Anthocyanidins inhibit epithelial-mesenchymal transition through a TGFß/Smad2 signaling pathway in glioblastoma cells.

Epidemiological studies have convincingly demonstrated that diets rich in fruits and vegetables play an important role in preventing cancer due to their polyphenol content. Among polyphenols, the anthocyanidins are known to possess anti-inflammatory, cardioprotective, anti-angiogenic, and anti-carcinogenic properties. Despite the well-known role of transforming growth factor-ß (TGF-ß) in high grade gliomas, the impact of anthocyanidins on TGF-ß-induced epithelial-mesenchymal transition (EMT), a process that allows benign tumor cells to infiltrate surrounding tissues, remains poorly understood. The objective of this study is to investigate the impact of anthocyanidins such as cyanidin (Cy), delphinidin (Dp), malvidin (Mv), pelargonidin (Pg), and petunidin (Pt) on TGF-ß-induced EMT and to determine the mechanism(s) underlying such action. Human U-87 glioblastoma (U-87 MG) cells were treated with anthocyanidins prior to, along with or following the addition of TGF-ß. We found that anthocyanidins differently affected TGF-ß-induced EMT, depending on the treatment conditions. Dp was the most potent EMT inhibitor through its inhibitory effect on the TGF-ß Smad and non-Smad signaling pathways. These effects altered expression of the EMT mesenchymal markers fibronectin and Snail, as well as markedly reducing U-87 MG cell migration. Our study highlights a new action of anthocyanidins against EMT that supports their beneficial health and chemopreventive effects in dietary-based strategies against cancer. © 2016 Wiley Periodicals, Inc.

Biphasic effects of luteolin on interleukin-1ß-induced cyclooxygenase-2 expression in glioblastoma cells.

Success in developing therapeutic approaches to target brain tumor-associated inflammation in patients has been limited. Given that the inflammatory microenvironment is a hallmark signature of solid tumor development, anti-inflammatory targeting strategies have been envisioned as preventing glioblastoma initiation or progression. Consumption of foods from plant origin is associated with reduced risk of developing cancers, a chemopreventive effect that is, in part, attributed to their high content of phytochemicals with potent anti-inflammatory properties. We explored whether luteolin, a common flavonoid in many types of plants, may inhibit interleukin (IL)-1ß function induction of the inflammation biomarker cyclooxygenase (COX)-2. We found that IL-1ß triggered COX-2 expression in U-87 glioblastoma cells and synergized with luteolin to potentiate or inhibit that induction in a biphasic manner. Luteolin pretreatment of cells inhibited IL-1ß-mediated phosphorylation of inhibitor of κB, nuclear transcription factor-κB (NF-κB) p65, extracellular signal-regulated kinase-1/2, and c-Jun amino-terminal kinase in a concentration-dependent manner. Luteolin also inhibited AKT phosphorylation and survivin expression, while it triggered both caspase-3 cleavage and expression of glucose-regulated protein 78. These effects were all potentiated by IL-1ß, in part through increased nuclear translocation of NF-κB p65. Finally, luteolin was able to reduce IL-1 receptor gene expression, and treatment with IL-1 receptor antagonist or gene silencing of IL-1 receptor prevented IL-1ß/luteolin-induced COX-2 expression. Our results document a novel adaptive cellular response to luteolin, which triggers anti-survival and anti-inflammatory mechanisms that contribute to the chemopreventive properties of this diet-derived molecule.

Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation.

Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention.

Epigallocatechin gallate targeting of membrane type 1 matrix metalloproteinase-mediated Src and Janus kinase/signal transducers and activators of transcription 3 signaling inhibits transcription of colony-stimulating factors 2 and 3 in mesenchymal stromal cells.

BACKGROUND: CSF-2 and CSF-3 confer proangiogenic and immunomodulatory properties to mesenchymal stromal cells (MSCs). RESULTS: Transcriptional regulation of CSF-2 and CSF-3 in concanavalin A-activated MSCs requires MT1-MMP signaling and is inhibited by EGCG. CONCLUSION: The chemopreventive properties of diet-derived EGCG alter MT1-MMP-mediated intracellular signaling. SIGNIFICANCE: Pharmacological targeting of MSCs proangiogenic functions may prevent their contribution to tumor development. Epigallocatechin gallate (EGCG), a major form of tea catechins, possesses immunomodulatory and antiangiogenic effects, both of which contribute to its chemopreventive properties. In this study, we evaluated the impact of EGCG treatment on the expression of colony-stimulating factors (CSF) secreted from human bone marrow-derived mesenchymal stromal cells (MSCs), all of which also contribute to the immunomodulatory and angiogenic properties of these cells. MSCs were activated with concanavalin A (ConA), a Toll-like receptor (TLR)-2 and TLR-6 agonist as well as a membrane type 1 matrix metalloproteinase (MT1-MMP) inducer, which increased granulocyte macrophage-CSF (GM-CSF, CSF-2), granulocyte CSF (G-CSF, CSF-3), and MT1-MMP gene expression. EGCG antagonized the ConA-induced CSF-2 and CSF-3 gene expression, and this process required an MT1-MMP-mediated sequential activation of the Src and JAK/STAT pathways. Gene silencing of MT1-MMP expression further demonstrated its requirement in the phosphorylation of Src and STAT3, whereas overexpression of a nonphosphorylatable MT1-MMP mutant (Y573F) abrogated CSF-2 and CSF-3 transcriptional increases. Given that MSCs are recruited within vascularizing tumors and are believed to contribute to tumor angiogenesis, possibly through secretion of CSF-2 and CSF-3, our study suggests that diet-derived polyphenols such as EGCG may exert chemopreventive action through pharmacological targeting of the MT1-MMP intracellular signaling.

Diet-derived polyphenols inhibit angiogenesis by modulating the interleukin-6/STAT3 pathway.

Several epidemiological studies have indicated that abundant consumption of foods from plant origin is associated with a reduced risk of developing several types of cancers. This chemopreventive effect is related to the high content of these foods in phytochemicals, such as polyphenols, that interfere with several processes involved in cancer progression including tumor cell growth, survival and angiogenesis. In addition to the low intake of plant-based foods, increased body mass and physical inactivity have recently emerged as other important lifestyle factors influencing cancer risk, leading to the generation of low-grade chronic inflammatory conditions which are a key process involved in tumor progression. The objectives of the current study are to investigate the inhibitory effects of these polyphenols on angiogenesis triggered by an inflammatory cytokine (IL-6) and to determine the mechanisms underlying this action. We found that, among the tested polyphenols, apigenin and luteolin were the most potent angiogenesis inhibitors through their inhibitory effect on the inflammatory cytokine IL-6/STAT3 pathway. These effects resulted in modulation of the activation of extracellular signal-regulated kinase-1/2 signaling triggered by IL-6, as well as in a marked reduction in the proliferation, migration and morphogenic differentiation of endothelial cells. Interestingly, these polyphenols also modulated the expression of IL-6 signal transducing receptor (IL-6Rα) and the secretion of the extracellular matrix degrading enzyme MMP-2 as well as the expression of suppressor of cytokine signaling (SOCS3) protein. Overall, these results may provide important new information on the role of diet in cancer prevention.

The stem cell marker CD133 (prominin-1) is phosphorylated on cytoplasmic tyrosine-828 and tyrosine-852 by Src and Fyn tyrosine kinases.

CD133 (prominin-1) is a transmembrane glycoprotein expressed at the surface of normal and cancer stem cells, progenitor cells, rod photoreceptor cells, and a variety of epithelial cells. Although CD133 is widely used as a marker of various somatic and putative cancer stem cells, its contribution to fundamental properties of stem cells such as self-renewal and differentiation remains unknown. CD133 contains a short C-terminal cytoplasmic domain with five tyrosine residues, including a consensus tyrosine phosphorylation site that has not yet been investigated. In this study, we show that CD133 is phosphorylated in human medulloblastoma D283 and Daoy cells, in a Src family kinase-dependent manner. The cytoplasmic domain of CD133 is tyrosine phosphorylated in Daoy cells overexpressing Src and Fyn tyrosine kinases, as well as in vitro using recombinant proteins. Deletion of the C-terminal cytoplasmic domain of CD133 considerably reduced its phosphorylation by Src. To identify the tyrosine phosphorylation sites in CD133, we used matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI Q-TOF) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of tyrosine-phosphorylated CD133 by mass spectrometry and site-directed mutagenesis identified tyrosine-828 and the nonconsensus tyrosine-852 as the major tyrosine phosphorylation sites both in vitro and in intact cells. Identification of CD133 as a substrate for Src-family tyrosine kinases suggests that the cytoplasmic domain of CD133 might play an important role in the regulation of its functions.

The flavonols quercetin, kaempferol, and myricetin inhibit hepatocyte growth factor-induced medulloblastoma cell migration.

Medulloblastoma, the most common malignant brain tumor in children, is a highly metastatic disease, with up to 30% of children having evidence of disseminated disease at presentation. Recently, the hepatocyte growth factor (HGF) and its receptor, the tyrosine kinase Met, have emerged as key components of human medulloblastoma growth and metastasis, suggesting that inhibition of this pathway may represent an attractive target for the prevention and treatment of this disease. Using immunoblotting procedures, we observed that the dietary-derived flavonols quercetin, kaempferol, and myricetin inhibited HGF/Met signaling in a medulloblastoma cell line (DAOY), preventing the formation of actin-rich membrane ruffles and resulting in the inhibition of Met-induced cell migration in Boyden chambers. Furthermore, quercetin and kaempferol also strongly diminished HGF-mediated Akt activation. Interestingly, the inhibitory effects of quercetin on the tyrosine kinase receptor Met [half-maximal inhibitory effect (IC(50)) of 12 micromol/L] or on the Met-induced activation of Akt (IC(50) of 2.5 micromol/L) occurred at concentrations achievable through dietary approaches. These results highlight quercetin, kaempferol, and myricetin as dietary-derived inhibitors of Met activity and suggest that this inhibitory effect may contribute to the chemopreventive properties of these molecules.

Targeting Ovarian Cancer Cell Cytotoxic Drug Resistance Phenotype with Xanthium strumarium L. Extract.

Emerging drugs aim at targeting the genomic integrity and replication machinery in ovarian cancer. While the antiproliferative activity of Xanthium strumarium L. extract (XFC), a traditional herbal medicine, is believed to alter the mitotic apparatus of Chinese hamster ovary epithelial cells, its capacity to target and overcome the chemoresistance phenotype in ovarian cancer is unknown. Among the cancer cell lines tested, we found that the best proliferation inhibitory effect for XFC was against ovarian cancer cells and ranged from 30 to 35 µg/mL. XFC efficiently targeted both the cytotoxic drug chemoresistance phenotype of SKOV-3 cells and of the chemosensitive ES-2 cells. Early apoptosis and late apoptosis were effectively induced by XFC extract in ES-2 cells, whereas late apoptosis and necrosis events were triggered in SKOV-3 cells. Cell cycling regulation was trapped by XFC extract in the G2/M phase in both the ES-2 and SKOV-3 cell models. This effect was, in part, attributable to increased dose-dependent tubulin polymerization, which was increased in SKOV-3 cells. Whereas XFC extract triggered poly (ADP-Ribose) polymerase (PARP) cleavage in both ES-2 and SKOV-3 cells, it only lowered Nrf2 in ES-2 cells and phosphorylated Akt levels in SKOV-3 cells. Interestingly, cell cycling regulators Cdk4, Cyclin D3, and p27 were all decreased in SKOV-3 cells. XFC extracts were effective in inhibiting in vitro migration in both ovarian cancer cell models. Our data support the potential anticancer targeting of chemoresistant human ovarian cancer cells phenotype by XFC extract.

Delphinidin, a dietary anthocyanidin, inhibits platelet-derived growth factor ligand/receptor (PDGF/PDGFR) signaling.

Most cancers are dependent on the growth of tumor blood vessels and inhibition of tumor angiogenesis may thus provide an efficient strategy to retard or block tumor growth. Recently, tumor vascular targeting has expanded to include not only endothelial cells (ECs) but also smooth muscle cells (SMCs), which contribute to a mature and functional vasculature. We have reported previously that delphinidin, a major biologically active constituent of berries, inhibits the vascular endothelial growth factor-induced phosphorylation of vascular endothelial growth factor receptor-2 and blocks angiogenesis in vitro and in vivo. In the present study, we show that delphinidin also inhibits activation of the platelet-derived growth factor (PDGF)-BB receptor-beta [platelet-derived growth factor receptor-beta (PDGFR-beta)] in SMC and that this inhibition may contribute to its antitumor effect. The inhibitory effect of delphinidin on PDGFR-beta was very rapid and led to the inhibition of PDGF-BB-induced activation of extracellular signal-regulated kinase (ERK)-1/2 signaling and of the chemotactic motility of SMC, as well as the differentiation and stabilization of EC and SMC into capillary-like tubular structures in a three-dimensional coculture system. Using an anthocyan-rich extract of berries, we show that berry extracts were able to suppress the synergistic induction of vessel formation by basic fibroblast growth factor-2 and PDGF-BB in the mouse Matrigel plug assay. Oral administration of the berry extract also significantly retarded tumor growth in a lung carcinoma xenograft model. Taken together, these results provide new insight into the molecular mechanisms underlying the antiangiogenic activity of delphinidin that will be helpful for the development of dietary-based chemopreventive strategies.

Periostin, a signal transduction intermediate in TGF-ß-induced EMT in U-87MG human glioblastoma cells, and its inhibition by anthocyanidins.

Periostin is a secreted protein that is highly expressed in glioblastoma cells as compared to normal brain tissue, and is therefore considered as a potential biomarker in therapeutic modalities. Its contribution in the cancer cells invasive phenotype is, however, poorly understood. This work investigates the role of periostin in U-87 MG glioblastoma cell invasion, cell migration and in Transforming Growth Factor ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT). Periostin gene silencing, using small interfering RNA, decreased TGF-ß-induced mesenchymal marker expression of fibronectin and vimentin, partly through reduced Smad2, Akt and Fak phosphorylation as well as U-87 MG cell invasion and migration. The effects of anthocyanidins, the most abundant diet-derived flavonoids, were examined on periostin-mediated downstream signaling pathways. Anthocyanidins were found to decrease periostin expression whether added under pre-, co- or post-treatment conditions along with TGF-ß, and altered the Akt and Fak signaling pathways. These effects were similar to Galunisertib (LY2157299), a small molecule inhibitor of the TGF-ß receptor I kinase. Taken together, our data demonstrate that periostin acts as a central element in TGF-ß-induced EMT, which can be prevented by diet-derived anthocyanidins.

Biophysical evidence for differential gallated green tea catechins binding to membrane type-1 matrix metalloproteinase and its interactors.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP which triggers intracellular signaling and regulates extracellular matrix proteolysis, two functions that are critical for tumor-associated angiogenesis and inflammation. While green tea catechins, particularly epigallocatechin gallate (EGCG), are considered very effective in preventing MT1-MMP-mediated functions, lack of structure-function studies and evidence regarding their direct interaction with MT1-MMP-mediated biological activities remain. Here, we assessed the impact in both cellular and biophysical assays of four ungallated catechins along with their gallated counterparts on MT1-MMP-mediated functions and molecular binding partners. Concanavalin-A (ConA) was used to trigger MT1-MMP-mediated proMMP-2 activation, expression of MT1-MMP and of endoplasmic reticulum stress biomarker GRP78 in U87 glioblastoma cells. We found that ConA-mediated MT1-MMP induction was inhibited by EGCG and catechin gallate (CG), that GRP78 induction was inhibited by EGCG, CG, and gallocatechin gallate (GCG), whereas proMMP-2 activation was inhibited by EGCG and GCG. Surface plasmon resonance was used to assess direct interaction between catechins and MT1-MMP interactors. We found that gallated catechins interacted better than their ungallated analogs with MT1-MMP as well as with MT1-MMP binding partners MMP-2, TIMP-2, MTCBP-1 and LRP1-clusterIV. Overall, current structure-function evidence supports a role for the galloyl moiety in both direct and indirect interactions of green tea catechins with MT1-MMP-mediated oncogenic processes.

Inhibition of sphingosine-1-phosphate- and vascular endothelial growth factor-induced endothelial cell chemotaxis by red grape skin polyphenols correlates with a decrease in early platelet-activating factor synthesis.

Vascular endothelial growth factor (VEGF) and platelet-derived lipid sphingosine-1-phosphate (S1P) are two proinflammatory mediators which contribute to angiogenesis, in part through the synthesis of platelet-activating factor (PAF). The red grape skin polyphenolic extract (SGE) both prevents and inhibits angiogenesis in the Matrigel model, decreases the basal motility of endothelial and cancer cells, and reverses the chemotactic effect of S1P and VEGF on bovine aortic endothelial cells (BAECs) as well as the chemotactic effect of conditioned medium on human HT-1080 fibrosarcoma, human U-87 glioblastoma, and human DAOY medulloblastoma cells. Inhibition of VEGF- and S1P-mediated chemotaxis by SGE is associated with a down-regulation of ERK and p38/MAPK phosphorylation and a decreased in acute PAF synthesis. Notably, as do extracellular inhibitors of PAF receptor, SGE prevents S1P-induced PAF synthesis and the resulting activation of the S1P/endothelial differentiation gene-1 cascade. Given the key role of VEGF and S1P in inflammation, angiogenesis, and tumor invasion, SGE may therefore contribute to prevent (or to delay) the development of diseases associated with angiogenesis dysregulation, including cancer. The dual inhibition of S1P- and VEGF-mediated migration of endothelial cell and of serum-stimulated migration of U-87 cells suggests a usefulness of SGE against highly invasive human glioblastoma.

Green tea catechins inhibit vascular endothelial growth factor receptor phosphorylation.

Vascular endothelial growth factor (VEGF) receptors (VEGFR) play a major role in tumor angiogenesis and, thus, represent attractive targets for the development of novel anticancer therapeutics. In this work, we report that green tea catechins are novel inhibitors of VEGFR-2 activity. Physiological concentrations (0.01-1 microM) of epigallocatechin-3 gallate, catechin-3 gallate, and, to a lesser extent, epicatechin-3 gallate induce a rapid and potent inhibition of VEGF-dependent tyrosine phosphorylation of VEGFR-2. The inhibition of VEGFR-2 by epigallocatechin-3 gallate was similar to that induced by Semaxanib (SU5416), a specific VEGFR-2 inhibitor. The inhibition of VEGFR-2 activity by the catechins displayed positive correlation with the suppression of in vitro angiogenesis. These observations suggest that the anticancer properties of green tea extracts may be related to their inhibition of VEGF-dependent angiogenesis.

Olive oil compounds inhibit the paracrine regulation of TNF-α-induced endothelial cell migration through reduced glioblastoma cell cyclooxygenase-2 expression.

The established causal relationship between the chronic inflammatory microenvironment, tumor development and cancer recurrence has provided leads for developing novel preventive strategies. Accumulating experimental, clinical and epidemiological data has provided support for the chemopreventive properties of olive oil compounds traditionally found within the Mediterranean diet. In this study, we investigated whether tyrosol (Tyr), hydroxytyrosol, oleuropein and oleic acid (OA), four compounds contained in extra virgin olive oil, can prevent tumor necrosis factor (TNF)-α-induced expression of cyclooxygenase (COX)-2 (an inflammation biomarker) in a human glioblastoma cell (U-87 MG) model. We found that Tyr and OA significantly inhibited TNF-α-induced COX-2 gene and protein expression, as well as PGE2 secretion. Both compounds also inhibited TNF-α-induced JNK and ERK phosphorylation, whereas only Tyr inhibited TNF-α-induced NF-κB phosphorylation. Paracrine-regulated migration of human brain microvascular endothelial cells (HBMECs) was assessed using growth factor-enriched conditioned media (CM) isolated from U-87 MG cells. We found that while PGE2 triggered HBMEC migration, the CM isolated from U-87 MG cells, where either COX-2 or NF-κB had been silenced or had been treated with Tyr or OA, exhibited decreased chemotactic properties. These observations demonstrate that olive oil compounds inhibit the effect of the chronic inflammatory microenvironment on glioblastoma progression through TNF-α actions and may be useful in cancer chemoprevention.

The antiangiogenic agent neovastat (AE-941) inhibits vascular endothelial growth factor-mediated biological effects.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, which exerts direct effects on vascular endothelial cells, including endothelial cell proliferation and survival, tubulogenesis, and vascular permeability. In this study, we examined whether Neovastat, a naturally occurring multifunctional antiangiogenic drug, could inhibit the endothelial cell response to VEGF stimulation. RESULTS: We demonstrated that Neovastat was able to block the VEGF-dependent microvessel sprouting from Matrigel-embedded rat aortic rings, and it also blocked the VEGF-induced endothelial cell tubulogenesis in vitro. In vivo studies showed that Neovastat was able to specifically inhibit VEGF-induced plasma extravasation in numerous tissues, including pancreas and skin. The mechanism of action of Neovastat on VEGF-mediated effects was also evaluated at the molecular level. Neovastat was shown to compete against the binding of VEGF to its receptor in endothelial cells and significantly inhibited the VEGF-dependent tyrosine phosphorylation of VEGF receptor-2, whereas it had no significant effect on VEGF receptor-1 activity. Moreover, the inhibition of receptor phosphorylation was correlated with a marked decrease in the ability of VEGF to induce pERK activation. Neovastat does not compete against the binding of basic fibroblast growth factor, indicating a preferential inhibitory effect on the VEGF receptor. CONCLUSIONS: Because Neovastat was shown previously to inhibit metalloproteinase activities, these results suggest that Neovastat is able to target multiple steps in tumor neovascularization, further emphasizing its use as a pleiotropic, multifunctional antiangiogenic drug.

Green tea catechins as novel antitumor and antiangiogenic compounds.

The concept of cancer prevention by use of naturally occuring substances that could be included in the diet is under investigation as a practical approach towards reducing cancer incidence, and therefore the mortality and morbidity associated with this disease. Tea, which is the most popularly consumed beverage aside from water, has been particularly associated with decreased risk of various proliferative diseases such as cancer and atherosclerosis in humans. Various studies have provided evidence that polyphenols are the strongest biologically active agents in green tea. Green tea polyphenols (GTPs) mainly consist of catechins (3-flavanols), of which (-)-epigallocatechin gallate is the most abundant and the most extensively studied. Recent observations have raised the possibility that green tea catechins, in addition to their antioxidative properties, also affect the molecular mechanisms involved in angiogenesis, extracellular matrix degradation, regulation of cell death and multidrug resistance. This article will review the effects and the biological activities of green tea catechins in relation to these mechanisms, each of which plays a crucial role in the development of cancer in humans. The extraction of polyphenols from green tea, as well as their bioavailability, are also discussed since these two important parameters affect blood and tissue levels of the GTPs and consequently their biological activities. In addition, general perspectives on the application of dietary GTPs as novel antiangiogenic and antitumor compounds are also presented.

Transcriptional targeting of sphingosine-1-phosphate receptor S1P2 by epigallocatechin-3-gallate prevents sphingosine-1-phosphate-mediated signaling in macrophage-differentiated HL-60 promyelomonocytic leukemia cells.

BACKGROUND: Macrophage chemotaxis followed by blood-brain barrier transendothelial migration is believed to be associated with inflammation in the central nervous system. Antineuroinflammatory strategies have identified the dietary-derived epigallocatechin-3-gallate (EGCG) as an efficient agent to prevent neuroinflammation-associated neurodegenerative diseases by targeting proinflammatory mediator signaling. METHODS: Given that high levels of sphingosine kinase and its product, sphingosine-1-phosphate (S1P), are present in brain tumors, we used quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunoblotting to test whether EGCG may impact on S1P receptor gene expression and prevent S1P response in undifferentiated and in terminally differentiated macrophages. RESULTS: Promyelomonocytic human leukemia (HL)-60 cells were differentiated into macrophages, and S1P triggered phosphorylation in extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and P38 mitogen-activated protein kinase (MAPK) intracellular signaling, as shown by Western blot analysis. Pretreatment of cells with EGCG prior to differentiation inhibited the response to S1P in all three pathways, while EGCG abrogated P38 MAPK phosphorylation when present only during differentiation. Terminally-differentiated macrophages were, however, insensitive to EGCG treatment. Using qRT-PCR, gene expression of the S1P receptors S1P1, S1P2, and S1P5 was predominantly induced in terminally-differentiated macrophages, while the S1P2 was decreased by EGCG treatment. CONCLUSION: Our data suggest that diet-derived EGCG achieves efficient effects as a preventive agent, targeting signaling pathways prior to cell terminal differentiation. Such properties could impact on cell chemotaxis through the blood-brain barrier and prevent cancer-related neuroinflammation.

Propranolol suppresses angiogenesis in vitro: inhibition of proliferation, migration, and differentiation of endothelial cells.

Propranolol, a non-selective ß-adrenergic blocking drug, was recently reported to control the growth of hemangiomas, the most common vascular tumor of infancy. However, the mechanisms involved in this effect remain unknown. Here, we demonstrate that propranolol dose-dependently inhibited growth factor-induced proliferation of cultured human umbilical vein endothelial cells (HUVECs) through a G0/G1 phase cell cycle arrest. This was correlated to decreased cyclin D1, cyclin D3, and cyclin-dependent kinase CDK6 protein levels, while increases in the CDK inhibitors p15(INK4B), p21(WAF1/Cip1) and p27(Kip1) were observed. Chemotactic motility and differentiation of HUVECs into capillary-like tubular structures in Matrigel were also inhibited by propranolol. Furthermore, inhibition by propranolol of vascular endothelial growth factor (VEGF)-induced tyrosine phosphorylation of VEGF receptor-2 lead to inhibition of downstream signaling such as the activation of the extracellular signal-regulated kinase-1/2 and the secretion of the extracellular matrix degrading enzyme MMP-2. Taken together, these results demonstrate that propranolol interferes with several essential steps of neovascularization and opens up novel therapeutic opportunities for the use of ß-blockers in the treatment of angiogenesis-dependent human diseases.

The dietary flavones apigenin and luteolin impair smooth muscle cell migration and VEGF expression through inhibition of PDGFR-beta phosphorylation.

Platelet-derived growth factor (PDGF)-dependent recruitment of mural cells such as pericytes and smooth muscle cells plays a central role in the maturation and stabilization of newly formed vasculature during angiogenesis. In this work, we show that the dietary flavones apigenin and luteolin may interfere with this event through their inhibitory effect on PDGF-dependent phosphorylation of PDGF receptor beta (PDGFR-beta) in smooth muscle cells. Inhibition of PDGFR-beta activity by apigenin and luteolin occurred at low concentrations of the molecules and resulted in the inhibition of extracellular signal-regulated kinase and Akt phosphorylation triggered by PDGF, as well as in a marked reduction of the migratory and invasive properties of these cells. Apigenin and luteolin also strongly inhibit the PDGF-dependent increase in vascular endothelial growth factor (VEGF) mRNA levels and the secretion of VEGF by smooth muscle cells as well as vessel formation in the mouse Matrigel plug assay, suggesting that the inhibitory effects of both molecules on smooth muscle cell function result in impaired angiogenesis. Overall, these results identify apigenin and luteolin as dietary-derived inhibitors of PDGFR-beta activity and suggest that this inhibitory effect may contribute to the chemopreventive properties of these molecules.

Anthocyanidins inhibit migration of glioblastoma cells: structure-activity relationship and involvement of the plasminolytic system.

Complete resection of malignant glioblastomas is usually impossible because of diffuse and widespread invasion of tumor cells, and complementary approaches need to be developed in order to improve the efficacy of current treatments. Consumption of fruits and berries has been associated with decreased risk of developing cancer and there is great interest in the use of molecules from dietary origin to improve anticancer therapies. In this work, we report that the aglycons of the most abundant anthocyanins in fruits, cyanidin (Cy), delphinidin (Dp), and petunidin (Pt), act as potent inhibitors of glioblastoma cell migration. Dp clearly exhibited the highest inhibitory potency, this effect being related to the ortho-dihydroxyphenyl structure on the B-ring and the presence of a free hydroxyl group at position 3. Dp decreases the expression of both urokinase-type plasminogen activator receptor (uPAR) and the low-density lipoprotein receptor-related protein (LRP), acting at the transcriptional levels. In addition, Dp upregulated urokinase-type plasminogen activator (uPA) and downregulated the plasminogen activator inhibitor-1 (PAI-1) but decreased, in a concentration-dependent manner, the uPA-dependent conversion of plasminogen to plasmin, indicating that the upregulation of uPA observed with these compounds was not associated with induction of the plasminolytic activity. Overall, these results demonstrate that Dp, Pt, and Cy affect plasminogen activation, thus leading to the inhibition of glioblastoma cell migration and therefore they may be helpful for the development of new strategies for cancer prevention and therapy.