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The pro-survival role of autophagy depends on Bcl-2 under nutrition stress conditions.

Xu HD, Wu D, Gu JH, Ge JB, Wu JC, Han R, Liang ZQ, Qin ZH - PLoS ONE (2013)

Bottom Line: In contrast, overexpression of Bcl-2 inhibited autophagy and blocked cell death in response to serum deprivation.These data suggest that Bcl-2 plays an essential role in limiting autophagy activation and preventing initiation of programmed cell death.Thus Bcl-2 may be an important mechanism for balancing beneficial and detrimental impacts of autophagy on cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science, Suzhou, China.

ABSTRACT
Autophagy can be induced under nutrition stress conditions. Bcl-2 is a pro-survival protein which inhibits apoptosis and autophagy. However, the role of Bcl-2 in autophagy regulation and cell survival under nutrition deprivation has not been fully understood. This study sought to investigate if Bcl-2 upregulation is essential in limiting autophagic activity and prevent cell death under nutrition deprivation conditions. Autophagic activity was monitored by the changes in GFP-LC3 localization and protein levels of Beclin1, LC3-II, cathepsin D and p62 in neuroblastoma SH-SY5Y cells underwent serum deprivation. Manipulation of Bcl-2 function was achieved with siRNAs and small molecular inhibitors. The cell viability and apoptosis were assessed with MTT assay and Annexin V/PI staining. The results showed that serum starvation increased protein levels of LC3-II and Beclin1 but decreased autophagy substrate p62. Autophagy activation induced by serum deprivation and rapamycin was accompanied by an upregulation of Bcl-2 protein levels. When Bcl-2 was knocked down with siRNA or inhibited with HA 14-1 or ABT-737, serum starvation induced profound cell death and enhanced autophagic flux under nutrition deprivation conditions, while knockdown of autophagic gene Beclin1 or autophagy inhibitors (bafilomycin A1 and E64D), rescued cell death. In contrast, overexpression of Bcl-2 inhibited autophagy and blocked cell death in response to serum deprivation. These data suggest that Bcl-2 plays an essential role in limiting autophagy activation and preventing initiation of programmed cell death. Thus Bcl-2 may be an important mechanism for balancing beneficial and detrimental impacts of autophagy on cell survival.

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Autophagic flux induced by serum deprivation was enhanced when Bcl-2 was down-regulated and inhibitors of autophagy and apoptosis rescue cell from death.A: Western blot analysis of LC3-II and p62 expression in SH-SY5Y cells. Seventy-two hours after transfection with indicated oligonucleotides, the cells were subjected to starvation for 12 hrs in the present or absence of 50 nM Baf A1. B: Quantitative analysis of optical densities of the LC3-II/LC3-I protein bands with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). Statistical analysis was carried out with unpaired t-test (*p<0.05, Bcl-2 siRNA #6 group vs negative control group). C: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were treated with Bcl-2 siRNA #6 for 72 hrs, and then were treated with 10 µg/ml E64D, 50 nM Baf A1 and 50 µM Z-VAD. Cells were either maintained in normal medium or subjected to 12 hrs or 24 hrs of starvation prior to cell viability assay. **p<0.01, ***P<0.001 represent the indicated groups vs Bcl-2 RNAi #6 starvation 12 hrs group; ##p<0.01 represent the indicated groups vs Bcl-2 RNAi #6 starvation 24 hrs group. D: Representative western blot image of LC3 in SH-SY5Y cells subjected to normal or serum-free medium in the presence or absence of 10 µg/ml E64D for 12 hrs. E and F: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were pre-treated with HA 14-1 or ABT-737, then the cells were either maintained in normal or starvation medium for 12 hrs or 24 hrs with E64D, Baf A1 or Z-VAD. **p<0.01 represent the indicated groups vs HA 14-1/ABT-737 starvation 12 hrs groups; #p<0.05, ##p<0.01 represent the indicated groups vs HA 14-1/ABT-737 starvation 24 hrs groups. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. Data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.
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pone-0063232-g005: Autophagic flux induced by serum deprivation was enhanced when Bcl-2 was down-regulated and inhibitors of autophagy and apoptosis rescue cell from death.A: Western blot analysis of LC3-II and p62 expression in SH-SY5Y cells. Seventy-two hours after transfection with indicated oligonucleotides, the cells were subjected to starvation for 12 hrs in the present or absence of 50 nM Baf A1. B: Quantitative analysis of optical densities of the LC3-II/LC3-I protein bands with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). Statistical analysis was carried out with unpaired t-test (*p<0.05, Bcl-2 siRNA #6 group vs negative control group). C: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were treated with Bcl-2 siRNA #6 for 72 hrs, and then were treated with 10 µg/ml E64D, 50 nM Baf A1 and 50 µM Z-VAD. Cells were either maintained in normal medium or subjected to 12 hrs or 24 hrs of starvation prior to cell viability assay. **p<0.01, ***P<0.001 represent the indicated groups vs Bcl-2 RNAi #6 starvation 12 hrs group; ##p<0.01 represent the indicated groups vs Bcl-2 RNAi #6 starvation 24 hrs group. D: Representative western blot image of LC3 in SH-SY5Y cells subjected to normal or serum-free medium in the presence or absence of 10 µg/ml E64D for 12 hrs. E and F: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were pre-treated with HA 14-1 or ABT-737, then the cells were either maintained in normal or starvation medium for 12 hrs or 24 hrs with E64D, Baf A1 or Z-VAD. **p<0.01 represent the indicated groups vs HA 14-1/ABT-737 starvation 12 hrs groups; #p<0.05, ##p<0.01 represent the indicated groups vs HA 14-1/ABT-737 starvation 24 hrs groups. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. Data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.

Mentions: To investigate the dynamic process of autophagic flux induced by serum deprivation when Bcl-2 is down-regulated, SH-SY5Y cells were treated with serum-free medium after Bcl-2 siRNA treatment, and then autophagosome marker LC3-II was determined in the presence of Baf A1. As the data shown, a conversion of LC3-I to LC3-II induced by serum starvation was robustly accumulated in the presence of Baf A1 in both negative control and Bcl-2 siRNA groups (Fig. 5A). However, when Bcl-2 was knocked down, the ratio of LC3-II/LC3-I was further enhanced compared with the negative siRNA-treated control group (Fig. 5B), indicating that starvation-induced autophagic flux was further increased when Bcl-2 was inhibited. In addition, autophagy substrate p62 was further accumulated in the presence of Baf A1 compared with the control group, consistent with the enhanced autophagic flux after down-regulation of Bcl-2. These data indicate that serum starvation induced autophagy can be further enhanced when Bcl-2 is knocked down.


The pro-survival role of autophagy depends on Bcl-2 under nutrition stress conditions.

Xu HD, Wu D, Gu JH, Ge JB, Wu JC, Han R, Liang ZQ, Qin ZH - PLoS ONE (2013)

Autophagic flux induced by serum deprivation was enhanced when Bcl-2 was down-regulated and inhibitors of autophagy and apoptosis rescue cell from death.A: Western blot analysis of LC3-II and p62 expression in SH-SY5Y cells. Seventy-two hours after transfection with indicated oligonucleotides, the cells were subjected to starvation for 12 hrs in the present or absence of 50 nM Baf A1. B: Quantitative analysis of optical densities of the LC3-II/LC3-I protein bands with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). Statistical analysis was carried out with unpaired t-test (*p<0.05, Bcl-2 siRNA #6 group vs negative control group). C: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were treated with Bcl-2 siRNA #6 for 72 hrs, and then were treated with 10 µg/ml E64D, 50 nM Baf A1 and 50 µM Z-VAD. Cells were either maintained in normal medium or subjected to 12 hrs or 24 hrs of starvation prior to cell viability assay. **p<0.01, ***P<0.001 represent the indicated groups vs Bcl-2 RNAi #6 starvation 12 hrs group; ##p<0.01 represent the indicated groups vs Bcl-2 RNAi #6 starvation 24 hrs group. D: Representative western blot image of LC3 in SH-SY5Y cells subjected to normal or serum-free medium in the presence or absence of 10 µg/ml E64D for 12 hrs. E and F: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were pre-treated with HA 14-1 or ABT-737, then the cells were either maintained in normal or starvation medium for 12 hrs or 24 hrs with E64D, Baf A1 or Z-VAD. **p<0.01 represent the indicated groups vs HA 14-1/ABT-737 starvation 12 hrs groups; #p<0.05, ##p<0.01 represent the indicated groups vs HA 14-1/ABT-737 starvation 24 hrs groups. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. Data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.
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pone-0063232-g005: Autophagic flux induced by serum deprivation was enhanced when Bcl-2 was down-regulated and inhibitors of autophagy and apoptosis rescue cell from death.A: Western blot analysis of LC3-II and p62 expression in SH-SY5Y cells. Seventy-two hours after transfection with indicated oligonucleotides, the cells were subjected to starvation for 12 hrs in the present or absence of 50 nM Baf A1. B: Quantitative analysis of optical densities of the LC3-II/LC3-I protein bands with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). Statistical analysis was carried out with unpaired t-test (*p<0.05, Bcl-2 siRNA #6 group vs negative control group). C: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were treated with Bcl-2 siRNA #6 for 72 hrs, and then were treated with 10 µg/ml E64D, 50 nM Baf A1 and 50 µM Z-VAD. Cells were either maintained in normal medium or subjected to 12 hrs or 24 hrs of starvation prior to cell viability assay. **p<0.01, ***P<0.001 represent the indicated groups vs Bcl-2 RNAi #6 starvation 12 hrs group; ##p<0.01 represent the indicated groups vs Bcl-2 RNAi #6 starvation 24 hrs group. D: Representative western blot image of LC3 in SH-SY5Y cells subjected to normal or serum-free medium in the presence or absence of 10 µg/ml E64D for 12 hrs. E and F: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were pre-treated with HA 14-1 or ABT-737, then the cells were either maintained in normal or starvation medium for 12 hrs or 24 hrs with E64D, Baf A1 or Z-VAD. **p<0.01 represent the indicated groups vs HA 14-1/ABT-737 starvation 12 hrs groups; #p<0.05, ##p<0.01 represent the indicated groups vs HA 14-1/ABT-737 starvation 24 hrs groups. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. Data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.
Mentions: To investigate the dynamic process of autophagic flux induced by serum deprivation when Bcl-2 is down-regulated, SH-SY5Y cells were treated with serum-free medium after Bcl-2 siRNA treatment, and then autophagosome marker LC3-II was determined in the presence of Baf A1. As the data shown, a conversion of LC3-I to LC3-II induced by serum starvation was robustly accumulated in the presence of Baf A1 in both negative control and Bcl-2 siRNA groups (Fig. 5A). However, when Bcl-2 was knocked down, the ratio of LC3-II/LC3-I was further enhanced compared with the negative siRNA-treated control group (Fig. 5B), indicating that starvation-induced autophagic flux was further increased when Bcl-2 was inhibited. In addition, autophagy substrate p62 was further accumulated in the presence of Baf A1 compared with the control group, consistent with the enhanced autophagic flux after down-regulation of Bcl-2. These data indicate that serum starvation induced autophagy can be further enhanced when Bcl-2 is knocked down.

Bottom Line: In contrast, overexpression of Bcl-2 inhibited autophagy and blocked cell death in response to serum deprivation.These data suggest that Bcl-2 plays an essential role in limiting autophagy activation and preventing initiation of programmed cell death.Thus Bcl-2 may be an important mechanism for balancing beneficial and detrimental impacts of autophagy on cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science, Suzhou, China.

ABSTRACT
Autophagy can be induced under nutrition stress conditions. Bcl-2 is a pro-survival protein which inhibits apoptosis and autophagy. However, the role of Bcl-2 in autophagy regulation and cell survival under nutrition deprivation has not been fully understood. This study sought to investigate if Bcl-2 upregulation is essential in limiting autophagic activity and prevent cell death under nutrition deprivation conditions. Autophagic activity was monitored by the changes in GFP-LC3 localization and protein levels of Beclin1, LC3-II, cathepsin D and p62 in neuroblastoma SH-SY5Y cells underwent serum deprivation. Manipulation of Bcl-2 function was achieved with siRNAs and small molecular inhibitors. The cell viability and apoptosis were assessed with MTT assay and Annexin V/PI staining. The results showed that serum starvation increased protein levels of LC3-II and Beclin1 but decreased autophagy substrate p62. Autophagy activation induced by serum deprivation and rapamycin was accompanied by an upregulation of Bcl-2 protein levels. When Bcl-2 was knocked down with siRNA or inhibited with HA 14-1 or ABT-737, serum starvation induced profound cell death and enhanced autophagic flux under nutrition deprivation conditions, while knockdown of autophagic gene Beclin1 or autophagy inhibitors (bafilomycin A1 and E64D), rescued cell death. In contrast, overexpression of Bcl-2 inhibited autophagy and blocked cell death in response to serum deprivation. These data suggest that Bcl-2 plays an essential role in limiting autophagy activation and preventing initiation of programmed cell death. Thus Bcl-2 may be an important mechanism for balancing beneficial and detrimental impacts of autophagy on cell survival.

Show MeSH
Related in: MedlinePlus