy, flow cytometry and western blot. The effect of small interfering (si) RNA-mediated knockdown and plasmid-mediated overexpression of endoglin on fibroblast responsiveness to TGF-beta1 was assessed by examining smad phosphorylation, smad binding element (SBE) promoter activity, connective tissue growth factor (CTGF) expression and ability to contract collagen. Crohn's stricture fibroblasts expressed increased constitutive cell-surface and whole-cell endoglin relative to control cells. Endoglin co-localized with filamentous actin. Fibroblasts treated with siRNA directed against endoglin exhibited enhanced TGF-beta1-mediated smad-3 phosphorylation, and collagen contraction. Cells transfected with an endoglin plasmid did not respond to TGF-beta1 by exhibiting SBE promoter activity or producing CTGF. Fibroblasts from strictures in CD express increased constitutive endoglin. Endoglin is a negative regulator of TGF-beta1 signalling in the intestinal fibroblast, modulating smad-3 phosphorylation, SBE promoter activity, CTGF production and collagen contraction.

Role of extracellular signal-regulated kinase and AKT cascades in regulating hypoxia-induced angiogenic factors produced by a trophoblast-derived cell line

During human pregnancy, trophoblasts play an important role in embryo implantation and placental development. Cytotrophoblast cells invade the uterine spiral arteries and differentiate into extravillous trophoblasts, resulting in the remodeling of the uterine vessels and fetoplacental vasculature. During early pregnancy, a physiologically hypoxic environment induces the production of angiogenic factors, such as vascular endothelial growth factor (VEGF), which are suggested to locally control the vascular remodeling. Endoglin, a cell-surface coreceptor for transforming growth factor-beta1, is highly expressed in endothelial cells and syncytiotrophoblasts, and can be associated with endothelial nitric oxide synthase and vascular homeostasis. Several studies have recently suggested that some pregnancy-related complications, such as preeclampsia, have their origins early in pregnancy as a result of abnormalities in implantation and placental development. Although angiogenic factors are recognized as key molecules in placental development, little is known about the mechanism(s) of their regulation in trophoblasts. In this study, we elucidated the mechanisms underlying the regulation of VEGF and endoglin production under hypoxic conditions in the trophoblast-derived cell line, BeWo. We evaluated the role of the AKT-MTOR cascade and ERK kinase in the expression of VEGF and endoglin in response to hypoxia using various kinase inhibitors and small interfering RNA targeted against hypoxia-inducible factor (HIF)-1alpha (listed as HIF1A in Hugo Database). Our results suggest that both the phosphatidylinositol 3-kinase-AKT-MTOR-HIF-1alpha and ERK-HIF-1alpha signaling pathways are crucial for increasing VEGF and endoglin expression in response to hypoxia in BeWo cells.

A New Application for the Drug Tranilast: Effects on Breast Cancer Cell Proliferation, Migration, and Invasion

BackgroundTranilast (Rizaben®) has been used to treat allergic and auto‐immune diseases for the last 26 years. The objective of this study was to investigate whether tranilast also has an effect on breast cancer cell biological features relevant to cancer progression, based on the its mechanism of action which involves TGFβsignaling.MethodsCultures of the BT‐474 human breast cancer cell line were used. Cell proliferation, clonogenicity, cell migration, endoglin and MMP‐9 expression were assessed by: MTT assay, soft agar colony formation, wound assay and immunocytochemistry respectively.ResultsTranilast was found to inhibit breast cancer cell proliferation by 70%. In addition, the drug decreased by 54% the number and size of cell colonies grown in soft agar, consistent with a decrease in clonogenicity. The closure of wounds made in cancer cell monolayers was slowed (55%) by Tranilast, consistent with an inhibitory effect on tumor cell migration. Endoglin, which plays a role in TGFβ signaling, was downregulated by the drug. MMP‐9, which is a metalloproteinase involved in tumor invasion and metastasis was also decreased by tranilast.ConclusionsThese results indicate that tranilast exerts inhibitory effects on key biological features of human breast cancer cells. The results are consistent with an anti‐tumor effect which can be exploited in preclinical and clinical therapeutic trials.