| Malignant gliomas are common primary tumors within the central nervous system, which are characterized by invasive growth, aberrant neovascularizion and rapid expansion. The active neovascularization not only contributes to the rapid growth of tumor cells by providing them nutrient and oxygen, but also facilitates these cells to invade into normal brain tissues, resulting in the difficulties in malignat glioma therapies. Thus, it is of great importance to further investigate the mechanism of angiogenesis for developing new antiangiogenesis strategy to treat such kind of malignancy.Tumor angiogenesis is regulated by aberrant production of proangiogenic and antiangiogenic factors produced by malignant tumor cells as well as the infiltrating leukocytes. VEGF and IL-8 are two important angiogenic factors. They promote endothelial cell proliferation, migration and tubule formation, which are cruiel steps in angiogenesis. Recently, it was reported that the CXC chemokine receptor CXCR4 regulated pathological angiogenesis, tumor invasion and metastasis. Our previous studies showed that CXCR4 expression was correlated directly with the degree of malignancy and microvessel density (MVD) of human gliomas. However, the machenism of CXCR4-induced angiogenesis remains to be clarified.Cancer stem cells (CSCs), or tumor initiating cells, are becoming fascinating because of their special biologic behaviors and the important roles in carcinogenesis. Although these cells account for only a small fraction of cancer cell population, they exclusively exhibit capacities of self-renewal, multipotent differentiation and tumor initiation. CSCs are also resistant to chemo- and radiotherapy, thus possibly responsible for cancer recurrence after treatment and metastasis. The great progress of study on CSCs is enormously challenging traditional theory of tumor vascularization, for instance, whether and how CSCs initiate angiogenesis. Our previous results showed that glioma stem cells (GSCs) produced higher levels of VEGF and IL-8 compared to the differentiated tumor cells. However, it is unclear whether GSCs produce VEGF and IL-8 constitutively or the production is induced by factors present in tumor microenvironment. Recently, we found high level of CXCR4 expression by GSCs with unknown function.In this study, we hypothezed that CXCR4 activation might initiate GSCs-mediated angiogenesis by promoting the production of VEGF and IL-8. We firstly decteted the effect of CXCR4 activation on a malignant glioma cell line U87 cell invasion and the production of angiogenic factors, and explored the inhibitory effect of Nordy, a sythesized chiral compound based on the structure of natural nordihydroguaiaretic acid (NDGA), on the response of U87 cells to CXCR4 agonist CXCL12. We then detected the distribution of GSCs in primary glioma tissues, isolated and characterized GSCs from the tissues, glioma cell lines and their xenografts. We further investigated the activation of CXCR4 on GSCs-induced angiogenesis and the potential mechanisms. Finally, we explored the therapeutic significance of inhibiting CXCR4 with GSCs xenograft model. The main results and conclusions are as follows:1. CXCR4 activation promoted U87 cells to migrate and produce VEGF and IL-8. (1) Stimulation of U87 cells with CXCL12, the CXCR4 ligand, caused a rapid increase in intracellular calcium mobilization with optimal effect of CXCL12 at 50 ng/ml. The effect of CXCL12 was abolished by the CXCR4 antagonist AMD3100. (2) CXCL12 promoted but AMD3100 inhibited the chemotactic response of U87, which might be attributed to the actin polymerization induced by CXCL12. (3) CXCL12 increased VEGF and IL-8 in U87 cells at both protein and mRNA levels. The effect of CXCL12 was bolcked by AMD3100.2. Nordy inhibited functional expression of CXCR4 on malignant glioma cells. (1) Immunofluorescence staining showed the decreased expression of CXCR4 after treatment with Nordy in a time-dependent manner. However, Nordy had no significant effects on CXCR4 mRNA expression. Futhermore, CXCR4 expression in U87 xenografts with Nordy treatment decreased as compared with control xenografts. (2) CXCL12-induced migration and actin polymerization of glioma cells were inhibited after pretreatment with Nordy. (3) Nordy also inhibited production of IL-8 and VEGF at either protein or mRNA level induced by CXCL12.3. CD133~+/nestin~+ GSCs were found in both primary and xengrafted gliomas. (1) CD133~+ and/or nestin~+ tumor cells were observed in primary human gliomas, human glioma cell lines and their xenografts. (2) CD133~+/nestin~+glioma cells were GSCs. When the CD133~+ cells were cultured in neural stem cell medium, they could form neurosphere-like spheroids expressing CD133~+/nestin~+. When seeded in differentiation conditions (medium containing serum), the spheroids generated differentiated cells with expression of GFAP, MBP and/orβ-tubulin III. Moreover, CD133~+/nestin~+ glioma cells initiated xenografts in nude mice. (3) Such kind of GSCs could be isolated and enriched with other methods, including colony heterogeneity-based and invasive potential-based methods for enrichment, and drug resistance-based selection. Moreover, increasing the concentration of serum-free neural stem cell medium was used as a new strategy for enrichment of GSCs.4. GSCs expressed higher levels of functional CXCR4, which promoted angiogenesis by producing VEGF and IL-8. (1) Compared with committed tumor cells, the GSCs expressed higher levels of CXCR4. (2) Stimulation of GSCs with CXCL12 caused a rapid increase of intracellular calcium mobilization with optimal effect of CXCL12 at 50 ng/ml, which could be abolished by pretreament with AMD3100. (3) The colony forming efficacy (CFE) of GSCs was increased when CXCR4 on the GSCs was activated. In the absence of AMD3100, the CFE of GSCs was 8.2±1.3 %, and increased to 17.4±4.8% after CXCL12 stimulation. After treatment with AMD3100, CFE of GSCs with or without CXCL12 stimulation was significantly inhibited. (4) CXCL12 could induce VEGF and IL-8 at both mRNA and protein levels produced by GSCs, while AMD3100 inhibited the effects. (5) The conditioned medium of GSCs presented more prowerful capability of promoting proliferation of umbilical vein endothelial cells than the CD133- conterparter. (6) CD133~+/nestin~+ cells were detected in the tissues adjacent to capillaries, which were revealed by CD31, in human primary glioma tissues. (7) The differential expression of microRNA was found between GSCs and committed differentiated cells, which might be responsible for their differential CXCR4 expression.5. AMD3100 suppressed growth and angiogenesis of xenografts formed by GSCs. (1) The xenografts in the mice treated with AMD3100 were significantly smaller (0.23±0.06 cm3) than those in the mice without any treatment (2.21±0.32 cm3) or with PBS treatment group (2.16±0.37 cm3). (2) Immunofluorescent staining with anti-CD31 antibody indicated that tumor specimens from AMD3100-treated mice contained significantly reduced MVD. This was associated with lower levels of VEGF and IL-8 in tumors from AMD3100 treatment group. (3) There was no significant difference in the expression of either CXCL12 or CXCR4 between AMD3100 treatment group and the control group. (4) AMD3100 did not change the ratio of CD133~+ or nestin~+ cells in GSCs xenografts in vivo.In summary, these results suggest that (1) activation of CXCR4 promoted U87 cells to migrate and produce VEGF and IL-8. Nordy inhibited the expression and effect of CXCR4 in U87 cells. (2) There were GSCs in human primary gliomas, human glioma cell lines and glioma xenografts. These cells expressed higher levels of CXCR4, activation of which promoted angiogenesis by producing VEGF and IL-8. A CXCR4 inhibitor AMD3100 suppressed GSCs xenograft growth and angiogenesis. Thus, CXCR4/CXCL12 axis is crucial for neovascularization of gliomas and may represent therapeutic targets for developing novel anti-GSCs agents to improve glioma treatment.
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