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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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Effects of EMAP II on the expression and distribution of LC3 and p62/SQSTM1 in human GBM cells and GSCs. (A) Western blot analysis was performed to detect the expression levels of LC3-II/LC3-I in U87, U118, and GSCs after treatment with 0.005 nM, 0.05 nM, 0.5 nM, and 5 nM EMAP II. Relative integrated density value (IDV) of LC3-II/LC3-I are shown. (B) Western blot analysis was performed to detect the expression of LC3-II/LC3-I and p62/SQSTM1 in U87, U118, and GSCs after treatment with 0.05 nM EMAP II for 0.5, 1, 2, 3, and 6 h, and EMAP II pretreatment with 3-MA. IDV of LC3-II/LC3-I and p62/SQSTM1 are shown. Values represented the means ± SD (n = 5, each). ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group. (C) The up-regulation of LC3 and down-regulation of p62/SQSTM1 in U87, U118, and GSCs were observed after treated with EMAP II alone or EMAP II plus 3-MA. Scale bar = 20 μm.
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Figure 3: Effects of EMAP II on the expression and distribution of LC3 and p62/SQSTM1 in human GBM cells and GSCs. (A) Western blot analysis was performed to detect the expression levels of LC3-II/LC3-I in U87, U118, and GSCs after treatment with 0.005 nM, 0.05 nM, 0.5 nM, and 5 nM EMAP II. Relative integrated density value (IDV) of LC3-II/LC3-I are shown. (B) Western blot analysis was performed to detect the expression of LC3-II/LC3-I and p62/SQSTM1 in U87, U118, and GSCs after treatment with 0.05 nM EMAP II for 0.5, 1, 2, 3, and 6 h, and EMAP II pretreatment with 3-MA. IDV of LC3-II/LC3-I and p62/SQSTM1 are shown. Values represented the means ± SD (n = 5, each). ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group. (C) The up-regulation of LC3 and down-regulation of p62/SQSTM1 in U87, U118, and GSCs were observed after treated with EMAP II alone or EMAP II plus 3-MA. Scale bar = 20 μm.

Mentions: Effects of EMAP II on the expression and distribution of LC3-II and p62/SQSTM1 were detected by Western blot assay and immunofluorescence assay, respectively. As shown in Figure 3A, Western blot assay was used to investigate the effects of EMAP II (0.005, 0.05, 0.5, and 5 nM) on the expression of LC3-I and LC3-II in U87, U118 and GSCs at 0.5 h. Compared with the control group, LC3-II/LC3-I expression significantly increased in 0.05, 0.5, and 5 nM EMAP II groups (P < 0.01), whereas it did not change in 0.005 nM group. There was no significant difference among these three groups (P > 0.05). Further, cells were treated with EMAP II (0.05 nM) at indicated time and pretreated with 3-MA to examine the LC3 and p62/SQSTM1 expression. As shown in Figure 3B, LC3-II/LC3-I expression was significantly increased at 0.5, 1, and 2 h after 0.05 nM EMAP II treatment compared with the control group (P < 0.01). In addition, LC3-II/LC3-I expression was decreased in EMAPII+3-MA group compared with EMAP II group (P < 0.01), suggesting that 3-MA blocked the effect of EMAP II on the LC3-II/LC3-I expression. Meanwhile, the p62/SQSTM1 expression was significantly decreased at 0.5, 1, and 2 h after 0.05 nM EMAP II treatment (P < 0.01). In addition, the p62/SQSTM1 expression was increased in EMAPII+3-MA group compared with EMAP II group (P < 0.01), suggesting that 3-MA blocked the inhibitory effect of EMAP II on the p62/SQSTM1 expression. The immunofluorescence assay of LC3-II and p62/SQSTM1 displayed similar results as above (Figure 3C). These results suggested that EMAP II induced cell autophagy by regulating the expression of LC3 and p62/SQSTM1 in human GBM cells and GSCs.


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)

Effects of EMAP II on the expression and distribution of LC3 and p62/SQSTM1 in human GBM cells and GSCs. (A) Western blot analysis was performed to detect the expression levels of LC3-II/LC3-I in U87, U118, and GSCs after treatment with 0.005 nM, 0.05 nM, 0.5 nM, and 5 nM EMAP II. Relative integrated density value (IDV) of LC3-II/LC3-I are shown. (B) Western blot analysis was performed to detect the expression of LC3-II/LC3-I and p62/SQSTM1 in U87, U118, and GSCs after treatment with 0.05 nM EMAP II for 0.5, 1, 2, 3, and 6 h, and EMAP II pretreatment with 3-MA. IDV of LC3-II/LC3-I and p62/SQSTM1 are shown. Values represented the means ± SD (n = 5, each). ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group. (C) The up-regulation of LC3 and down-regulation of p62/SQSTM1 in U87, U118, and GSCs were observed after treated with EMAP II alone or EMAP II plus 3-MA. Scale bar = 20 μm.
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Related In: Results  -  Collection

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Figure 3: Effects of EMAP II on the expression and distribution of LC3 and p62/SQSTM1 in human GBM cells and GSCs. (A) Western blot analysis was performed to detect the expression levels of LC3-II/LC3-I in U87, U118, and GSCs after treatment with 0.005 nM, 0.05 nM, 0.5 nM, and 5 nM EMAP II. Relative integrated density value (IDV) of LC3-II/LC3-I are shown. (B) Western blot analysis was performed to detect the expression of LC3-II/LC3-I and p62/SQSTM1 in U87, U118, and GSCs after treatment with 0.05 nM EMAP II for 0.5, 1, 2, 3, and 6 h, and EMAP II pretreatment with 3-MA. IDV of LC3-II/LC3-I and p62/SQSTM1 are shown. Values represented the means ± SD (n = 5, each). ∗∗P < 0.01 vs. control group, ##P < 0.01 vs. EMAP II group. (C) The up-regulation of LC3 and down-regulation of p62/SQSTM1 in U87, U118, and GSCs were observed after treated with EMAP II alone or EMAP II plus 3-MA. Scale bar = 20 μm.
Mentions: Effects of EMAP II on the expression and distribution of LC3-II and p62/SQSTM1 were detected by Western blot assay and immunofluorescence assay, respectively. As shown in Figure 3A, Western blot assay was used to investigate the effects of EMAP II (0.005, 0.05, 0.5, and 5 nM) on the expression of LC3-I and LC3-II in U87, U118 and GSCs at 0.5 h. Compared with the control group, LC3-II/LC3-I expression significantly increased in 0.05, 0.5, and 5 nM EMAP II groups (P < 0.01), whereas it did not change in 0.005 nM group. There was no significant difference among these three groups (P > 0.05). Further, cells were treated with EMAP II (0.05 nM) at indicated time and pretreated with 3-MA to examine the LC3 and p62/SQSTM1 expression. As shown in Figure 3B, LC3-II/LC3-I expression was significantly increased at 0.5, 1, and 2 h after 0.05 nM EMAP II treatment compared with the control group (P < 0.01). In addition, LC3-II/LC3-I expression was decreased in EMAPII+3-MA group compared with EMAP II group (P < 0.01), suggesting that 3-MA blocked the effect of EMAP II on the LC3-II/LC3-I expression. Meanwhile, the p62/SQSTM1 expression was significantly decreased at 0.5, 1, and 2 h after 0.05 nM EMAP II treatment (P < 0.01). In addition, the p62/SQSTM1 expression was increased in EMAPII+3-MA group compared with EMAP II group (P < 0.01), suggesting that 3-MA blocked the inhibitory effect of EMAP II on the p62/SQSTM1 expression. The immunofluorescence assay of LC3-II and p62/SQSTM1 displayed similar results as above (Figure 3C). These results suggested that EMAP II induced cell autophagy by regulating the expression of LC3 and p62/SQSTM1 in human GBM cells and GSCs.

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