Consensus guidelines for the use and interpretation of angiogenesis assays.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Histone deacetylase 10 promotes autophagy-mediated cell survival.

Tumor cells activate autophagy in response to chemotherapy-induced DNA damage as a survival program to cope with metabolic stress. Here, we provide in vitro and in vivo evidence that histone deacetylase (HDAC)10 promotes autophagy-mediated survival in neuroblastoma cells. We show that both knockdown and inhibition of HDAC10 effectively disrupted autophagy associated with sensitization to cytotoxic drug treatment in a panel of highly malignant V-MYC myelocytomatosis viral-related oncogene, neuroblastoma derived-amplified neuroblastoma cell lines, in contrast to nontransformed cells. HDAC10 depletion in neuroblastoma cells interrupted autophagic flux and induced accumulation of autophagosomes, lysosomes, and a prominent substrate of the autophagic degradation pathway, p62/sequestosome 1. Enforced HDAC10 expression protected neuroblastoma cells against doxorubicin treatment through interaction with heat shock protein 70 family proteins, causing their deacetylation. Conversely, heat shock protein 70/heat shock cognate 70 was acetylated in HDAC10-depleted cells. HDAC10 expression levels in high-risk neuroblastomas correlated with autophagy in gene-set analysis and predicted treatment success in patients with advanced stage 4 neuroblastomas. Our results demonstrate that HDAC10 protects cancer cells from cytotoxic agents by mediating autophagy and identify this HDAC isozyme as a druggable regulator of advanced-stage tumor cell survival. Moreover, these results propose a promising way to considerably improve treatment response in the neuroblastoma patient subgroup with the poorest outcome.

The rise and fall of mandrake in medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Mandrake (Mandragora sp.) is one of the most famous medicinal plants. It has been in continuous medical use throughout written history and is still in use today in popular medicine. AIM OF THE STUDY: Mandrake derived drugs once played an important role in medicine and in magical practices. Today, the role of mandrake in popular medicine is marginal. However, natural products present in mandrake such as atropine and scopolamine, as well as their semi synthetic derivatives continue to hold and important role in medicine. Here we aim to trace the development of historical rationales and scientific events that led to the abandonment of mandrake as a medicine. MATERIALS AND METHODS: We review the medicinal uses of mandrake drugs since antiquity in an attempt to pinpoint use patterns that were popular in certain periods of time and others that are more general. We compare the uses from the native territories to those from regions where the plant got introduced and use literature reporting mandrake's chemistry and pharmacology in order to explain the diachronic changes of use patterns. RESULTS AND CONCLUSION: We found information about 88 different medicinal uses for mandrake, grouped into 39 conditions. According to the number of different medicinal uses, the most versatile period was the medieval (37), followed by the Renaissance (31), the classical (27), and the modern period (21). Considering the higher number of textual sources and use-records collected for the Renaissance period, the decrease of versatility in comparison to the medieval period appears robust. This seems to indicate a more consolidated use pattern, that might be conditioned by the reproduction of classic textual sources as well as by a less experimental approach and reduced popularity of mandrake in medicine. The introduction of the volatile anaesthetics with more reliable narcotic effects set the seal on using mandrake in surgery but opened the way for atropine being used as a prophylactic and antidote during surgical interventions.

Migration inhibition of mammary epithelial cells by Syk is blocked in the presence of DDR1 receptors.

The non-receptor tyrosine kinase Syk is a well-characterized hematopoietic signal transducer, which is also expressed in non-hematopoietic cells. In epithelial cells, the function of Syk is not wholly known. It interacts with the receptor tyrosine kinase DDR1 and is frequently lost from metastatic mammary tumors. Here, using genetic tracing, we demonstrate Syk expression in murine mammary epithelium, myoepithelium and skin epithelium, but not in intestinal or lung epithelia. Investigating possible functions of Syk, we found a substantial suppression of cell mobility that depended on Syk kinase activity in trans-well migration and wounding assays. Co-expression of DDR1 resulted in constitutive interaction and strong activation of Syk kinase. Most importantly, Syk-mediated migration inhibition was blocked in the presence of DDR1, while conversely DDR1 knockdown restored migration inhibition. Our study identifies Syk as a potent inhibitor of epithelial migration and describes a first functional consequence of the interaction with the collagen receptor DDR1.

Midkine Promotes Metastasis and Therapeutic Resistance via mTOR/RPS6 in Uveal Melanoma.

Uveal melanoma is a rare form of melanoma that originates in the eye, exerts widespread therapeutic resistance, and displays an inherent propensity for hepatic metastases. Because metastatic disease is characterized by poor survival, there is an unmet clinical need to identify new therapeutic targets in uveal melanoma. Here, we show that the pleiotropic cytokine midkine is expressed in uveal melanoma. Midkine expression in primary uveal melanoma significantly correlates with poor survival and is elevated in patients that develop metastatic disease. Monosomy 3 and histopathologic staging parameters are associated with midkine expression. In addition, we demonstrate that midkine promotes survival, migration across a barrier of hepatic sinusoid endothelial cells and resistance to AKT/mTOR inhibition. Furthermore, midkine is secreted and mediates mTOR activation by maintaining phosphorylation of the mTOR target RPS6 in uveal melanoma cells. Therefore, midkine is identified as a uveal melanoma cell survival factor that drives metastasis and therapeutic resistance, and could be exploited as a biomarker as well as a new therapeutic target. IMPLICATIONS: Midkine is identified as a survival factor that drives liver metastasis and therapeutic resistance in melanoma of the eye.

The PEA-15 protein regulates autophagy via activation of JNK.

PEA-15/PED (phosphoprotein enriched in astrocytes 15 kDa/phosphoprotein enriched in diabetes) is a death effector domain-containing protein which is known to modulate apoptotic cell death. The mechanism by which PEA-15 inhibits caspase activation and increases ERK (extracellular-regulated kinase) activity is well characterized. Here, we demonstrate that PEA-15 is not only pivotal in the activation of the ERK pathway but also modulates JNK (c-Jun N-terminal kinase) signaling. Upon overexpression of PEA-15 in malignant glioma cells, JNK is potently activated. The PEA-15-induced JNK activation depends on the phosphorylation of PEA-15 at both phosphorylation sites (serine 104 and serine 116). The activation of JNK is substantially inhibited by siRNA-mediated down-regulation of endogenous PEA-15. Moreover, we demonstrate that glioma cells overexpressing PEA-15 show increased signs of autophagy in response to classical autophagic stimuli such as ionizing irradiation, serum deprivation, or rapamycin treatment. In contrast, the non-phosphorylatable mutants of PEA-15 are not capable of promoting autophagy. The inhibition of JNK abrogates the PEA-15-mediated increase in autophagy. In conclusion, our data show that PEA-15 promotes autophagy in glioma cells in a JNK-dependent manner. This might render glioma cells more resistant to adverse stimuli such as starvation or ionizing irradiation.

Medicinal plants of the Bible-revisited.

BACKGROUND: Previous lists number from 55 to 176 plant species as "Biblical Medicinal Plants." Modern studies attest that many names on these lists are no longer valid. This situation arose due to old mistranslations and/or mistakes in botanical identification. Many previously recognized Biblical plants are in no way related to the flora of the Bible lands. Accordingly, the list needs revision. METHODS: We re-examine the list of possible medicinal plants in the Bible based on new studies in Hebrew Biblical philology and etymology, new studies on the Egyptian and Mesopotamian medicinal use of plants, on ethnobotany and on archaeobotany. RESULTS: In our survey, we suggest reducing this list to 45 plant species. Our contribution comprises 20 "newly" suggested Biblical Medicinal Plants. Only five species are mentioned directly as medicinal plants in the Bible: Fig (Ficus carica), Nard (Nardostachys jatamansi), Hyssop (Origanum syriacum), balm of Gilead (Commiphora gileadensis) and Mandrake (Mandragora officinarum). No fewer than 18 medicinal plants are mentioned in old Jewish post-Biblical sources, in addition to those in the Bible. Most of these plants (15) are known also in Egypt and Mesopotamia while three are from Egypt only. Seven of our suggested species are not mentioned in the Bible or in the Jewish post-Biblical literature but were recorded as medicinal plants from Egypt, as well as from Mesopotamia. It is quite logical to assume that they can be included as Biblical Medicinal Plants. CONCLUSIONS: All our suggested Biblical Medicinal Plants are known as such in Ancient Egypt and/or Mesopotamia also. Examination of our list shows that all these plants have been in continuous medicinal use in the Middle East down the generations, as well as being used in the Holy Land today. Precisely in King Solomon's words, "That which has been is what will be, that which is done is what will be done. And there is nothing new under the sun" (Ecclesiastes 1:9).

Characterization of CD4+ T cells primed and boosted by MHCII primary uveal melanoma cell-based vaccines.

Uveal melanoma is the most common primary malignancy of the eye in adults. Despite significant improvements in treatment of the primary tumor, to date none of these therapies prevent metastatic disease or improve overall survival. We are exploring immunotherapeutic options for metastatic uveal melanoma using MHC II uveal melanoma cell-based vaccines that target the activation of tumor-reactive CD4+ T cells. Previously, we showed that these uveal melanoma cell-based vaccines activate CD4+ T cells within total peripheral blood lymphocytes (PBMC). Since PBMC include professional antigen presenting cells, we now demonstrate that Mel202/DR1/CD80 vaccine cells directly activate a diverse repertoire of purified, naïve CD4+ T cells. The activated CD4+ T cells proliferated, secreted high amounts of interferon gamma (IFNγ) and produced a heterogeneous profile of Th1, Th2 and Th17 cytokines. Analysis of the TCR-Vß-repertoire showed that a polyclonal T cell response was induced, suggesting the capacity of vaccine-activated CD4+ T cells to target multiple tumor (neo)antigens. In addition, a subset of the responding CD4+ T cells expressed forkhead box protein P3 (FoxP3), indicating that although a regulatory component of the vaccine-activated CD4+ T cell response was induced, the anti-tumor vaccine response was not limited by these regulatory CD4+ T cells. Finally, Mel202/DR1/CD80 uveal melanoma vaccine cells expressed the intercellular adhesion molecule 1 (ICAM-1) that was pivotal for CD4+ T cell activation via lymphocyte function-associated antigen 1(LFA-1). In conclusion, MHC II uveal melanoma vaccines activate purified CD4+ T cells and may serve as a novel immunotherapy for uveal melanoma patients.

Basal caspase activity promotes migration and invasiveness in glioblastoma cells.

Glioblastomas, the most malignant of all brain tumors, are characterized by cellular resistance to apoptosis and a highly invasive growth pattern. These factors contribute to the poor response of glioblastomas to radiochemotherapy and prevent their complete neurosurgical resection. However, the driving force behind the distinct motility of glioma cells is only partly understood. Here, we report that in the absence of cellular stress and proapoptotic stimuli, human glioblastoma cells exhibit a constitutive activation of caspases in vivo and in vitro. The inhibition of caspases by various peptide inhibitors decreases the migration of cells in scrape motility assays and the invasiveness of cells in spheroid assays. Similarly, specific small interfering RNA- or antisense-mediated down-regulation of caspase-3 and caspase-8 results in an inhibition of the migratory potential of glioma cells. The constitutive caspase-dependent motility of glioblastoma cells is independent of CD95 activation and it is not mediated by mitogen-activated protein/extracellular signal-regulated kinase kinase signaling. The basal caspase activity is accompanied by a constant cleavage of the motility-associated gelsolin protein, which may contribute to the caspase-mediated promotion of migration and invasiveness in glioblastoma cells. Our results suggest that the administration of low doses of caspase inhibitors that block glioma cell motility without affecting the execution of apoptotic cell death may be exploited as a novel strategy for the treatment of glioblastomas.

Mouse models of human cancer.

The Helmholtz Alliance Preclinical Comprehensive Cancer Center (PCCC; www.helmholtz-pccc.de) hosted the "1st International Kloster Seeon Meeting on Mouse Models of Human Cancer" in the Seeon monastery (Germany) from March 8 to 11, 2014. The meeting focused on the development and application of novel mouse models in tumor research and high-throughput technologies to overcome one of the most critical bottlenecks in translational bench-to-bedside tumor biology research. Moreover, the participants discussed basic molecular mechanisms underlying tumor initiation, progression, metastasis, and therapy resistance, which are the prerequisite for the development of novel treatment strategies and clinical applications in cancer therapy.

Regulation of developmental lymphangiogenesis by Syk(+) leukocytes.

Lymphatic vessels are essential for tissue homeostasis and immune surveillance and contribute to pathological conditions. Lymphatic endothelium differentiates from veins and forms an independent vascular tree with only few connections to the venous circulation. Failure of blood and lymphatic vessel separation results in hemorrhage and edema. VEGF-C and -D are strong inducers of lymphangiogenesis and have essential (VEGF-C) and modulatory (VEGF-D) roles during developmental lymphangiogenesis. We describe here a myeloid population that is defined by expression of the tyrosine kinase Syk, comprises largely M2-polarized mononuclear cells, and robustly expresses angiogenic factors, including VEGF-C/-D and chemokines. These cells stimulate lymphangiogenesis in vivo. Deletion of Syk causes increased chemotractant production, enhanced transmigration, and accumulation in the skin. Ensuing lymphatic hyperplasia and vessel dilation cause the formation of blood-lymphatic shunts. This mechanism does not involve circulating endothelial progenitor cells and demonstrates the potential of hematopoietic cells to control developmental lymphangiogenesis.

Troglitazone sensitizes tumor cells to TRAIL-induced apoptosis via down-regulation of FLIP and Survivin.

Induction of apoptosis by the death ligand TRAIL might be a promising therapeutic approach in cancer therapy. However, since not all tumor cells are sensitive to TRAIL, there is a need for the development of strategies to overcome TRAIL-resistance. The results of the present study show that the anti-diabetic drug troglitazone sensitizes human glioma and neuroblastoma cells to TRAIL-induced apoptosis. This process is accompanied by a substantial increase of active caspase 8 and active caspase 3, but it is independent of troglitazone's effects on the nuclear receptor PPAR-gamma. Troglitazone induces a pronounced reduction in protein expression levels of the anti-apoptotic FLICE-inhibitory protein (FLIP) without affecting FLIP mRNA levels. Further, protein and mRNA expression levels of the anti-apoptotic protein Survivin significantly decrease upon treatment with troglitazone. Moreover, sensitization to TRAIL is partly accompanied by an up-regulation of the TRAIL receptor, TRAIL-R2. A combined treatment with troglitazone and TRAIL might be a promising experimental therapy because troglitazone sensitizes tumor cells to TRAIL-induced apoptosis via various mechanisms, thereby minimizing the risk of acquired tumor cell resistance.

Cdc42 induces activation loop phosphorylation and membrane targeting of mixed lineage kinase 3.

Mixed lineage kinase 3 (MLK3) functions as a mitogen-activated protein kinase kinase kinase to activate multiple mitogen-activated protein kinase pathways. Our current studies demonstrate that lack of MLK3 blocks signaling of activated Cdc42 to c-Jun N-terminal kinase, giving strong support for the idea that Cdc42 is a physiological activator of MLK3. We show herein that Cdc42, in a prenylation-dependent manner, targets MLK3 from a perinuclear region to membranes, including the plasma membrane. Cdc42-induced membrane targeting of MLK3 is independent of MLK3 catalytic activity but depends upon an intact Cdc42/Rac-interactive binding motif, consistent with MLK3 membrane translocation being mediated through direct binding of Cdc42. Phosphorylation of the activation loop of MLK3 requires MLK3 catalytic activity and is induced by Cdc42 in a prenylation-independent manner, arguing that Cdc42 binding is sufficient for activation loop autophosphorylation of MLK3. However, membrane targeting is necessary for full activation of MLK3 and maximal signaling to JNK. We previously reported that MLK3 is autoinhibited through an interaction between its N-terminal SH3 domain and a proline-containing sequence found between the leucine zipper and the CRIB motif of MLK3. Thus we propose a model in which GTP-bound Cdc42/Rac binds MLK3 and disrupts SH3-mediated autoinhibition leading to dimerization and activation loop autophosphorylation. Targeting of this partially active MLK3 to membranes likely results in additional phosphorylation events that fully activate MLK3 and its ability to maximally signal through the JNK pathway.

Mixed lineage kinase 3 inhibits platelet-derived growth factor-stimulated DNA synthesis and matrix mRNA expression in mesangial cells.

Mixed lineage kinase 3 (MLK 3) is a recently described member of the MLK subfamily of Ser/Thr protein kinases that interacts with MAPK pathways. The aim of this study was to test the potential interaction of MLK 3 with signaling pathways stimulated by PDGF in rat mesangial cells. We have established a stable cell line expressing human MLK 3 in rat glomerular mesangial cells. The effects of PDGF on proliferation and matrix mRNA expression were examined. In control (vector-transfected) mesangial cells PDGF increased [(3)H]-thymidine incorporation significantly in a concentration-dependent manner. In mesangial cells expressing MLK 3, PDGF-induced increase in DNA synthesis was significantly reduced. PDGF also induced fibronectin and collagen I mRNA expression in control cells, the effects of which were also significantly blocked in MLK 3-transfected cells. To understand the potential interaction of MLK 3 over expression with the MAPK pathways and to examine the potential mechanism of the effects of MLK 3 over expression on proliferation and matrix expression, activation of ERK2, JNK1 and p38 were examined. ERK2 activation was increased several fold by PDGF in control cells but was attenuated significantly in MLK 3 expressing cells. PDGF did not have any effect on JNK and p38 activation, in either cell types. Using the same stable-transfected cell line, identical results were obtained on proliferation and matrix expression with sarafotoxin-s6b (endothelin receptor agonist) another potent mitogenic and sclerotic agent for mesangial cells. These results indicate an important role for MLK 3 in the regulation of growth and matrix expression in mesangial cells.

Mixed lineage kinase 3 inhibits phorbol myristoyl acetate-induced DNA synthesis but not osteopontin expression in rat mesangial cells.

Mixed lineage kinase 3 (MLK 3) (also called SPRK or PTK-1) is a recently described member of the family of the mixed lineage kinase subfamily of Ser/Thr protein kinases that interacts with mitogen-activated protein kinase pathways. In order to test the biological relevance and potential interaction of MLK 3 with protein kinase C-mediated signaling pathways, human MLK 3 was stably expressed in rat glomerular mesangial cells using a retroviral vector (LXSN) and the effects of phorbol myristoyl acetate (PMA) on DNA synthesis and osteopontin mRNA expression were examined. In control (vector-transfected) mesangial cells PMA increased [3H]-thymidine incorporation in a concentration-dependent manner. In mesangial cells stably expressing MLK 3, the PMA-induced increase in [3H]-thymidine incorporation was significantly reduced (> 50%). However, the PMA-induced increase in osteopontin mRNA was not affected by MLK 3 expression. To determine the mechanisms of these effects, activation of ERK2, JNK1 and p38 in response to PMA was examined in both vector and MLK 3 transfected cells. ERK2 activation was increased several fold by PMA in control cells but was attenuated significantly in MLK 3 expressing cells, suggesting that MLK 3 expression in mesangial cells can negatively regulate the ERK pathway. PMA had no significant effect on JNK and P38 activation, in either vector- or MLK 3-expressing cells. PD98059, a MEK inhibitor blocked PMA-induced DNA synthesis without affecting osteopontin expression. These results suggest that while protein kinase C activation increases cellular proliferation and osteopontin mRNA expression, over-expression of MLK 3 affects only the PKC-induced DNA synthesis, probably through inhibition of ERK. These results also indicate a novel mechanism of growth regulation by a member of the mixed-lineage kinase family that might have significant therapeutic implications in proliferative glomerulonephritis.