Effects Of Marchantin C And Fucoidan On Angiogenesis In Glioma Microenvironment And The Mechanical Study

BackgroundMalignant glioma is the most often-occurred primary intracranial tumor in adults and also one of the most refractory tumors. Despite various therapeutic methods such as surgery resection, radiotherapy and chemotherapy, no more than3%of patients with glioblastoma can survive five years. One of the main reasons for this recalcitrance is the excessive pathological angiogenesis in glioma tissue. Neovascularization is a hallmark of glioma biology and relates with its malignancy. In the highly complicated process of angiogenesis, vascular endothelial growth factor (VEGF) is studied extensively as a pro-angiogenic factor which believed to occupy a central position. Similar with neovascularization, VEGF concentration within a glioma was reported to correlate directly with its malignancy degree. In the microenvironment of glioma tissue, glioma cells and infiltrating myeloid cells are both important sources of VEGF. Although myeloid cells account for a much smaller fraction compared with tumor cells, they play an irreplaceable role in tumorigenic angiogenesis, probably because of the unique effect of myeloid-derived VEGF-A on phosphorylation of VEGF receptor2which is the major mediator of VEGF-A's effects on endothelial cells. Over past few decades, researchers have taken anti-angiogenic therapy as an effective anti-tumor method and several products have shown promising effects on tumor treatment. The products, mainly targeting VEGF signal transduction, include VEGF antibody, e.g. bevacizumab, VEGF receptor tyrosine kinase inhibitors, e.g. sunitinib, cediranib, and decoy ligand, i.e. VEGF-TRAP. Anti-angiogenic agents influence tumor growth mainly through two paths. Firstly, they starve tumor cells by depriving their delivery of nutrients and oxygen. Secondly, they can transiently normalize the tortuous tumor vasculature and thus enhance the efficacy of concurrent radiotherapy and chemotherapy. Despite the evidences of their efficiency in improving patients' prognosis, the effects of fore-mentioned products are still unsatisfactory and toxicity such as hypertension, proteinuria and thromboembolic events are frequently observed. Therefore, a novel endogenous anti-angiogenic factor—soluble VEGF receptor1(sFlt-1)—comes into the focus of many researches.sFlt-1was originally cloned in1993by Kendall et al. and is an alternatively spliced variant of membrane-binding VEGF receptor1. Compared with membrane-binding VEGF receptors, sFlt-1keeps the ligand-binding extracellular domain, but lacks transmembrane and intracellular domains. As the affinity of sFlt-1to VEGF-A is much stronger than that of VEGF receptor2, sFlt-1competitively inhibits the binding of VEGF ligands to VEGF receptor2. Another mechanism by which sFlt-1inhibits VEGF-dependent signaling is heterodimerization with membrane-binding receptors to form dominant-negative complexes. In human beings, sFlt-1mainly comes from vascular endothelial cells, but other cells such as glioma cells and monocytes are also sources of sFlt-1. With regard to gliomas, sFlt-1was reported to correlate with VEGF level, microvessel density, tumor malignancy and prognosis. Several studies have shown its efficacy in glioma treatment through gene delivery.Marchantin C, a family member of macrocyclic bisbibenzyls, is exclusively derived from liverworts. This class of compounds has shown a variety of biological functions, e.g. cytotoxicity, anti-tumor and anti-fungal activities. Marchantin C was reported to inhibit proliferation and invasion of glioma cell lines U87and T98G, or promote the apoptosis of A172glioma cell line. Fucoidan, a sulphated polysaccharide rich in L-fucose and ester groups, is extracted from brown seaweed and some marine invertebrates like sea urchins. It is also called fucan, fucosan or sulfated fucan. Fucoidan has more complicated biological functions than marchantin C, including anti-coagulant, anti-virus, anti-tumor, immunomodulatory, anti-oxidant, anti-complementary and anti-inflammatory activities. In TNF-alpha-and IFN-gamma-stimulated C6glioma cells, fucoidan suppresses NO production and iNOS expression through p38MAPK, JAK/STAT, AP-1and IRF-1pathways. Up to now, no study of marchantin C or fucoidan on sFlt-1production has been reported. In this study, we compared functions of marchantin C and fucoidan on sFlt-1secretion from glioma cells and monocytes and subsequent angiogenesis.Part I:Effects of marchantin C on angiogenesis in glioma microenvironment and the mechanical studyObjectives1. To study the effect of marchantin Con sFlt-1secretion from T98G or THP1cells.2. To study the effect of marchantin Con angiogenesis induced by T98G or THP1cells.3. To study the effect of sFlt-1on marchantin C's effect on T98G or THP1cells induced angiogenesis.4. To study the relative cytokine and mechanism in the process of marchantin C's effect on sFlt-1secretion from T98G or THP1cells.Methods1. The effect of marchantin Con sFlt-1secretion from T98G or THP1cells.Add marchantin C in the culture medium of T98G and THP1cells at the concentration of1μM,5μM,10μM and continue cell culture for6h,12h,24h. Collect the conditioned mediums and perform ELISA for sFlt-1detection.2. The effect of marchantin Con angiogenesis induced by T98G or THP1cells.Perform tube formation assay of HUVEC with conditioned mediums. After6h, observe and take pictures of HUVEC under a microscope. The level of tube formation was determined by counting branch points from tubes formed between discrete endothelial cells in three random fields per well.3. The effect of sFlt-1on marchantin C's effect on T98G or THP1cells induced angiogenesis.Design3groups:control group, drug group and antibody group. For control group, add the same amount of marchantin C into unconditioned medium. For antibody group, pretreat conditioned mediums with2μg/ml sFlt-1antibody for0.5hour at4℃. After6h, observe and take pictures of HUVEC under a microscope. The level of tube formation was determined by counting branch points from tubes formed between discrete endothelial cells in three random fields per well.4. The relative cytokine and mechanism in the process of marchantin C's effect on sFlt-1secretion from T98G or THP1cells.1) After stimulation of T98G and THP1cells with5μ M marchantin C for24h, collect cells and perform Real-time PCR for sFlt-1mRNA detection.2) After stimulation of T98G and THP1cells with5μ M marchantin C for24h, collect cells and perform cytometry assay for VEGFR1expression.3) After stimulation of T98G and THP1cells with5μ M marchantin C for24h, collect cells and perform Real-time PCR for MMP-2,7,9,12mRNA detection.4) Pretreat T98G and THP1cells with20μ M U0126or10μ M SB203580for24h, then collect cells and perform Real-time PCR for MMP-7mRNA detection.Results1. The effect of marchantin Con sFlt-1secretion from T98G or THP1cells.For stimulation of24h, sFlt-1level in the supernatant of T98G cells initially increased and then decreased as the concentration of marchantin C increased. At the concentration of5μ M, sFlt-1level in the supernatant of T98G cells increased as time increased. The sFlt-1level in the supernatant of THP1cells was not influenced by marchantin C at any concentration or time point.2. The effect of marchantin Con angiogenesis induced by T98G or THP1cells.Marchantin C significantly inhibited T98G cells induced angiogenesis while THP1cells induced angiogenesis was not significantly influenced.3. The effect of sFlt-1on marchantin C's effect on T98G or THP1cells inducedangiogenesis. Conditioned mediums significantly inhibited tube formation ability of HUVEC, sFlt-1antibody significantly restored the tube formation level. This effect was further confirmed by quantification of the tube formation level.4. The relative cytokine and mechanism in the process of marchantin C's effect on sFlt-1secretion from T98G or THP1cells.1) Marchantin C significantly decreased sFlt-1mRNA in T98G glioma cells.2) VEGFR1was scarcely expressed on the membrane of T98G cells.3) Marchantin C significantly decreased MMP-7mRNA in T98G cells while MMP-2,9,12were not influenced.4) ERK1/2signal pathway inhibitor U0126significantly inhibited MMP-7mRNA in T98G cells while p38inhibitor SB203580have no influence.Conclusion1. Marchantin C promotes sFlt-1secretion from T98G glioma cells but not THP1monocytes.2. Marchantin C inhibits angiogenesis induced by T98G glioma cells but not THP1monocytes.3. The inhibition of marchantin C on T98G cells induced angiogenesis is attributed to sFlt-1.4. Marchantin C inhibits MMP-7transcription in T98G cells, which is related with ERK1/2signal pathway.Part Ⅱ:Effects of fucoidan on angiogenesis in glioma microenvironment and the mechanical studyObjectives1. To study the effect of fucoidan on sFlt-1secretion from T98G or THP1cells.2. To study the effect of fucoidan on angiogenesis induced by T98G or THP1cells.3. To study the effect of sFlt-1on fucoidan's effect on T98G or THP1cells induced angiogenesis.4. To study the relative mechanisms in the process of fucoidan's effect on sFlt-1secretion from T98G or THP1cells. Methods1. The effect of fucoidan on sFlt-1secretion from T98G or THP1cells.Add fucoidan in the culture medium of T98G and THP1cells at the concentration of10μg/ml,50μg/ml,100μg/ml and continue cell culture for6h,12h,24h. Collect the conditioned mediums and perform ELISA for sFlt-1detection.2. The effect of fucoidan on angiogenesis induced by T98G or THP1cells.Perform tube formation assay of HUVEC with conditioned mediums. After6h, observe and take pictures of HUVEC under a microscope. The level of tube formation was determined by counting branch points from tubes formed between discrete endothelial cells in three random fields per well.3. The effect of sFlt-1on fucoidan's effect on T98G or THP1cells induced angiogenesis.Design3groups:control group, drug group and antibody group. For control group, add the same amount of fucoidan into unconditioned medium. For antibody group, pretreat conditioned mediums with2μg/ml sFlt-1antibody for0.5hour at4℃. After6h, observe and take pictures of HUVEC under a microscope. The level of tube formation was determined by counting branch points from tubes formed between discrete endothelial cells in three random fields per well.4. The relative cytokine and mechanism in the process of fucoidan's effect on sFlt-1secretion from T98G or THP1cells.1) After stimulation of T98G and THP1cells with100μg/ml fucoidan for24h, collect cells and perform Real-time PCR for sFlt-1mRNA detection.2) After stimulation of T98G and THP1cells with100μg/ml fucoidan for24h, collect cells and perform cytometry assay for VEGFR1expression.Results1. The effect of fucoidanon sFlt-1secretion from T98G or THP1cells. For stimulation of24h, sFlt-1level in the supernatant of T98G cells or THP1cells increased as the concentration of fucoidan increased. At the concentration of100μg/ml, sFlt-1level in the supernatant of T98G cells or THP1cells increased as time increased.2. The effect of fucoidanon angiogenesis induced by T98G or THP1cells.Fucoidan significantly inhibited T98G cells or THP1cells induced angiogenesis. This effect was further confirmed by quantification of the tube formation level.3. The effect of sFlt-1on fucoidan's effect on T98G or THP1cells induced angiogenesis.Conditioned mediums significantly inhibited tube formation ability of HUVEC, sFlt-1antibody significantly restored the tube formation level. This effect was further confirmed by quantification of the tube formation level.4. The relative mechanisms in the process of fucoidan's effect on sFlt-1secretion from T98G or THP1cells.1) Fucoidan significantly increased sFlt-1mRNA in T98G glioma cells while sFlt-1mRNA in THP1monocytes was not significantly influenced.2) VEGFR1was scarcely expressed on the membrane of T98G cells. Fucoidan significantly decreased expression of VEGFR1on THP1cells.Conclusion1. Fucoidan promotes sFlt-1secretion from T98G glioma cells and THP1monocytes.2. Fucoidan inhibits angiogenesis induced by T98G glioma cells or THP1monocytes.3. The inhibition of fucoidan on T98G cells or THP1cells induced angiogenesis is attributed to sFlt-1.4. Fucoidan increases sFlt-1transcription in T98G cells.5. Fucoidan decreases VEGFR1expression on THP1cells.