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Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages.

Chen F, Li Q, Zhang Z, Lin P, Lei L, Wang A, Jin Y - Int J Mol Sci (2015)

Bottom Line: Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay.Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes.Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest Agriculture and Forestry University, Yangling 712100, Shaanxi, China. cfl0604114114@163.com.

ABSTRACT
Zearalenone (ZEA) is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER) stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

No MeSH data available.


Related in: MedlinePlus

ZEA induces cell death in RAW 264.7 macrophages. (A) ZEA reduced the viability of RAW 264.7 macrophages in a dose-dependent manner. Cells were treated with 0, 10, 20, 30, 40, 50, 80 and 100 µM ZEA for 24 h and then processed for the MTT assay; (B,C) Apoptosis was detected via flow cytometry. B1 is the part of cell death caused by mechanical damage, B2 is the part of late apoptotic or necrotic cells, B3 is the part of the normal cells, and B4 is the part of early apoptotic cells. After exposure to 0, 30, and 50 µM ZEA for 24 h, RAW 264.7 macrophages were collected for Annexin V-PE/7-AAD staining followed by flow cytometric analysis. The statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05); and (D) Representative photomicrographs of RAW 264.7 macrophages stained with Hoechst 33342 and PI fluorescent dye after exposure of the cells to 0 µM ZEA for 12 h (a); 30 µM ZEA for 12 h (b); 50 µM ZEA for 12 h (c); 0 µM ZEA for 24 h (d); 30 µM ZEA for 24 h (e); and 50 µM ZEA for 24 h (f). Apoptotic cells were characterized as having condensed or fragmented nuclei that stained deep blue, while necrotic cells were stained deep blue and deep red. Bar = 20 µm.
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ijms-16-19780-f001: ZEA induces cell death in RAW 264.7 macrophages. (A) ZEA reduced the viability of RAW 264.7 macrophages in a dose-dependent manner. Cells were treated with 0, 10, 20, 30, 40, 50, 80 and 100 µM ZEA for 24 h and then processed for the MTT assay; (B,C) Apoptosis was detected via flow cytometry. B1 is the part of cell death caused by mechanical damage, B2 is the part of late apoptotic or necrotic cells, B3 is the part of the normal cells, and B4 is the part of early apoptotic cells. After exposure to 0, 30, and 50 µM ZEA for 24 h, RAW 264.7 macrophages were collected for Annexin V-PE/7-AAD staining followed by flow cytometric analysis. The statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05); and (D) Representative photomicrographs of RAW 264.7 macrophages stained with Hoechst 33342 and PI fluorescent dye after exposure of the cells to 0 µM ZEA for 12 h (a); 30 µM ZEA for 12 h (b); 50 µM ZEA for 12 h (c); 0 µM ZEA for 24 h (d); 30 µM ZEA for 24 h (e); and 50 µM ZEA for 24 h (f). Apoptotic cells were characterized as having condensed or fragmented nuclei that stained deep blue, while necrotic cells were stained deep blue and deep red. Bar = 20 µm.

Mentions: The toxic effect of ZEA on RAW 264.7 macrophages was examined by treatment with 0–100 µM ZEA for 24 h. Then the effect of this treatment on their proliferation was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) assay. ZEA was found to have very little effect at low doses (10 µM), while 20 µM ZEA significantly inhibited the growth of cells (Figure 1A, p < 0.05). These results showed that ZEA treatment clearly inhibited cell viability in a dose-dependent manner. The results of flow cytometry analysis also revealed that the apoptotic percentage of RAW 264.7 macrophages was significantly increased by ZEA treatment in a time-dependent manner compared with the control (Figure 1B,C, p < 0.05). The results of the Hoechst 33342 and propidium iodide (PI) staining assays showed that the early apoptotic cells were mainly observed after exposure to 30 and 50 µM ZEA for 12 h, but late apoptotic and necrotic cells were mainly observed after exposure to 30 and 50 µM ZEA for 24 h (Figure 1D).


Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages.

Chen F, Li Q, Zhang Z, Lin P, Lei L, Wang A, Jin Y - Int J Mol Sci (2015)

ZEA induces cell death in RAW 264.7 macrophages. (A) ZEA reduced the viability of RAW 264.7 macrophages in a dose-dependent manner. Cells were treated with 0, 10, 20, 30, 40, 50, 80 and 100 µM ZEA for 24 h and then processed for the MTT assay; (B,C) Apoptosis was detected via flow cytometry. B1 is the part of cell death caused by mechanical damage, B2 is the part of late apoptotic or necrotic cells, B3 is the part of the normal cells, and B4 is the part of early apoptotic cells. After exposure to 0, 30, and 50 µM ZEA for 24 h, RAW 264.7 macrophages were collected for Annexin V-PE/7-AAD staining followed by flow cytometric analysis. The statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05); and (D) Representative photomicrographs of RAW 264.7 macrophages stained with Hoechst 33342 and PI fluorescent dye after exposure of the cells to 0 µM ZEA for 12 h (a); 30 µM ZEA for 12 h (b); 50 µM ZEA for 12 h (c); 0 µM ZEA for 24 h (d); 30 µM ZEA for 24 h (e); and 50 µM ZEA for 24 h (f). Apoptotic cells were characterized as having condensed or fragmented nuclei that stained deep blue, while necrotic cells were stained deep blue and deep red. Bar = 20 µm.
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Related In: Results  -  Collection

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ijms-16-19780-f001: ZEA induces cell death in RAW 264.7 macrophages. (A) ZEA reduced the viability of RAW 264.7 macrophages in a dose-dependent manner. Cells were treated with 0, 10, 20, 30, 40, 50, 80 and 100 µM ZEA for 24 h and then processed for the MTT assay; (B,C) Apoptosis was detected via flow cytometry. B1 is the part of cell death caused by mechanical damage, B2 is the part of late apoptotic or necrotic cells, B3 is the part of the normal cells, and B4 is the part of early apoptotic cells. After exposure to 0, 30, and 50 µM ZEA for 24 h, RAW 264.7 macrophages were collected for Annexin V-PE/7-AAD staining followed by flow cytometric analysis. The statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05); and (D) Representative photomicrographs of RAW 264.7 macrophages stained with Hoechst 33342 and PI fluorescent dye after exposure of the cells to 0 µM ZEA for 12 h (a); 30 µM ZEA for 12 h (b); 50 µM ZEA for 12 h (c); 0 µM ZEA for 24 h (d); 30 µM ZEA for 24 h (e); and 50 µM ZEA for 24 h (f). Apoptotic cells were characterized as having condensed or fragmented nuclei that stained deep blue, while necrotic cells were stained deep blue and deep red. Bar = 20 µm.
Mentions: The toxic effect of ZEA on RAW 264.7 macrophages was examined by treatment with 0–100 µM ZEA for 24 h. Then the effect of this treatment on their proliferation was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) assay. ZEA was found to have very little effect at low doses (10 µM), while 20 µM ZEA significantly inhibited the growth of cells (Figure 1A, p < 0.05). These results showed that ZEA treatment clearly inhibited cell viability in a dose-dependent manner. The results of flow cytometry analysis also revealed that the apoptotic percentage of RAW 264.7 macrophages was significantly increased by ZEA treatment in a time-dependent manner compared with the control (Figure 1B,C, p < 0.05). The results of the Hoechst 33342 and propidium iodide (PI) staining assays showed that the early apoptotic cells were mainly observed after exposure to 30 and 50 µM ZEA for 12 h, but late apoptotic and necrotic cells were mainly observed after exposure to 30 and 50 µM ZEA for 24 h (Figure 1D).

Bottom Line: Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay.Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes.Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest Agriculture and Forestry University, Yangling 712100, Shaanxi, China. cfl0604114114@163.com.

ABSTRACT
Zearalenone (ZEA) is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER) stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

No MeSH data available.


Related in: MedlinePlus