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Autophagy Induction by Endothelial-Monocyte Activating Polypeptide II Contributes to the Inhibition of Malignant Biological Behaviors by the Combination of EMAP II with Rapamycin in Human Glioblastoma.

Ma J, Meng F, Li S, Liu L, Zhao L, Liu Y, Hu Y, Li Z, Yao Y, Xi Z, Teng H, Xue Y - Front Mol Neurosci (2015)

Bottom Line: In this study, EMAP II inhibited the cell viability and decreased the mitochondrial membrane potential in human GBM cells and GSCs, and autophagy inhibitor 3-methyl adenine (3-MA) blocked these effects.In addition, the up-regulation of microtubule-associated protein-1 light chain-3 (LC3)-II and the down-regulation of autophagic degraded substrate p62/SQSTM1 caused by EMAP II were observed.The combination of EMAP II with rapamycin demonstrated the inhibitory effect on the malignant biological behaviors of human GBM cells and GSCs in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, College of Basic Medicine, China Medical University Shenyang, China ; Institute of Pathology and Pathophysiology, China Medical University Shenyang, China.

ABSTRACT
This study aims to investigate the effect of endothelial-monocyte activating polypeptide II (EMAP II) on human glioblastoma (GBM) cells and glioblastoma stem cells (GSCs) as well as its possible mechanisms. In this study, EMAP II inhibited the cell viability and decreased the mitochondrial membrane potential in human GBM cells and GSCs, and autophagy inhibitor 3-methyl adenine (3-MA) blocked these effects. Autophagic vacuoles were formed in these cells after EMAP II treatment and this phenomenon was blocked by 3-MA. In addition, the up-regulation of microtubule-associated protein-1 light chain-3 (LC3)-II and the down-regulation of autophagic degraded substrate p62/SQSTM1 caused by EMAP II were observed. Cells treated with EMAP-II inhibited the PI3K/Akt/mTOR signal pathway, and PI3K/Akt agonist insulin-like growth factor-1 (IGF-1) blocked the effect of EMAP II on the expression of LC3-II and p62/SQSTM1. Cells exposed to EMAP-II experienced mitophagy and ER stress. Furthermore, the inhibition of cell proliferation, migration and invasion of GBM cells and GSCs were more remarkable by the combination of EMAP II and rapamycin than either agent alone in vitro and in vivo. The current study demonstrated that the cytotoxicity of EMAP II in human GBM cells and GSCs was induced by autophagy, accompanied by the inhibition of PI3K/Akt/mTOR signal pathway, mitophagy and ER stress. The combination of EMAP II with rapamycin demonstrated the inhibitory effect on the malignant biological behaviors of human GBM cells and GSCs in vitro and in vivo.

No MeSH data available.


Related in: MedlinePlus

Effect of EMAP II and 3-MA on the ultrastructural change in human GBM cells and GSCs. (A) Effect of EMAP II and 3-MA on the ultrastructural change in U87, U118, and GSCs. Arrows show autophagic vacuoles. (B) The colocalization of LC3 and LysoTracker Red in U87, U118, and GSCs. Relative mean optical density of LC3 and LysoTracker Red, and the co-localization of LC3 and LysoTracker Red quantified. Pictures are respective magnification (n = 4, each). Scale bar = 20 μm. ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group.
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Figure 2: Effect of EMAP II and 3-MA on the ultrastructural change in human GBM cells and GSCs. (A) Effect of EMAP II and 3-MA on the ultrastructural change in U87, U118, and GSCs. Arrows show autophagic vacuoles. (B) The colocalization of LC3 and LysoTracker Red in U87, U118, and GSCs. Relative mean optical density of LC3 and LysoTracker Red, and the co-localization of LC3 and LysoTracker Red quantified. Pictures are respective magnification (n = 4, each). Scale bar = 20 μm. ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group.

Mentions: Due to the fact that the cell viability and MMP were not changed in cells treated with 3-MA alone, the following experiments were performed without cells treated with 3-MA alone. TEM was used to observe the effects of EMAP II on the ultrastructural changes in human GBM cells and GSCs. As shown in Figure 2A, the autophagic vacuoles were positive in U87 cells in the 0.05 nM EMAP II for 0.5 h group and negative in the control group and the EMAP II+3-MA group. The same results were also observed in U118 cells and GSCs. As shown in Figure 2B, cells were stained with anti-LC3 and LysoTracker Red by immunofluorescence. EMAP II significantly increased the punctate distribution and density of LC3 as well as autophagic vacuoles numbers in U87, U118, and GSCs (P < 0.01). Co-treatment with 3-MA reduced the punctate distribution and density of LC3 as well as autophagic vacuoles numbers (P < 0.01). In addition, we found that there was a significant overlap between LC3 and lysosomal signals in EMAP II treated cells (P < 0.01), suggesting that autophagosome–lysosome fusion was not inhibited by EMAP II. These results suggested that EMAP II could induce autophagic vacuoles formation in human GBM cells and GSCs, and 3-MA could block this effect.


Autophagy Induction by Endothelial-Monocyte Activating Polypeptide II Contributes to the Inhibition of Malignant Biological Behaviors by the Combination of EMAP II with Rapamycin in Human Glioblastoma.

Ma J, Meng F, Li S, Liu L, Zhao L, Liu Y, Hu Y, Li Z, Yao Y, Xi Z, Teng H, Xue Y - Front Mol Neurosci (2015)

Effect of EMAP II and 3-MA on the ultrastructural change in human GBM cells and GSCs. (A) Effect of EMAP II and 3-MA on the ultrastructural change in U87, U118, and GSCs. Arrows show autophagic vacuoles. (B) The colocalization of LC3 and LysoTracker Red in U87, U118, and GSCs. Relative mean optical density of LC3 and LysoTracker Red, and the co-localization of LC3 and LysoTracker Red quantified. Pictures are respective magnification (n = 4, each). Scale bar = 20 μm. ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664732&req=5

Figure 2: Effect of EMAP II and 3-MA on the ultrastructural change in human GBM cells and GSCs. (A) Effect of EMAP II and 3-MA on the ultrastructural change in U87, U118, and GSCs. Arrows show autophagic vacuoles. (B) The colocalization of LC3 and LysoTracker Red in U87, U118, and GSCs. Relative mean optical density of LC3 and LysoTracker Red, and the co-localization of LC3 and LysoTracker Red quantified. Pictures are respective magnification (n = 4, each). Scale bar = 20 μm. ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group.
Mentions: Due to the fact that the cell viability and MMP were not changed in cells treated with 3-MA alone, the following experiments were performed without cells treated with 3-MA alone. TEM was used to observe the effects of EMAP II on the ultrastructural changes in human GBM cells and GSCs. As shown in Figure 2A, the autophagic vacuoles were positive in U87 cells in the 0.05 nM EMAP II for 0.5 h group and negative in the control group and the EMAP II+3-MA group. The same results were also observed in U118 cells and GSCs. As shown in Figure 2B, cells were stained with anti-LC3 and LysoTracker Red by immunofluorescence. EMAP II significantly increased the punctate distribution and density of LC3 as well as autophagic vacuoles numbers in U87, U118, and GSCs (P < 0.01). Co-treatment with 3-MA reduced the punctate distribution and density of LC3 as well as autophagic vacuoles numbers (P < 0.01). In addition, we found that there was a significant overlap between LC3 and lysosomal signals in EMAP II treated cells (P < 0.01), suggesting that autophagosome–lysosome fusion was not inhibited by EMAP II. These results suggested that EMAP II could induce autophagic vacuoles formation in human GBM cells and GSCs, and 3-MA could block this effect.

Bottom Line: In this study, EMAP II inhibited the cell viability and decreased the mitochondrial membrane potential in human GBM cells and GSCs, and autophagy inhibitor 3-methyl adenine (3-MA) blocked these effects.In addition, the up-regulation of microtubule-associated protein-1 light chain-3 (LC3)-II and the down-regulation of autophagic degraded substrate p62/SQSTM1 caused by EMAP II were observed.The combination of EMAP II with rapamycin demonstrated the inhibitory effect on the malignant biological behaviors of human GBM cells and GSCs in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, College of Basic Medicine, China Medical University Shenyang, China ; Institute of Pathology and Pathophysiology, China Medical University Shenyang, China.

ABSTRACT
This study aims to investigate the effect of endothelial-monocyte activating polypeptide II (EMAP II) on human glioblastoma (GBM) cells and glioblastoma stem cells (GSCs) as well as its possible mechanisms. In this study, EMAP II inhibited the cell viability and decreased the mitochondrial membrane potential in human GBM cells and GSCs, and autophagy inhibitor 3-methyl adenine (3-MA) blocked these effects. Autophagic vacuoles were formed in these cells after EMAP II treatment and this phenomenon was blocked by 3-MA. In addition, the up-regulation of microtubule-associated protein-1 light chain-3 (LC3)-II and the down-regulation of autophagic degraded substrate p62/SQSTM1 caused by EMAP II were observed. Cells treated with EMAP-II inhibited the PI3K/Akt/mTOR signal pathway, and PI3K/Akt agonist insulin-like growth factor-1 (IGF-1) blocked the effect of EMAP II on the expression of LC3-II and p62/SQSTM1. Cells exposed to EMAP-II experienced mitophagy and ER stress. Furthermore, the inhibition of cell proliferation, migration and invasion of GBM cells and GSCs were more remarkable by the combination of EMAP II and rapamycin than either agent alone in vitro and in vivo. The current study demonstrated that the cytotoxicity of EMAP II in human GBM cells and GSCs was induced by autophagy, accompanied by the inhibition of PI3K/Akt/mTOR signal pathway, mitophagy and ER stress. The combination of EMAP II with rapamycin demonstrated the inhibitory effect on the malignant biological behaviors of human GBM cells and GSCs in vitro and in vivo.

No MeSH data available.


Related in: MedlinePlus