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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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Serum deprivation induced cell death when Bcl-2 function was suppressed.A: Flow cytometry analysis of SH-SY5Y control cells transfected with negative control, Bcl-2 RNAi #6 and Bcl-2 RNAi #7. The cells were either growing in normal medium or subjected to 12 hrs starvation. B: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining. Cells were either maintained in normal medium or subjected to 12 hrs of starvation prior to cell death analysis. Statistical analysis was either carried with Student t-test or carried out with ANOVA followed by Dunnett t-test (##p<0.01, ###p<0.001, Bcl-2 RNAi #6 + starvation, or #7 + starvation group vs NC+starvation group). C: Nutrient deprivation induced activation of caspase-3 (p19 and p17) and PARP cleavage in SH-SY5Y cells when Bcl-2 was knocked down. The results are representative of three experiments. D: Flow cytometry analysis of SH-SY5Y.control cells treated with DMSO, HA 14-1 and ABT-737. E: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining in SH-SY5Y cells treated with DMSO, HA 14-1 and ABT-737. Cells were either maintained in normal medium or subjected to 12 hrs of starvation prior to cell death analysis. Statistical analysis was either carried out with Student t-test or carried out with ANOVA followed by Dunnett t-test (###p<0.001, HA 14-1 + starvation, or ABT-737 + starvation group vs DMSO + starvation group). For B and E, data represent mean ± SD for combined data from three independent experiments.
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pone-0063232-g004: Serum deprivation induced cell death when Bcl-2 function was suppressed.A: Flow cytometry analysis of SH-SY5Y control cells transfected with negative control, Bcl-2 RNAi #6 and Bcl-2 RNAi #7. The cells were either growing in normal medium or subjected to 12 hrs starvation. B: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining. Cells were either maintained in normal medium or subjected to 12 hrs of starvation prior to cell death analysis. Statistical analysis was either carried with Student t-test or carried out with ANOVA followed by Dunnett t-test (##p<0.01, ###p<0.001, Bcl-2 RNAi #6 + starvation, or #7 + starvation group vs NC+starvation group). C: Nutrient deprivation induced activation of caspase-3 (p19 and p17) and PARP cleavage in SH-SY5Y cells when Bcl-2 was knocked down. The results are representative of three experiments. D: Flow cytometry analysis of SH-SY5Y.control cells treated with DMSO, HA 14-1 and ABT-737. E: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining in SH-SY5Y cells treated with DMSO, HA 14-1 and ABT-737. Cells were either maintained in normal medium or subjected to 12 hrs of starvation prior to cell death analysis. Statistical analysis was either carried out with Student t-test or carried out with ANOVA followed by Dunnett t-test (###p<0.001, HA 14-1 + starvation, or ABT-737 + starvation group vs DMSO + starvation group). For B and E, data represent mean ± SD for combined data from three independent experiments.

Mentions: To test if nutrient deprivation induces apoptotic cell death when the function of Bcl-2 is inhibited, the cells were stained with Annexin V and PI and subjected to flow cytometry examination. Culture cells in starvation medium induced minor cell death. In contrast, cell death of the Bcl-2 siRNA groups significantly increased comparing with negative siRNA-treated control group (Fig 4, A and B). Furthermore, the active forms of caspase-3 and cleaved PARP (Fig. 4C) were detected in nutrient-starved SH-SY5Y cells when Bcl-2 was knocked down, indicating that apoptotic cell death was induced. The present study also examined apoptotic cell death of SH-SY5Y cells after treatment with HA 14-1, ABT-737 and DMSO (vehicle control). HA 14-1 and ABT-737 did not induced substantial cell death, but when the function of Bcl-2 was inhibited by the inhibitors, nutrient deprivation led to significantly more cell death as compared with the DMSO treatment group (Fig. 4D and E). These data suggest that Bcl-2 partially blocks the apoptotic cell death of SH-SY5Y cells under nutrient deprivation conditions.


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)

Serum deprivation induced cell death when Bcl-2 function was suppressed.A: Flow cytometry analysis of SH-SY5Y control cells transfected with negative control, Bcl-2 RNAi #6 and Bcl-2 RNAi #7. The cells were either growing in normal medium or subjected to 12 hrs starvation. B: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining. Cells were either maintained in normal medium or subjected to 12 hrs of starvation prior to cell death analysis. Statistical analysis was either carried with Student t-test or carried out with ANOVA followed by Dunnett t-test (##p<0.01, ###p<0.001, Bcl-2 RNAi #6 + starvation, or #7 + starvation group vs NC+starvation group). C: Nutrient deprivation induced activation of caspase-3 (p19 and p17) and PARP cleavage in SH-SY5Y cells when Bcl-2 was knocked down. The results are representative of three experiments. D: Flow cytometry analysis of SH-SY5Y.control cells treated with DMSO, HA 14-1 and ABT-737. E: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining in SH-SY5Y cells treated with DMSO, HA 14-1 and ABT-737. Cells were either maintained in normal medium or subjected to 12 hrs of starvation prior to cell death analysis. Statistical analysis was either carried out with Student t-test or carried out with ANOVA followed by Dunnett t-test (###p<0.001, HA 14-1 + starvation, or ABT-737 + starvation group vs DMSO + starvation group). For B and E, data represent mean ± SD for combined data from three independent experiments.
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pone-0063232-g004: Serum deprivation induced cell death when Bcl-2 function was suppressed.A: Flow cytometry analysis of SH-SY5Y control cells transfected with negative control, Bcl-2 RNAi #6 and Bcl-2 RNAi #7. The cells were either growing in normal medium or subjected to 12 hrs starvation. B: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining. Cells were either maintained in normal medium or subjected to 12 hrs of starvation prior to cell death analysis. Statistical analysis was either carried with Student t-test or carried out with ANOVA followed by Dunnett t-test (##p<0.01, ###p<0.001, Bcl-2 RNAi #6 + starvation, or #7 + starvation group vs NC+starvation group). C: Nutrient deprivation induced activation of caspase-3 (p19 and p17) and PARP cleavage in SH-SY5Y cells when Bcl-2 was knocked down. The results are representative of three experiments. D: Flow cytometry analysis of SH-SY5Y.control cells treated with DMSO, HA 14-1 and ABT-737. E: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining in SH-SY5Y cells treated with DMSO, HA 14-1 and ABT-737. Cells were either maintained in normal medium or subjected to 12 hrs of starvation prior to cell death analysis. Statistical analysis was either carried out with Student t-test or carried out with ANOVA followed by Dunnett t-test (###p<0.001, HA 14-1 + starvation, or ABT-737 + starvation group vs DMSO + starvation group). For B and E, data represent mean ± SD for combined data from three independent experiments.
Mentions: To test if nutrient deprivation induces apoptotic cell death when the function of Bcl-2 is inhibited, the cells were stained with Annexin V and PI and subjected to flow cytometry examination. Culture cells in starvation medium induced minor cell death. In contrast, cell death of the Bcl-2 siRNA groups significantly increased comparing with negative siRNA-treated control group (Fig 4, A and B). Furthermore, the active forms of caspase-3 and cleaved PARP (Fig. 4C) were detected in nutrient-starved SH-SY5Y cells when Bcl-2 was knocked down, indicating that apoptotic cell death was induced. The present study also examined apoptotic cell death of SH-SY5Y cells after treatment with HA 14-1, ABT-737 and DMSO (vehicle control). HA 14-1 and ABT-737 did not induced substantial cell death, but when the function of Bcl-2 was inhibited by the inhibitors, nutrient deprivation led to significantly more cell death as compared with the DMSO treatment group (Fig. 4D and E). These data suggest that Bcl-2 partially blocks the apoptotic cell death of SH-SY5Y cells under nutrient deprivation conditions.

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