Xuguang Zheng

Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth

Exosomes are 30-150 nm vesicles secreted by a wide range of mammalian cells that can contain microRNA (miRNA). To test if marrow stromal cell (MSC) exosomes could be used as a vehicle for delivery of anti-tumor miRNAs, we transfected MSCs with a miR-146b expression plasmid, and harvested exosomes released by the MSCs. Intra-tumor injection of exosomes derived from miR-146-expressing MSCs significantly reduced glioma xenograft growth in a rat model of primary brain tumor.

MiR-146b-5p suppresses EGFR expression and reduces in vitro migration and invasion of glioma.

ABSTRACT Human miR-146b-5p is located on chromosome 10q24.3. Loss of the 10q24–26 region is frequently observed in gliomas. Here, we report that miR-146b-5p suppresses expression of epidermal growth factor receptor (EGFR) in human glioblastoma cell lines. Introduction of miR-146b-5p decreases cell invasion, migration, and phosphorylation of protein kinase B (AKT). MiR-146b-5p suppresses translation of EGFR, and binds to the EGFR 3′-UTR. Furthermore, analysis of U87-MG laser-capture microdissected cells in tumor-bearing mice indicated that expression of miR-146b-5p was inversely correlated with distance from the tumor core. These findings suggest that reconstitution of miR-146b-5p may be useful for the treatment of this invasive tumor.

Gallic acid suppresses cell viability, proliferation, invasion and angiogenesis in human glioma cells

Gallic acid, an organic acid, also known as 3,4,5-trihydroxybenzoic acid, is cytotoxic against certain cancer cells, without harming normal cells. The objective of this study is to evaluate whether gallic acid can inhibit glioma cell viability, proliferation, invasion and reduce glioma cell mediated angiogenesis. Treatment of U87 and U251n glioma cells with gallic acid inhibited cell viability in a dose-dependent manner. BrdU and tube formation assays indicated that gallic acid significantly decreased glioma cell proliferation and tube formation in mouse brain endothelial cells, respectively. In addition, gallic acid decreased U87 cell invasion in vitro. Western blot analysis showed that expression of ADAM17, p-Akt and p-Erk was suppressed by gallic acid in both U87 and U251n cell lines. These data suggest that suppression of ADAM17 and downregulation of PI3K/Akt and Ras/MAPK signaling pathways may contribute to gallic acid-induced decrease of invasiveness. Gallic acid may be a valuable candidate for treatment of brain tumor.

MiR-15b and miR-152 reduce glioma cell invasion and angiogenesis via NRP-2 and MMP-3

We tested invasion and angiogenesis related mRNA expression and miRNA profiles of glioma. Genes with mRNA expression that changed significantly were selected to predict possible miRNAs that regulate mRNA expression, and were then matched with miRNA results. NRP-2 with the matching miRNA miR-15b, and MMP-3 with the matching miRNA miR-152 were selected for further study. Luciferase activity assay confirmed that miR-15b and miR-152 attenuate expression of NRP-2 and MMP-3 protein by binding to NRP-2 and MMP-3 transcript, respectively. In vitro invasion assay data showed that miR-15b and miR-152 significantly decreased 9L cell invasiveness. In vitro tube formation assay data showed that miR-15b reduced tube formation. A preliminary pathway study indicated that miR-15b and miR-152 deactivated the MEK-ERK pathway via NRP-2 and MMP-3 in 9L cells, respectively.

Inhibition of ADAM17 reduces hypoxia-induced brain tumor cell invasiveness

The membrane‐anchored metalloproteinase tumor necrosis factor‐α‐converting enzyme (TACE/a disintegrin and metalloproteinase [ADAM] 17) is key in proteolytic ectodomain shedding of several membrane‐bound growth factors, cytokines and receptors. The expression and activity of ADAM17 increases under some pathological conditions including stroke, and promotes neural progenitor cell migration and contributes to stroke‐induced neurogenesis. Hypoxia initiates cellular invasive processes that occur under both physiological and pathological conditions such as invasion and metastasis of some tumors. In the present study, we sought to elucidate whether ADAM17 contributes to brain tumor invasion. To this end, we examined the role of ADAM17 in the invasiveness of two different brain tumor cell lines, 9L rat gliosarcoma and U87 human glioma, under normoxic and hypoxic conditions. Additionally, we tested the effects of ADAM17 suppression on in vitro tumor cell invasion by means of ADAM17 proteolytic inhibitors and specific small interfering RNA. We found that tumor cells upregulated ADAM17 expression under hypoxia, and that ADAM17 activity correlated with increased tumor cell invasion. Conversely, suppression of ADAM17 proteolysis decreased invasiveness induced by hypoxia in 9L and U87 cells. Furthermore, the contribution of ADAM17 to tumor invasion was independent of matrix metalloproteinase (MMP)‐2 and MMP‐9 activity. ADAM17 was also found to activate the epidermal growth factor/phosphoinositide‐3 kinase/serine/threonine kinase signal transduction pathway. Our data suggest that hypoxia‐induced ADAM17 contributes to glioma cell invasiveness through activation of the EGFR signal pathway. (Cancer Sci 2007; 98: 674–684)

ADAM17 promotes breast cancer cell malignant phenotype through EGFR-PI3K-AKT activation

A disintegrin and metalloproteinase-17 (ADAM17) is involved in proteolytic ectodomain shedding of several membrane-bound growth factors and cytokines. The expression and activity of ADAM17 increase under some pathological conditions such as stroke and glioma. ADAM17 promotes neural progenitor cell migration and contributes to stroke-induced neurogenesis after stroke and brain tumor growth and invasion. In the present study, we sought to elucidate whether ADAM17 contributes to breast cancer progression and its mechanisms. To this end, we examined the role of ADAM17 in the proliferation, invasion, and tube formation of MDA-MB-231 breast cancer cells in vitro. Stable transfection of the MDA-MB-231 cell line with either a plasmid for over-expression of human ADAM17, or a siRNA to ADAM17 was employed in this study to establish high or low ADAM17 expression in breast cancer cells, respectively. For study of mechanism, the ADAM17 inhibitor TAPI-2 and the PI3K-AKT inhibitor LY294002 were used to counteract high ADAM17 expression or the activated PI3K-AKT pathway. Proliferation of MDA-MB-231 breast cancer cells were tested by MTT, Bromodeoxyuridine incorporation assay, growth curve, and sulforhodamine B assay. Matrigel invasion assays were used to assess the ability of MDA-MB-231 cells to penetrate the Extra Cellular Matrix. A Matrigel tube formation assay was performed to test capillary tube formation ability. EGFR-PI3K-Akt pathway activation in MDA-MB-231 cells under different ADAM17 expression levels were tested by Western blot and ELISA. Our data show that ADAM17 promotes the MDA-MB-231 malignant phenotype by increased proliferation, invasion and angiogenesis. TGF-α, VEGF secretion and VEGF expression was increasing by ADAM17 and counteracted by ADAM17 siRNA, TAPI-2, and LY294002 in MDA-MB-231 cells. ADAM17 activated, whereas ADAM17 siRNA, TAPI-2, and LY294002 deactivated the EGFR-PI3K-AKT signal pathway, which correlated with MDA-MB-231 cell malignant phenotype changes. This study suggests ADAM17 contributes to breast cancer progression through activation of the EGFR-PI3K-AKT signal pathway.

Transcription factor Sp1 induces ADAM17 and contributes to tumor cell invasiveness under hypoxia

BackgroundExpression of the Sp1 transcription factor is induced by hypoxia, and the ADAM17 promoter contains predicted Sp1 binding sites. ADAM17 contributes to hypoxic-induce invasiveness of glioma. In this study, we investigated whether Sp1 transcription factor induces ADAM17 and/or contributes to tumor cell invasiveness in hypoxia.MethodsEmploying RT-PCR and Western blot, we examined the role of Sp1 in ADAM17 transcription/expression under normoxic and hypoxic conditions, and whether it binds to the ADAM17 GC-rich promoter region using a chromatin immunoprecipitation assay. Additionally, we tested the effect of Sp1 suppression in tumor cell invasion and migration, using Matrigel basement membrane invasion chambers, a scratch wound-healing assay, and small interfering RNA.ResultsHere, we found that Sp1 binds to the ADAM17 promoter, and that Sp1 regulates ADAM17 expression under hypoxia. Furthermore, suppression of Sp1 decreases invasiveness and migration in U87 tumor cells.ConclusionOur findings suggest the Sp1 transcription factor mediates ADAM17 expression under hypoxia, regulates glioma invasiveness, and thus, may be a target for anti-invasion therapies.

ADAM17 promotes glioma cell malignant phenotype

A disintegrin and metalloproteinase‐17 (ADAM17) is involved in proteolytic ectodomain shedding of several membrane‐bound growth factors and cytokines. The expression and activity of ADAM17 increase under some pathological conditions such as stroke and cancer. ADAM17 promotes neural progenitor cell migration and contributes to neurogenesis after stroke and breast cancer growth and invasion. In the present study, we sought to elucidate whether ADAM17 contributes to glioma progression. To this end, we examined the role of ADAM17 in the proliferation, invasion, and tube formation of U87 human glioma cells in vitro and tumor growth in vivo. Stable transfection of the U87 cell line with either a plasmid for over‐expression of human ADAM17, or a siRNA to ADAM17 was employed in this study to establish high‐ or low‐ADAM17 expression in glioma cells, respectively. For study of mechanism, the ADAM17 inhibitor TAPI‐2 and the PI3K‐AKT inhibitor LY294002 were used to counteract high‐ADAM17 expression and the activated PI3K‐AKT pathway, respectively. Proliferation of glioma cells were tested by thiazolyl blue tetrazolium bromide (MTT) assay, bromodeoxyuridine incorporation assay, growth curve, and sulforhodamine B assay. Matrigel invasion assays were used to assess the ability of U87 cells to penetrate the extracellular matrix (ECM). A Matrigel tube formation assay was performed to test capillary tube formation ability. EGFR‐PI3K‐Akt pathway activation in U87 cells under different ADAM17 expression levels were tested by Western blot. Our data show that ADAM17 promotes the U87 malignant phenotype by increased proliferation, invasion, angiogenesis, and in vivo tumor growth. Tumor growth in nude mice was significantly inhibited by ADAM17 inhibitor and A17‐shRNA in vivo transfection. TGF‐α, VEGF secretion, and VEGF expression was increased by ADAM17 and counteracted by ADAM17 siRNA, TAPI‐2, and LY294002 in U87 cells. ADAM17 activated, whereas ADAM17 siRNA, TAPI‐2, and LY294002 deactivated the EGFR‐PI3K‐AKT signal pathway, which correlated with U87 cell malignant phenotype changes. This study suggests ADAM17 contributes to glioma progression through activation of the EGFR‐PI3K‐AKT signal pathway. Mol. Carcinog.

Bone marrow stromal cell therapy reduces proNGF and p75 expression in mice with experimental autoimmune encephalomyelitis.

Demyelination is prominent in experimental autoimmune encephalomyelitis (EAE). The receptor p75 and its high affinity ligand proNGF are required for oligodendrocyte death after injury. We hypothesize that bone marrow stromal cells (BMSCs) provide therapeutic benefit in EAE mice by reducing proNGF/p75 expression. PBS or BMSCs (2 x 10(circumflex)6) were administered intravenously on the day of EAE onset. Neurological function and demyelination areas were measured. Immunohistochemical staining was used to measure apoptotic oligodendrocytes, expression of proNGF and p75, and the relationship between proNGF and p75 in neural cells. proNGF was used to treat oligodendrocytes in culture with or without BMSCs. EAE mice exhibited neurological function deficit and demyelination, and expression of proNGF and p75 was increased. BMSC treatment improved functional recovery, reduced demyelination area and apoptotic oligodendrocytes, decreased expression of proNGF and p75 compared with PBS treatment. proNGF(+) cells colocalized with neural cell markers, while p75 colocalized with an oligodendrocytic marker, and proNGF colocalized with p75. proNGF induced apoptosis of oligodendrocytes in vitro, and p75 antibody blocked this apoptotic activity. BMSCs reduced p75 expression and apoptotic activity in oligodendrocytes with proNGF treatment. BMSC treatment benefits on EAE mice may be fostered by decreasing the cellular expression of proNGF and p75, thereby reducing oligodendrocyte death.

Combination Therapy with Antiangiogenic Treatment and Photodynamic Therapy for the Nude Mouse Bearing U87 Glioblastoma

The objective of this study was to evaluate the effects of combination therapy with photodynamic therapy (PDT) and a novel antiangiogenic regimen using monoclonal antibodies against both vascular endothelial growth factor receptors (VEGFR)‐1 (MF1) and VEGFR‐2 (DC101) on intracranial glioblastoma xenografts in nude mice. Nude mice bearing intracerebral U87 glioblastoma were treated with PDT and the antiangiogenic regimen (MF1 and DC101) either alone or in combination, while those left untreated served as tumor controls. Tumor volume and animal survival time were analyzed to evaluate the outcome of different treatment modalities. In addition, the immunohistochemical expression of VEGF in the brain adjacent to the tumor, von Willebrand factor (vWF), apoptotic, and proliferative markers in the tumor area were examined. PDT or MF1 + DC101 alone significantly reduced the tumor volume and prolonged the survival time of glioma‐implanted animals. Combined therapy markedly reduced tumor volume and increased survival time with significantly better outcomes than both monotherapies. Both vWF and VEGF levels significantly increased after PDT while they both significantly decreased after antiangiogenic treatment, compared with no treatment. PDT plus antiangiogenic treatment led to significant decreases in both vWF and VEGF expression, compared with PDT alone. Either PDT or antiangiogenic treatment alone significantly increased tumor cell apoptosis compared with no treatment, while combination therapy resulted in further augmentation of apoptosis. Antiangiogenic treatment with or without PDT significantly decreased tumor cell proliferation, compared with either no treatment or PDT alone. In summary, we demonstrate both significant inhibition of tumor growth and extended survival of mice treated by the combination therapy with PDT and antiangiogenic agents, compared with each single treatment, suggesting that the combination therapy may be a promising strategy to improve clinical outcomes in glioblastoma.