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Mitochondrial Apoptotic Pathway Is Activated by H2O2-Mediated Oxidative Stress in BmN-SWU1 Cells from Bombyx mori Ovary.

Chen P, Hu YF, Wang L, Xiao WF, Bao XY, Pan C, Yi HS, Chen XY, Pan MH, Lu C - PLoS ONE (2015)

Bottom Line: H2O2-induced apoptosis also increased intracellular ROS level, changed mitochondrial distribution, reduced mitochondrial membrane potential and increased the release of cytochrome c from mitochondria.These results show that H2O2 treatment induced apoptosis in BmN-SWU1 cells via the mitochondrial apoptotic pathway.Taken together, these findings improve our knowledge of apoptosis in silkworm and the apoptotic pathways in insects.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716, China; Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing, 400716, China.

ABSTRACT
Apoptosis is a known regulator of morphogenetic events. In mammals, the critical role of oxidative stress-induced apoptosis has been well-studied; however, in insects the role of oxidative stress in apoptosis is not clear. In a previous study, we showed that apoptosis-related genes are present in the silkworm Bombyx mori, an important lepidopteran insect model. In this study, we evaluated the effect of H2O2-induced oxidative stress on apoptosis, reactive oxygen species (ROS) levels, mitochondrial response, cytochrome c release and apoptosis-related gene expression in the BmN-SWU1 cell line from B. mori ovaries. Our results showed that BmN-SWU1 cells exposed to H2O2 showed cell protuberances, cytoplasmic condensation, apoptotic bodies, DNA ladder formation and caspase activities indicating apoptosis. H2O2-induced apoptosis also increased intracellular ROS level, changed mitochondrial distribution, reduced mitochondrial membrane potential and increased the release of cytochrome c from mitochondria. Furthermore, western blot analysis revealed a significant increase in p53 and cytochrome c expression, and a decrease in Bcl-2 expression compared to the controls. Moreover, quantitative real-time PCR (qRT-PCR) showed an increase in the transcript levels of BmICE, Bmapaf-1 and BmEndoG by 439.5%, 423.9% and 42.2%, respectively, after treatment with 1 μM H2O2 for 24 h. However, the transcript levels of Bmbuffy declined by 41.4% after 24 h of exposure to 1 μM H2O2. These results show that H2O2 treatment induced apoptosis in BmN-SWU1 cells via the mitochondrial apoptotic pathway. Further, it appears that oxidative stress induced by H2O2 activates both caspase-dependent and caspase-independent mitochondrial apoptotic pathways in silkworm cells. Taken together, these findings improve our knowledge of apoptosis in silkworm and the apoptotic pathways in insects.

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Related in: MedlinePlus

Apoptosis assays in BmN-SWU1 cells after exposure to H2O2.(A) Effects of H2O2 exposure on the viability of BmN-SWU1 cells treated with different concentrations of H2O2 for 24 h (a) and for different time periods with 1 μM H2O2 (b). (B) Fluorescence microscopic images of morphological changes in the nuclei of BmN-SWU1 cells treated with H2O2. Arrows indicate apoptotic bodies in the DAPI stained images (12–24 h), and the arrow in the bright field image (8 h) indicates the vesicles. Scale bars = 50 μm. (C) Effects of 1 μM H2O2 on caspase-9 (a) and caspase-3/7 (b) activities in BmN-SWU1 cells. All treated groups were compared with the control (0 h). Values represent mean ± SD (n = 3). **, P < 0.01, Student’s t-test.
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pone.0134694.g001: Apoptosis assays in BmN-SWU1 cells after exposure to H2O2.(A) Effects of H2O2 exposure on the viability of BmN-SWU1 cells treated with different concentrations of H2O2 for 24 h (a) and for different time periods with 1 μM H2O2 (b). (B) Fluorescence microscopic images of morphological changes in the nuclei of BmN-SWU1 cells treated with H2O2. Arrows indicate apoptotic bodies in the DAPI stained images (12–24 h), and the arrow in the bright field image (8 h) indicates the vesicles. Scale bars = 50 μm. (C) Effects of 1 μM H2O2 on caspase-9 (a) and caspase-3/7 (b) activities in BmN-SWU1 cells. All treated groups were compared with the control (0 h). Values represent mean ± SD (n = 3). **, P < 0.01, Student’s t-test.

Mentions: In order to determine the optimal H2O2 required to induce apoptosis in BmN-SWU1 cells, we first treated the cells with various H2O2 concentrations (0, 0.1, 1.5, 10 μM) for different time points (0, 8, 12, 16, 24 h) and determined cell proliferation using the MTT assay. The results showed that H2O2 inhibited cell proliferation in a dose and time-dependent manner (Fig 1A). From these assays the median lethal dose (LD50) was estimated as 1 μM H2O2 with an exposure time of 24 h. Further, the morphology of BmN-SWU1 cells treated with H2O2 for 0, 8, 12, 16 and 24 h showed that the number of rounded and normal shaped cells was reduced after H2O2 treatment when compared to the control. In addition, when the treatment period was prolonged the number of abnormal cells increased, cell protrusions disappeared, and formation of vesicles and apoptotic bodies increased (Fig 1B). In addition, the effect of H2O2 on BmN-SWU1 cell apoptosis was also assessed by the Annexin V-FLUOS/PI flow cytometry and DNA ladder assays (S1 Fig). The results showed an increase in Annexin V staining and a longer smear of the DNA ladder in the H2O2 treated cells corroborating the morphological observations. Together, these findings indicate that H2O2 induces apoptosis in BmN-SWU1 cells.


Mitochondrial Apoptotic Pathway Is Activated by H2O2-Mediated Oxidative Stress in BmN-SWU1 Cells from Bombyx mori Ovary.

Chen P, Hu YF, Wang L, Xiao WF, Bao XY, Pan C, Yi HS, Chen XY, Pan MH, Lu C - PLoS ONE (2015)

Apoptosis assays in BmN-SWU1 cells after exposure to H2O2.(A) Effects of H2O2 exposure on the viability of BmN-SWU1 cells treated with different concentrations of H2O2 for 24 h (a) and for different time periods with 1 μM H2O2 (b). (B) Fluorescence microscopic images of morphological changes in the nuclei of BmN-SWU1 cells treated with H2O2. Arrows indicate apoptotic bodies in the DAPI stained images (12–24 h), and the arrow in the bright field image (8 h) indicates the vesicles. Scale bars = 50 μm. (C) Effects of 1 μM H2O2 on caspase-9 (a) and caspase-3/7 (b) activities in BmN-SWU1 cells. All treated groups were compared with the control (0 h). Values represent mean ± SD (n = 3). **, P < 0.01, Student’s t-test.
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Related In: Results  -  Collection

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pone.0134694.g001: Apoptosis assays in BmN-SWU1 cells after exposure to H2O2.(A) Effects of H2O2 exposure on the viability of BmN-SWU1 cells treated with different concentrations of H2O2 for 24 h (a) and for different time periods with 1 μM H2O2 (b). (B) Fluorescence microscopic images of morphological changes in the nuclei of BmN-SWU1 cells treated with H2O2. Arrows indicate apoptotic bodies in the DAPI stained images (12–24 h), and the arrow in the bright field image (8 h) indicates the vesicles. Scale bars = 50 μm. (C) Effects of 1 μM H2O2 on caspase-9 (a) and caspase-3/7 (b) activities in BmN-SWU1 cells. All treated groups were compared with the control (0 h). Values represent mean ± SD (n = 3). **, P < 0.01, Student’s t-test.
Mentions: In order to determine the optimal H2O2 required to induce apoptosis in BmN-SWU1 cells, we first treated the cells with various H2O2 concentrations (0, 0.1, 1.5, 10 μM) for different time points (0, 8, 12, 16, 24 h) and determined cell proliferation using the MTT assay. The results showed that H2O2 inhibited cell proliferation in a dose and time-dependent manner (Fig 1A). From these assays the median lethal dose (LD50) was estimated as 1 μM H2O2 with an exposure time of 24 h. Further, the morphology of BmN-SWU1 cells treated with H2O2 for 0, 8, 12, 16 and 24 h showed that the number of rounded and normal shaped cells was reduced after H2O2 treatment when compared to the control. In addition, when the treatment period was prolonged the number of abnormal cells increased, cell protrusions disappeared, and formation of vesicles and apoptotic bodies increased (Fig 1B). In addition, the effect of H2O2 on BmN-SWU1 cell apoptosis was also assessed by the Annexin V-FLUOS/PI flow cytometry and DNA ladder assays (S1 Fig). The results showed an increase in Annexin V staining and a longer smear of the DNA ladder in the H2O2 treated cells corroborating the morphological observations. Together, these findings indicate that H2O2 induces apoptosis in BmN-SWU1 cells.

Bottom Line: H2O2-induced apoptosis also increased intracellular ROS level, changed mitochondrial distribution, reduced mitochondrial membrane potential and increased the release of cytochrome c from mitochondria.These results show that H2O2 treatment induced apoptosis in BmN-SWU1 cells via the mitochondrial apoptotic pathway.Taken together, these findings improve our knowledge of apoptosis in silkworm and the apoptotic pathways in insects.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716, China; Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing, 400716, China.

ABSTRACT
Apoptosis is a known regulator of morphogenetic events. In mammals, the critical role of oxidative stress-induced apoptosis has been well-studied; however, in insects the role of oxidative stress in apoptosis is not clear. In a previous study, we showed that apoptosis-related genes are present in the silkworm Bombyx mori, an important lepidopteran insect model. In this study, we evaluated the effect of H2O2-induced oxidative stress on apoptosis, reactive oxygen species (ROS) levels, mitochondrial response, cytochrome c release and apoptosis-related gene expression in the BmN-SWU1 cell line from B. mori ovaries. Our results showed that BmN-SWU1 cells exposed to H2O2 showed cell protuberances, cytoplasmic condensation, apoptotic bodies, DNA ladder formation and caspase activities indicating apoptosis. H2O2-induced apoptosis also increased intracellular ROS level, changed mitochondrial distribution, reduced mitochondrial membrane potential and increased the release of cytochrome c from mitochondria. Furthermore, western blot analysis revealed a significant increase in p53 and cytochrome c expression, and a decrease in Bcl-2 expression compared to the controls. Moreover, quantitative real-time PCR (qRT-PCR) showed an increase in the transcript levels of BmICE, Bmapaf-1 and BmEndoG by 439.5%, 423.9% and 42.2%, respectively, after treatment with 1 μM H2O2 for 24 h. However, the transcript levels of Bmbuffy declined by 41.4% after 24 h of exposure to 1 μM H2O2. These results show that H2O2 treatment induced apoptosis in BmN-SWU1 cells via the mitochondrial apoptotic pathway. Further, it appears that oxidative stress induced by H2O2 activates both caspase-dependent and caspase-independent mitochondrial apoptotic pathways in silkworm cells. Taken together, these findings improve our knowledge of apoptosis in silkworm and the apoptotic pathways in insects.

No MeSH data available.


Related in: MedlinePlus