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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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Overexpression of Bcl-2 inhibited autophagy and blocked cell death.A: SH-SY5Y cells were transiently transfected with pCDNA3.1-Bcl-2 or empty control vector, and the cells were subjected to serum starvation for 12 hrs after 48 hrs transfection. 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). C: Quantitative analysis of cell viability with MTT assay in SH-SY5Y cells 24 hrs after transfection with control vector or Bcl-2 plasmid. Cells were either maintained in normal or starvation medium for 12 hrs prior to cell viability assay. D: Flow cytometry analysis of SH-SY5Y cells transfected with mock control and Bcl-2 plasmid for 48 hrs, then the cells were growing in normal medium or subjected to 24 hrs starvation. E: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining. Statistical analysis was carried out with ANOVA followed by Dunnett t-test (*p<0.05). Data represent mean ± SD for combined data from three independent experiments. For MTT assay, each experiment has six replicate wells.
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pone-0063232-g007: Overexpression of Bcl-2 inhibited autophagy and blocked cell death.A: SH-SY5Y cells were transiently transfected with pCDNA3.1-Bcl-2 or empty control vector, and the cells were subjected to serum starvation for 12 hrs after 48 hrs transfection. 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). C: Quantitative analysis of cell viability with MTT assay in SH-SY5Y cells 24 hrs after transfection with control vector or Bcl-2 plasmid. Cells were either maintained in normal or starvation medium for 12 hrs prior to cell viability assay. D: Flow cytometry analysis of SH-SY5Y cells transfected with mock control and Bcl-2 plasmid for 48 hrs, then the cells were growing in normal medium or subjected to 24 hrs starvation. E: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining. Statistical analysis was carried out with ANOVA followed by Dunnett t-test (*p<0.05). Data represent mean ± SD for combined data from three independent experiments. For MTT assay, each experiment has six replicate wells.

Mentions: To further address the role of autophagy in serum-starvation induced cell death, we downregulated essential autophagic gene Beclin1 with siRNA (Fig. 6, A and B). When Beclin1 was knocked down, serum deprivation did not robustly induce LC3-II up-regulation (Fig. 6, C and D), indicating that autophagic activity was severely impaired. Then, SH-SY5Y cells were co-transfected with Bcl-2 siRNA and Beclin1 siRNA. The transfection itself did not affect the cell viability. However, Beclin1 down-regulation rescued large numbers of cells from death induced by Bcl-2 knockdown under nutrient deprivation conditions (12 hrs and 24 hrs) (Fig. 6E). Moreover, apoptotic inhibitor, Z-VAD, did not further enhance cell viability under Beclin1 knockdown conditions. These data strongly suggest that autophagy plays an important role in these types of cell death. To evaluate the effects of Bcl-2 on autophagy, we transiently transfected Bcl-2 into SH-SY5Y cells. Overexpression of Bcl-2 itself did not cause significant autophagic flux change, while inhibited serum starvation induced LC3-II transformation (Fig. 7, A and B), indicating that the formation of autophagosomes was inhibited. Overexpression of Bcl-2 increased the cell viability under serum starvation conditions (Fig. 7C). Annexin V and PI staining also showed that starvation-induced cell death was prevented by overexpression of Bcl-2 (Fig. 7, D and E). Taken together, these results support the concept that Bcl-2 blocks autophagy and cell death 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)

Overexpression of Bcl-2 inhibited autophagy and blocked cell death.A: SH-SY5Y cells were transiently transfected with pCDNA3.1-Bcl-2 or empty control vector, and the cells were subjected to serum starvation for 12 hrs after 48 hrs transfection. 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). C: Quantitative analysis of cell viability with MTT assay in SH-SY5Y cells 24 hrs after transfection with control vector or Bcl-2 plasmid. Cells were either maintained in normal or starvation medium for 12 hrs prior to cell viability assay. D: Flow cytometry analysis of SH-SY5Y cells transfected with mock control and Bcl-2 plasmid for 48 hrs, then the cells were growing in normal medium or subjected to 24 hrs starvation. E: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining. Statistical analysis was carried out with ANOVA followed by Dunnett t-test (*p<0.05). Data represent mean ± SD for combined data from three independent experiments. For MTT assay, each experiment has six replicate wells.
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Related In: Results  -  Collection

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pone-0063232-g007: Overexpression of Bcl-2 inhibited autophagy and blocked cell death.A: SH-SY5Y cells were transiently transfected with pCDNA3.1-Bcl-2 or empty control vector, and the cells were subjected to serum starvation for 12 hrs after 48 hrs transfection. 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). C: Quantitative analysis of cell viability with MTT assay in SH-SY5Y cells 24 hrs after transfection with control vector or Bcl-2 plasmid. Cells were either maintained in normal or starvation medium for 12 hrs prior to cell viability assay. D: Flow cytometry analysis of SH-SY5Y cells transfected with mock control and Bcl-2 plasmid for 48 hrs, then the cells were growing in normal medium or subjected to 24 hrs starvation. E: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining. Statistical analysis was carried out with ANOVA followed by Dunnett t-test (*p<0.05). Data represent mean ± SD for combined data from three independent experiments. For MTT assay, each experiment has six replicate wells.
Mentions: To further address the role of autophagy in serum-starvation induced cell death, we downregulated essential autophagic gene Beclin1 with siRNA (Fig. 6, A and B). When Beclin1 was knocked down, serum deprivation did not robustly induce LC3-II up-regulation (Fig. 6, C and D), indicating that autophagic activity was severely impaired. Then, SH-SY5Y cells were co-transfected with Bcl-2 siRNA and Beclin1 siRNA. The transfection itself did not affect the cell viability. However, Beclin1 down-regulation rescued large numbers of cells from death induced by Bcl-2 knockdown under nutrient deprivation conditions (12 hrs and 24 hrs) (Fig. 6E). Moreover, apoptotic inhibitor, Z-VAD, did not further enhance cell viability under Beclin1 knockdown conditions. These data strongly suggest that autophagy plays an important role in these types of cell death. To evaluate the effects of Bcl-2 on autophagy, we transiently transfected Bcl-2 into SH-SY5Y cells. Overexpression of Bcl-2 itself did not cause significant autophagic flux change, while inhibited serum starvation induced LC3-II transformation (Fig. 7, A and B), indicating that the formation of autophagosomes was inhibited. Overexpression of Bcl-2 increased the cell viability under serum starvation conditions (Fig. 7C). Annexin V and PI staining also showed that starvation-induced cell death was prevented by overexpression of Bcl-2 (Fig. 7, D and E). Taken together, these results support the concept that Bcl-2 blocks autophagy and cell death 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