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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.


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EMAP II induced mitophagy in human GBM cells and GSCs. (A) The colocalization of LC3 and MitoTracker in U87, U118, and GSCs after cells treated with EMAP II and the combination of EMAP II and 3-MA. (B) Quantitative analysis of the co-localization of LC3 with MitoTracker. Pictures are respective magnification (n = 4, each). Scale bar = 20 μm. Western blot analysis of the expression of TOMM20 and TIMM23 in U87 (C), U118 (D) and GSCs (E) after cells treated with EMAP II and the combination of EMAP II and 3-MA. Values represented the means ± SD (n = 5, each). ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group.
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Figure 6: EMAP II induced mitophagy in human GBM cells and GSCs. (A) The colocalization of LC3 and MitoTracker in U87, U118, and GSCs after cells treated with EMAP II and the combination of EMAP II and 3-MA. (B) Quantitative analysis of the co-localization of LC3 with MitoTracker. Pictures are respective magnification (n = 4, each). Scale bar = 20 μm. Western blot analysis of the expression of TOMM20 and TIMM23 in U87 (C), U118 (D) and GSCs (E) after cells treated with EMAP II and the combination of EMAP II and 3-MA. Values represented the means ± SD (n = 5, each). ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group.

Mentions: Due to the MMP was affected by EMAP II, therefore we detected whether mitophagy was induced by EMAP II. As shown in Figures 6A,B, cells were stained with MitoTracker Deep Red FM and anti-LC3 by immunofluorescence, and results showed that there was a significant overlap between LC3 and MitoTracker signals in EMAP II treated cells (P < 0.01), and co-treatment with 3-MA blocked this effect (P < 0.01). In addition, the outer and inner mitochondrial membrane proteins (TOMM20 and TIMM23) was reduced by EMAP II, and co-treatment with 3-MA rescued the inhibitory effect induced by EMAP II (Figures 6C–E).


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)

EMAP II induced mitophagy in human GBM cells and GSCs. (A) The colocalization of LC3 and MitoTracker in U87, U118, and GSCs after cells treated with EMAP II and the combination of EMAP II and 3-MA. (B) Quantitative analysis of the co-localization of LC3 with MitoTracker. Pictures are respective magnification (n = 4, each). Scale bar = 20 μm. Western blot analysis of the expression of TOMM20 and TIMM23 in U87 (C), U118 (D) and GSCs (E) after cells treated with EMAP II and the combination of EMAP II and 3-MA. Values represented the means ± SD (n = 5, each). ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group.
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Related In: Results  -  Collection

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Figure 6: EMAP II induced mitophagy in human GBM cells and GSCs. (A) The colocalization of LC3 and MitoTracker in U87, U118, and GSCs after cells treated with EMAP II and the combination of EMAP II and 3-MA. (B) Quantitative analysis of the co-localization of LC3 with MitoTracker. Pictures are respective magnification (n = 4, each). Scale bar = 20 μm. Western blot analysis of the expression of TOMM20 and TIMM23 in U87 (C), U118 (D) and GSCs (E) after cells treated with EMAP II and the combination of EMAP II and 3-MA. Values represented the means ± SD (n = 5, each). ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group.
Mentions: Due to the MMP was affected by EMAP II, therefore we detected whether mitophagy was induced by EMAP II. As shown in Figures 6A,B, cells were stained with MitoTracker Deep Red FM and anti-LC3 by immunofluorescence, and results showed that there was a significant overlap between LC3 and MitoTracker signals in EMAP II treated cells (P < 0.01), and co-treatment with 3-MA blocked this effect (P < 0.01). In addition, the outer and inner mitochondrial membrane proteins (TOMM20 and TIMM23) was reduced by EMAP II, and co-treatment with 3-MA rescued the inhibitory effect induced by EMAP II (Figures 6C–E).

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