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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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Nutrient deprivation decreased cell viability when Bcl-2 was down-regulated.A: Western blot analysis of Bcl-2 expression in SH-SY5Y cells after transfection with negative control and Bcl-2 siRNA oligonucleotides. B: Quantitative analysis of optical densities of Bcl-2 protein bands (NC, negative control; Bcl-2 RNAi #6 and Bcl-2 RNAi #7) and normalized to the loading control. Bars represent Mean ± SD (n = 4). Statistical analysis was carried out with ANOVA followed by Dunnett t-test. **p<0.01 represent Bcl-2 RNAi groups (#6 and #7) vs negative control group. C: Quantitative analysis of cell viability with MTT assay in SH-SY5Y control cells and the cells after transfection with NC, #6 and 7# oligonucleotides. Cells were either maintained in normal medium or subjected to 12 hrs or 24 hrs of starvation prior to cell viability assay. ***p<0.001 represent the indicated groups vs NC starvation 12 hrs group; ###p<0.001 represent the indicated groups vs NC starvation 24 hrs group. D: Quantitative analysis of cell viability with MTT assay in SH-SY5Y control cells and the cells treated with DMSO, HA 14-1 and ABT-737. After pre-treated with HA 14-1 or ABT-737, cells were maintained either in normal medium or starvation medium containing DMSO, HA14-1 and ABT-737 for 12 hrs or 24 hrs prior to cell viability assay. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. ***p<0.001 represent HA 14-1/ABT-737 starvation 12 hrs groups vs DMSO starvation 12 hrs group; ###p<0.001 represent HA 14-1/ABT-737 starvation 24 hrs groups vs DMSO starvation 24 hrs group. For C and D, data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.
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pone-0063232-g003: Nutrient deprivation decreased cell viability when Bcl-2 was down-regulated.A: Western blot analysis of Bcl-2 expression in SH-SY5Y cells after transfection with negative control and Bcl-2 siRNA oligonucleotides. B: Quantitative analysis of optical densities of Bcl-2 protein bands (NC, negative control; Bcl-2 RNAi #6 and Bcl-2 RNAi #7) and normalized to the loading control. Bars represent Mean ± SD (n = 4). Statistical analysis was carried out with ANOVA followed by Dunnett t-test. **p<0.01 represent Bcl-2 RNAi groups (#6 and #7) vs negative control group. C: Quantitative analysis of cell viability with MTT assay in SH-SY5Y control cells and the cells after transfection with NC, #6 and 7# oligonucleotides. Cells were either maintained in normal medium or subjected to 12 hrs or 24 hrs of starvation prior to cell viability assay. ***p<0.001 represent the indicated groups vs NC starvation 12 hrs group; ###p<0.001 represent the indicated groups vs NC starvation 24 hrs group. D: Quantitative analysis of cell viability with MTT assay in SH-SY5Y control cells and the cells treated with DMSO, HA 14-1 and ABT-737. After pre-treated with HA 14-1 or ABT-737, cells were maintained either in normal medium or starvation medium containing DMSO, HA14-1 and ABT-737 for 12 hrs or 24 hrs prior to cell viability assay. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. ***p<0.001 represent HA 14-1/ABT-737 starvation 12 hrs groups vs DMSO starvation 12 hrs group; ###p<0.001 represent HA 14-1/ABT-737 starvation 24 hrs groups vs DMSO starvation 24 hrs group. For C and D, data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.

Mentions: Bcl-2 is a well-defined anti-apoptotic protein, which also inhibits autophagy. Therefore, we reasoned that the role of upregulated Bcl-2 is either to protect the cells from death, or prevent the overactivation of autophagy. To evaluate the actual role of Bcl-2 in serum deprivation-induced autophagy, Bcl-2 was knocked down in SH-SY5Y cells with siRNA. Among many siRNA duplexes tested, the number six and number seven acquired a ∼60% silencing efficiency (Fig. 3, A and B). We investigated whether Bcl-2 knockdown affected the survival of SH-SY5Y cells under starvation conditions. While serum deprivation (12 hrs and 24 hrs) slightly decreased cell viability in all treated groups, the cell viability was greatly reduced in nutrient-starved Bcl-2 siRNA groups (e.g., #6 and #7) as compared with nutrient-starved negative siRNA-treated control group (Fig. 3C). We also used two small molecular antagonists of Bcl-2, HA 14-1 and ABT-737, to inhibit the function of Bcl-2. The treatment of these two compounds did not significantly change cell viability in control and DMSO groups during growth in normal conditions. In contrast, nutrient deprivation (12 hrs and 24 hrs) decreased cell viability in HA 14-1 or ABT-737 pre-treatment groups (Fig. 3D). These data suggest that Bcl-2 plays an essential role to maintain the viability of SH-SY5Y cells under serum 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)

Nutrient deprivation decreased cell viability when Bcl-2 was down-regulated.A: Western blot analysis of Bcl-2 expression in SH-SY5Y cells after transfection with negative control and Bcl-2 siRNA oligonucleotides. B: Quantitative analysis of optical densities of Bcl-2 protein bands (NC, negative control; Bcl-2 RNAi #6 and Bcl-2 RNAi #7) and normalized to the loading control. Bars represent Mean ± SD (n = 4). Statistical analysis was carried out with ANOVA followed by Dunnett t-test. **p<0.01 represent Bcl-2 RNAi groups (#6 and #7) vs negative control group. C: Quantitative analysis of cell viability with MTT assay in SH-SY5Y control cells and the cells after transfection with NC, #6 and 7# oligonucleotides. Cells were either maintained in normal medium or subjected to 12 hrs or 24 hrs of starvation prior to cell viability assay. ***p<0.001 represent the indicated groups vs NC starvation 12 hrs group; ###p<0.001 represent the indicated groups vs NC starvation 24 hrs group. D: Quantitative analysis of cell viability with MTT assay in SH-SY5Y control cells and the cells treated with DMSO, HA 14-1 and ABT-737. After pre-treated with HA 14-1 or ABT-737, cells were maintained either in normal medium or starvation medium containing DMSO, HA14-1 and ABT-737 for 12 hrs or 24 hrs prior to cell viability assay. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. ***p<0.001 represent HA 14-1/ABT-737 starvation 12 hrs groups vs DMSO starvation 12 hrs group; ###p<0.001 represent HA 14-1/ABT-737 starvation 24 hrs groups vs DMSO starvation 24 hrs group. For C and D, data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.
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pone-0063232-g003: Nutrient deprivation decreased cell viability when Bcl-2 was down-regulated.A: Western blot analysis of Bcl-2 expression in SH-SY5Y cells after transfection with negative control and Bcl-2 siRNA oligonucleotides. B: Quantitative analysis of optical densities of Bcl-2 protein bands (NC, negative control; Bcl-2 RNAi #6 and Bcl-2 RNAi #7) and normalized to the loading control. Bars represent Mean ± SD (n = 4). Statistical analysis was carried out with ANOVA followed by Dunnett t-test. **p<0.01 represent Bcl-2 RNAi groups (#6 and #7) vs negative control group. C: Quantitative analysis of cell viability with MTT assay in SH-SY5Y control cells and the cells after transfection with NC, #6 and 7# oligonucleotides. Cells were either maintained in normal medium or subjected to 12 hrs or 24 hrs of starvation prior to cell viability assay. ***p<0.001 represent the indicated groups vs NC starvation 12 hrs group; ###p<0.001 represent the indicated groups vs NC starvation 24 hrs group. D: Quantitative analysis of cell viability with MTT assay in SH-SY5Y control cells and the cells treated with DMSO, HA 14-1 and ABT-737. After pre-treated with HA 14-1 or ABT-737, cells were maintained either in normal medium or starvation medium containing DMSO, HA14-1 and ABT-737 for 12 hrs or 24 hrs prior to cell viability assay. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. ***p<0.001 represent HA 14-1/ABT-737 starvation 12 hrs groups vs DMSO starvation 12 hrs group; ###p<0.001 represent HA 14-1/ABT-737 starvation 24 hrs groups vs DMSO starvation 24 hrs group. For C and D, data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.
Mentions: Bcl-2 is a well-defined anti-apoptotic protein, which also inhibits autophagy. Therefore, we reasoned that the role of upregulated Bcl-2 is either to protect the cells from death, or prevent the overactivation of autophagy. To evaluate the actual role of Bcl-2 in serum deprivation-induced autophagy, Bcl-2 was knocked down in SH-SY5Y cells with siRNA. Among many siRNA duplexes tested, the number six and number seven acquired a ∼60% silencing efficiency (Fig. 3, A and B). We investigated whether Bcl-2 knockdown affected the survival of SH-SY5Y cells under starvation conditions. While serum deprivation (12 hrs and 24 hrs) slightly decreased cell viability in all treated groups, the cell viability was greatly reduced in nutrient-starved Bcl-2 siRNA groups (e.g., #6 and #7) as compared with nutrient-starved negative siRNA-treated control group (Fig. 3C). We also used two small molecular antagonists of Bcl-2, HA 14-1 and ABT-737, to inhibit the function of Bcl-2. The treatment of these two compounds did not significantly change cell viability in control and DMSO groups during growth in normal conditions. In contrast, nutrient deprivation (12 hrs and 24 hrs) decreased cell viability in HA 14-1 or ABT-737 pre-treatment groups (Fig. 3D). These data suggest that Bcl-2 plays an essential role to maintain the viability of SH-SY5Y cells under serum 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