Ablation of PRMT6 reveals a role as a negative transcriptional regulator of the p53 tumor suppressor.

Arginine methylation of histones is a well-known regulator of gene expression. Protein arginine methyltransferase 6 (PRMT6) has been shown to function as a transcriptional repressor by methylating the histone H3 arginine 2 [H3R2(me2a)] repressive mark; however, few targets are known. To define the physiological role of PRMT6 and to identify its targets, we generated PRMT6(-/-) mouse embryo fibroblasts (MEFs). We observed that early passage PRMT6(-/-) MEFs had growth defects and exhibited the hallmarks of cellular senescence. PRMT6(-/-) MEFs displayed high transcriptional levels of p53 and its targets, p21 and PML. Generation of PRMT6(-/-); p53(-/-) MEFs prevented the premature senescence, suggesting that the induction of senescence is p53-dependent. Using chromatin immunoprecipitation assays, we observed an enrichment of PRMT6 and H3R2(me2a) within the upstream region of Trp53. The PRMT6 association and the H3R2(me2a) mark were lost in PRMT6(-/-) MEFs and an increase in the H3K4(me3) activator mark was observed. Our findings define a new regulator of p53 transcriptional regulation and define a role for PRMT6 and arginine methylation in cellular senescence.

Quercetin abrogates IL-6/STAT3 signaling and inhibits glioblastoma cell line growth and migration.

Evidence has suggested that STAT3 functions as an oncogene in gliomagenesis. As a consequence, changes in the inflammatory microenvironment are thought to promote tumor development. Regardless of its origin, cancer-related inflammation has many tumor-promoting effects, such as the promotion of cell cycle progression, cell proliferation, cell migration and cell survival. Given that IL-6, a major cancer-related inflammatory cytokine, regulates STAT3 activation and is upregulated in glioblastoma, we sought to investigate the inhibitory effects of the chemopreventive flavonoid quercetin on glioblastoma cell proliferation and migration triggered by IL-6, and to determine the underlying mechanisms of action. In this study, we show that quercetin is a potent inhibitor of the IL-6-induced STAT3 signaling pathway in T98G and U87 glioblastoma cells. Exposure to quercetin resulted in the reduction of GP130, JAK1 and STAT3 activation by IL-6, as well as a marked decrease of the proliferative and migratory properties of glioblastoma cells induced by IL-6. Interestingly, quercetin also modulated the expression of two target genes regulated by STAT3, i.e. cyclin D1 and matrix metalloproteinase-2 (MMP-2). Moreover, quercetin reduced the recruitment of STAT3 at the cyclin D1 promoter and inhibited Rb phosphorylation in the presence of IL-6. Overall, these results provide new insight into the role of quercetin as a blocker of the STAT3 activation pathway stimulated by IL-6, with a potential role in the prevention and treatment of glioblastoma.

Thrombospondin-1 is a transcriptional repression target of PRMT6.

Protein arginine methyltransferase 6 (PRMT6) is known to catalyze the generation of asymmetric dimethylarginine in polypeptides. Although the cellular role of PRMT6 is not well understood, it has been implicated in human immunodeficiency virus pathogenesis, DNA repair, and transcriptional regulation. PRMT6 is known to methylate histone H3 Arg-2 (H3R2), and this negatively regulates the lysine methylation of H3K4 resulting in gene repression. To identify in a nonbiased manner genes regulated by PRMT6 expression, we performed a microarray analysis on U2OS osteosarcoma cells transfected with control and PRMT6 small interfering RNAs. We identified thrombospondin-1 (TSP-1), a potent natural inhibitor of angiogenesis, as a transcriptional repression target of PRMT6. Moreover, we show that PRMT6-deficient U2OS cells exhibited cell migration defects that were rescued by blocking the secreted TSP-1 with a neutralizing peptide or blocking alpha-TSP-1 antibody. PRMT6 associates with the TSP-1 promoter and regulates the balance of methylation of H3R2 and H3K4, such that in PRMT6-deficient cells H3R2 was hypomethylated and H3K4 was trimethylated at the TSP-1 promoter. Using a TSP-1 promoter reporter gene, we further show that PRMT6 directly regulates the TSP-1 promoter activity. These findings show that TSP-1 is a transcriptional repression target of PRMT6 and suggest that neutralizing the activity of PRMT6 could inhibit tumor progression and therefore may be of cancer therapeutic significance.

Arginine methylation of the histone H3 tail impedes effector binding.

Histone tail post-translational modification results in changes in cellular processes, either by generating or blocking docking sites for histone code readers or by altering the higher order chromatin structure. H3K4me3 is known to mark the promoter regions of active transcription. Proteins bind H3K4 in a methyl-dependent manner and aid in the recruitment of histone-remodeling enzymes and transcriptional cofactors. The H3K4me3 binders harbor methyl-specific chromatin binding domains, including plant homeodomain, Chromo, and tudor domains. Structural analysis of the plant homeodomains present in effector proteins, as well as the WD40 repeats of WDR5, reveals critical contacts between residues in these domains and H3R2. The intimate contact between H3R2 and these domain types leads to the hypothesis that methylation of this arginine residue antagonizes the binding of effector proteins to the N-terminal tail of H3. Here we show that H3 tail binding effector proteins are indeed sensitive to H3R2 methylation and that PRMT6, not CARM1/PRMT4, is the primary methyltransferase acting on this site. We have tested the expression of a select group of H3K4 effector-regulated genes in PRMT6 knockdown cells and found that their levels are altered. Thus, PRMT6 methylates H3R2 and is a negative regulator of N-terminal H3 tail binding.

Plasminogen-dependent internalization of soluble melanotransferrin involves the low-density lipoprotein receptor-related protein and annexin II.

We investigated the effect of plasminogen (Plg) on the internalization of recombinant soluble melanotransferrin (sMTf) using U87 human glioblastoma cells and murine embryonic fibroblasts (MEF) deficient in the low-density lipoprotein receptor-related protein (LRP). Using biospecific interaction analysis, both Glu- and Lys-Plg were shown to interact with immobilized sMTf. The binding of sMTf at the cell surface increased in the presence of both forms of Plg in control and in LRP-deficient MEF cells, whereas the uptake was strongly stimulated only by Lys-Plg in control MEF and U87 cells. In addition, in the presence of Lys-Plg, the internalization of sMTf was a saturable process, sensitive to temperature and dependent on the integrity of lysine residues. The addition of the receptor-associated protein, lactoferrin and aprotinin, as well as a monoclonal antibody (mAb) directed against LRP, inhibited the Lys-Plg-dependent uptake of sMTf. These results suggest an important role for LRP in this process. In addition, using binding and uptake assays in the presence of anti-annexin II mAb, we showed that annexin II might be responsible for the initial binding of sMTf in the presence of Plg. Our results suggest a Plg-mediated internalization mechanism for the clearance of sMTf via annexin II and LRP.

Inhibition of tumor growth by a truncated and soluble form of melanotransferrin.

Melanotransferrin is a glycoprotein expressed at the cell membrane and secreted in the extracellular environment. Recombinant truncated form of membrane-bound melanotransferrin (sMTf) was reported to exert in vitro anti-angiogenic properties. Here we show that sMTf treatment leads to a 50% inhibition of neovascularization in Matrigel implants when stimulated by growth factors. Using a glioblastoma xenograft model, we demonstrate that sMTf delivery at 2.5 and 10 mg/kg/day by micro-osmotic pump inhibits tumor growth by 73% and 91%, respectively. In a lung carcinoma xenograft model, sMTf treatment at 2.5 and 10 mg/kg/day impeded tumor growth by 87% and 97%. Furthermore, subcutaneous glioblastoma and lung carcinoma tumors from mice treated with 10 mg/kg/day of sMTf present insignificant growth toward the study. In association with a reduction in endoglin mRNA expression, the hemoglobin content decreased by half in sMTf-treated glioblastoma tumors. In vitro experiments revealed that NCI-H460 cells treated with sMTf display an inhibition in their invasive capabilities with a concomitant reduction in the expression of the low-density lipoprotein receptor protein and urokinase plasminogen activator receptor. Altogether, our results demonstrate that sMTf exerts anti-cancer and anti-angiogenic activities, suggesting that its administration may provide novel therapeutic strategies for the treatment of cancer.

Lebectin, a Macrovipera lebetina venom-derived C-type lectin, inhibits angiogenesis both in vitro and in vivo.

Integrins play an essential role in endothelial cell motility processes during angiogenesis and thus present interesting targets for the development of new anti-angiogenic agents. Snake venoms naturally contain a variety of proteins that can affect integrin-ligand interactions. Recently, the C-type lectin proteins (CLPs) have been characterized as efficient modulators of integrin functions. In this study, we investigated the anti-angiogenic activity of lebectin, a newly discovered CLP from Macrovipera lebetina venom. Human brain microvascular endothelial cells (HBMEC), used as an in vitro model, express alphavbeta3, alphavbeta5, and alpha5beta1 integrins, as well as the alpha2, alpha3, alpha6, and beta4 subunits. Our data show that lebectin acts as a very potent inhibitor (IC(50) approximately 0.5 nM) of HBMEC adhesion and migration on fibronectin by blocking the adhesive functions of both the alpha5beta1 and alphaV integrins. In addition, lebectin strongly inhibits both HBMEC in vitro tubulogenesis on Matrigel trade mark (IC(50) = 0.4 nM) and proliferation. Finally, using both a chicken CAM assay and a Matrigel trade mark Plug assay in nude mice, our results show that lebectin displays potent anti-angiogenic activity in vivo. Lebectin thus represents a new C-type lectin with anti-angiogenic properties with great potential for the treatment of angiogenesis-related diseases.

Melanotransferrin induces human melanoma SK-Mel-28 cell invasion in vivo.

The expression of melanotransferrin (MTf), a membrane-bound glycoprotein highly expressed in melanomas, is correlated with tumor vascularization and progression, suggesting a proinvasive function associated with MTf in malignant tumors. To test this hypothesis, we silenced MTf in human melanoma SK-MEL-28 cells using small interfering RNA (siRNA) and examined the plasmin activity and invasiveness of MTf-silenced melanoma. In vitro, the siRNA-mediated MTf knockdown inhibited by 58% the cell surface activation of plasminogen into plasmin. In addition, decreased expression of MTf in melanoma cells reduced cell migration. In vivo, we used a nude mice invasion model in which tissue factor (TF) induces vascular [125I]-fibrin deposition following injection. Using this metastasis model, the invasive potential of MTf-silenced cells into the lungs was reduced by fivefold. Altogether, these findings strongly suggest that MTf overexpression in melanoma cells contributes to tumor progression by stimulating plasmin generation as well as cell migration and invasion.

In vivo inhibition of angiogenesis by a soluble form of melanotransferrin.

Melanotransferrin (MTf), the membrane-bound human melanoma antigen p97, binds to plasminogen and stimulates its activation, thus regulating a crucial step involved in angiogenesis. In our study, a truncated soluble form of MTf (sMTf) inhibits, in a dose-dependent manner, the in vitro tubulogenesis of human umbilical vessel endothelial cells (HUVEC) induced by media conditioned with MTf-expressing cells. Following these results, we used the in vivo Matrigel plug angiogenesis assay to investigate whether truncated sMTf could inhibit neovascularization in mice. We found that basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and MTf-expressing cells strongly stimulate in vivo Matrigel neovascularization. However, subcutaneous (s.c.) administration of truncated sMTf inhibits both bFGF- and VEGF- as well as human melanoma SK-Mel-28-induced angiogenesis. This inhibition was dependent on the truncated sMTf concentration and reached maximal inhibition at 40 mg/kg/week. Furthermore, we found that a single s.c. injection of truncated sMTf into nude mice at 5 mg/kg produced a maximal blood concentration of approximately 40 nM, which is comparable with the level required to inhibit in vitro HUVEC tubulogenesis. Overall, our results strongly suggest that s.c. administration of truncated sMTf may provide a novel therapeutic strategy for the treatment of angiogenesis-related disorders.

Delphinidin, a dietary anthocyanidin, inhibits vascular endothelial growth factor receptor-2 phosphorylation.

Epidemiological studies have shown that a diet rich in fruits and vegetables has a beneficial preventive effect on cardiovascular diseases and cancer by mechanisms that have not yet been elucidated. In this work, we investigated the antiangiogenic activities of anthocyanidins, a class of polyphenols present at high levels in fruits. Among the tested anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin), delphinidin was the most potent angiogenic inhibitor. In vitro, low concentrations of delphinidin inhibited vascular endothelial growth factor (VEGF)-induced tyrosine phosphorylation of VEGF receptor (VEGFR)-2, leading to the inhibition of downstream signaling triggered by VEGFR-2. Inhibition of VEGFR-2 by delphinidin inhibited the VEGF-induced activation of ERK-1/2 signaling and the chemotactic motility of human EC as well as their differentiation into capillary-like tubular structures in Matrigel and within fibrin gels. In vivo, delphinidin was able to suppress basic fibroblast growth factor-induced vessel formation in the mouse Matrigel plug assay. The identification of delphinidin as a naturally occurring inhibitor of VEGF receptors suggests that this molecule possesses important antiangiogenic properties that may be helpful for the prevention and treatment of cancer.

Stimulation of cell surface plasminogen activation by membrane-bound melanotransferrin: a key phenomenon for cell invasion.

The activation of plasminogen at the cell surface is a crucial step in cell migration and invasion. In the present study, the effect of membrane-bound melanotransferrin (mMTf), also known as human melanoma antigen p97, on cell surface plasminogen binding and activation was investigated by using Chinese Hamster Ovary (CHO) cells transfected with full-length melanotransferrin (MTf) cDNA and SK-MeL-28 melanoma cells. The expression of mMTf in CHO increased cell surface plasminogen binding by about 2-fold. In addition, application of the monoclonal antibody L235 against MTf as well as truncated, soluble MTf (sMTf) abolished plasminogen binding to MTf-transfected and SK-MeL-28 cells, indicating that mMTf is a potential cell surface plasminogen receptor. Moreover, mMTf expression in CHO cells stimulates plasminogen activation at the cell surface by about 2.5-fold. In addition to the induced binding and activation of plasminogen, cell motility, migration and invasion were about 3-fold higher in CHO cells expressing mMTf. Both monoclonal antibody L235 and truncated sMTf inhibited mMTf-stimulated CHO cell motility, migration and invasion. Overall, our results indicate a key role for mMTf in cell surface plasminogen binding and in activation processes involved during cell migration and invasion.

Inhibition of endothelial cell movement and tubulogenesis by human recombinant soluble melanotransferrin: involvement of the u-PAR/LRP plasminolytic system.

We have previously demonstrated that human recombinant soluble melanotransferrin (hr-sMTf) interacts with the single-chain zymogen pro urokinase-type plasminogen activator (scu-PA) and plasminogen. In the present work, the impact of exogenous hr-sMTf on endothelial cells (EC) migration and morphogenic differentiation into capillary-like structures (tubulogenesis) was assessed. hr-sMTF at 10 nM inhibited by 50% the migration and tubulogenesis of human microvessel EC (HMEC-1). In addition, in hr-sMTf-treated HMEC-1, the expression of both urokinase-type plasminogen activator receptor (u-PAR) and low-density lipoprotein receptor-related protein (LRP) are down-regulated. However, fluorescence-activated cell sorting analysis revealed a 25% increase in cell surface u-PAR in hr-sMTf-treated HMEC-1, whereas the binding of the urokinase-type plasminogen activator (u-PA)*plasminogen activator inhibitor-1 (PAI-1) complex is decreased. This reduced u-PA-PAI-1 binding is correlated with a strong inhibition of the HMEC-1 plasminolytic activity, indicating that exogenous hr-sMTf treatment alters the internalization and recycling processes of free and active u-PAR at the cellular surface. Overall, these results demonstrate that exogenous hr-sMTf affects plasminogen activation at the cell surface, thus leading to the inhibition of EC movement and tubulogenesis. These results are the first to consider the potential use of hr-sMTf as a possible therapeutic agent in angiogenesis-related pathologies.

Regulation of plasminogen activation: a role for melanotransferrin (p97) in cell migration.

We recently reported that human recombinant melanotransferrin (p97) presents a high transport rate across the blood-brain barrier that might involve the low-density lipoprotein receptor-related protein (LRP). We now report new interactions between p97 and another LRP ligand, the urokinase plasminogen activator (uPA) complex. By using biospecific interaction analysis, both pro-uPA and plasminogen are shown to interact with immobilized p97. Moreover, the activation of plasminogen by pro-uPA is increased by soluble p97. Because the uPA system plays a crucial role in cell migration, both in cancer and in angiogenesis, we also measured the impact of both endogenous membrane-bound and exogenous p97 on cell migration. The monoclonal antibody L235 (which recognizes a conformational epitope on p97) inhibited the migration of human microvascular endothelial cells (HMECs-1) and of human melanoma SK-MEL-28 cells, indicating that endogenous membrane-bound p97 could be associated with this process. In addition, low concentrations of exogenous p97 (10 and 100 nM) inhibited HMEC-1 and SK-MEL28 cell migration by more than 50%. These results indicate that membrane-bound and soluble p97 affect the migration capacity of endothelial and melanoma cells and suggest that p97 could be involved in the regulation of plasminogen activation by interacting with pro-uPA and plasminogen.

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.