抗凋亡蛋白Bcl-2在自噬调控以及细胞生存中的机制研究
摘要
目的：抗凋亡蛋白Bcl-2在急性饥饿时，通过与自噬关键蛋白Beclin1的相互作用，抑制Beclin1诱导的自噬，但是在某些情况下，Bcl-2的过表达并不能抑制自噬活性，因此抗凋亡蛋白Bcl-2和自噬之间的关系有待进一步研究。本研究的目的是探讨抗凋亡蛋白Bcl-2在自噬调控中的作用及其在细胞生存和死亡中的意义。
方法：利用血清饥饿和自噬激动剂雷帕霉素（Rapamycin）在人神经母细胞瘤SH-SY5Y中，激活自噬，Western检测自噬相关蛋白Beclin1，LC3，p62和CathepsinD，确定自噬激活模型成功；检测Bcl-2家族蛋白变化；利用siRNA Knockdown Bcl-2，或者应用Bcl-2的小分子抑制剂拮抗Bcl-2的功能，用MTT法检测细胞在饥饿下的活力变化，用Annexin-V/PI检测细胞凋亡的改变；加入自噬不同阶段的抑制剂，3-MA，E64D和Bafilomycin A1，检测在Bcl-2被下调或被小分子抑制剂拮抗时，血清饥饿诱导的自噬对细胞活力的影响。
结果：利用血清饥饿SH-SY5Y细胞6，12，24，36，48小时，Western检测自噬蛋白Beclin1，LC3，Cathepsin D和p62，结果显示Beclin1，LC3和Cathepsin D在血清饥饿时均上调，而自噬底物p62在6小时一过性下调，提示自噬被激活；用自噬激动剂雷帕霉素同样激活了自噬；在自噬被激活时，检测到抗凋亡蛋白Bcl-2上调；用siRNA干扰Bcl-2蛋白表达水平，检测到LC3无变化，加入溶酶体抑制剂氯化铵，LC3出现堆积，并且自噬底物p62被进一步降解，提示自噬流量进一步增强；在Bcl-2被下调时，或者应用小分子抑制剂HA14-1和ABT-737拮抗Bcl-2的功能活性时，MTT结果显示，血清饥饿进一步使细胞活力下降，Annexin V/PI结果显示细胞发生早期和晚期凋亡；泛caspase抑制剂Z-VAD阻断细胞死亡证实部分细胞死亡是凋亡性的；应用自噬不同阶段的抑制剂，E64D和巴普洛霉素（Bafilomycin A1）抑制自噬，可以挽救血清饥饿导致的部分细胞死亡。这些结果表明，伴随自噬激活而上调的Bcl-2蛋白能够限制自噬的过度激活和保护营养应激下的细胞死亡，当Bcl-2被下调时，自噬进一步激活，并且细胞发生死亡，这种死亡是凋亡性的，同时又是自噬性，因为caspase的抑制剂和自噬的抑制剂都能够挽救部分细胞死亡。
结论：营养剥夺诱导的自噬激活在生理条件下是保护性的，当Bcl-2下调时，抗凋亡蛋白与促凋亡蛋白，抗自噬蛋白与促自噬蛋白之间的力量对比发生变化，这时候，上调自噬活性可以导致营养应激条件下的细胞死亡,提示自噬的保护性作用依赖于抗凋亡蛋白Bcl-2，一旦Bcl-2下调或者功能受损，自噬的激活对细胞死亡有贡献。
Aims: Anti-apoptotic protein Bcl-2could inhibit essential autophagic proteinBeclin1-dependent autophagy through the interaction with Beclin1under acute starvationcondition. However, autophagy induction has also been observed under the condition ofBcl-2overexpression. Our aim was to study the mechanism of Bcl-2in autophagicregulation and cell survival under starvation condition.
Methods: Autophagy was induced with serum starvation and rapamycin inneuroblastoma SH-SY5Y cells. Western blot analysis was used to detect changes inautophagy related proteins Beclin1, LC3, Cathepsin D and p62and Bcl-2proteins. MTTassay was used to detect cell viability under serum starvation after knockdown of Bcl-2with siRNA duplex, or inhibition of Bcl-2function with small molecular inhibitors.Annexin V/PI and Western blot analysis were used to detect the change of apoptosis.Changes in autophagic flux when Bcl-2was knocked down were examined with Westernblot analysis. The contribution of autophagy to starvation-induced cell death was assessedwith the autophagic inhibitors,3-methyladenine, E64D or Bafilomycin A1and apoptoticinhibitor, Z-VAD.
Results: Autophagy was induced by serum starvation and Rapamycin inneuroblastoma SH-SY5Y cells. Western Blotting detected elevations of autophagy relatedproteins, including Beclin1, LC3and lysosomal enzyme Cathepsin D, and autophagicsubstrate p62was detected to be down-regulated, indicting autophagy was induced in thiscell line. Anti-apoptotic protein Bcl-2was also up-regulated accompanying autophagyinduction. When knockdown of Bcl-2by siRNA duplex or blockade of Bcl-2function withthe small molecular inhibitors, HA14-1and ABT-737, serum starvation induced apoptoticcell death, as evidenced by the reduction in cell viability (MTT), the eversion ofphosphatidylserine/increased membrane permeability (Annexin V/PI) and the blockage ofcell death by pan-caspase inhibitor, Z-VAD. Knockdown of Bcl-2did not increase LC3 turnover induced by serum starvation, but enhanced down-regulation of autophagicsubstrate p62, and LC3-II accumulated in the present of lysosome inhibitor, NH4CL, thesedata indicted that autophagic flux was further increased. Using different stages of inhibitorsof autophagy, E64D or Bafilomycin A1, to inhibit autophagy, MTT results showed thatstarvation-induced cell death under serum starvation could be partially rescued, indictingautophagy is involved in the process of cell death.
Conclusions: In normal conditions, serum starvation-induced autophagy wasprotective, however, when Bcl-2was down-regulated, and the balance betweenanti-autophagic and pro-autophagic was broken, in such case, enhance autophagic activitycould lead to cell death under starvation condition, suggesting Bcl-2plays an importantrole in preventing autophagy overactivation and promot cell survival by inhibitinginitiation of apoptosis.
方法：利用血清饥饿和自噬激动剂雷帕霉素（Rapamycin）在人神经母细胞瘤SH-SY5Y中，激活自噬，Western检测自噬相关蛋白Beclin1，LC3，p62和CathepsinD，确定自噬激活模型成功；检测Bcl-2家族蛋白变化；利用siRNA Knockdown Bcl-2，或者应用Bcl-2的小分子抑制剂拮抗Bcl-2的功能，用MTT法检测细胞在饥饿下的活力变化，用Annexin-V/PI检测细胞凋亡的改变；加入自噬不同阶段的抑制剂，3-MA，E64D和Bafilomycin A1，检测在Bcl-2被下调或被小分子抑制剂拮抗时，血清饥饿诱导的自噬对细胞活力的影响。
结果：利用血清饥饿SH-SY5Y细胞6，12，24，36，48小时，Western检测自噬蛋白Beclin1，LC3，Cathepsin D和p62，结果显示Beclin1，LC3和Cathepsin D在血清饥饿时均上调，而自噬底物p62在6小时一过性下调，提示自噬被激活；用自噬激动剂雷帕霉素同样激活了自噬；在自噬被激活时，检测到抗凋亡蛋白Bcl-2上调；用siRNA干扰Bcl-2蛋白表达水平，检测到LC3无变化，加入溶酶体抑制剂氯化铵，LC3出现堆积，并且自噬底物p62被进一步降解，提示自噬流量进一步增强；在Bcl-2被下调时，或者应用小分子抑制剂HA14-1和ABT-737拮抗Bcl-2的功能活性时，MTT结果显示，血清饥饿进一步使细胞活力下降，Annexin V/PI结果显示细胞发生早期和晚期凋亡；泛caspase抑制剂Z-VAD阻断细胞死亡证实部分细胞死亡是凋亡性的；应用自噬不同阶段的抑制剂，E64D和巴普洛霉素（Bafilomycin A1）抑制自噬，可以挽救血清饥饿导致的部分细胞死亡。这些结果表明，伴随自噬激活而上调的Bcl-2蛋白能够限制自噬的过度激活和保护营养应激下的细胞死亡，当Bcl-2被下调时，自噬进一步激活，并且细胞发生死亡，这种死亡是凋亡性的，同时又是自噬性，因为caspase的抑制剂和自噬的抑制剂都能够挽救部分细胞死亡。
结论：营养剥夺诱导的自噬激活在生理条件下是保护性的，当Bcl-2下调时，抗凋亡蛋白与促凋亡蛋白，抗自噬蛋白与促自噬蛋白之间的力量对比发生变化，这时候，上调自噬活性可以导致营养应激条件下的细胞死亡,提示自噬的保护性作用依赖于抗凋亡蛋白Bcl-2，一旦Bcl-2下调或者功能受损，自噬的激活对细胞死亡有贡献。
Aims: Anti-apoptotic protein Bcl-2could inhibit essential autophagic proteinBeclin1-dependent autophagy through the interaction with Beclin1under acute starvationcondition. However, autophagy induction has also been observed under the condition ofBcl-2overexpression. Our aim was to study the mechanism of Bcl-2in autophagicregulation and cell survival under starvation condition.
Methods: Autophagy was induced with serum starvation and rapamycin inneuroblastoma SH-SY5Y cells. Western blot analysis was used to detect changes inautophagy related proteins Beclin1, LC3, Cathepsin D and p62and Bcl-2proteins. MTTassay was used to detect cell viability under serum starvation after knockdown of Bcl-2with siRNA duplex, or inhibition of Bcl-2function with small molecular inhibitors.Annexin V/PI and Western blot analysis were used to detect the change of apoptosis.Changes in autophagic flux when Bcl-2was knocked down were examined with Westernblot analysis. The contribution of autophagy to starvation-induced cell death was assessedwith the autophagic inhibitors,3-methyladenine, E64D or Bafilomycin A1and apoptoticinhibitor, Z-VAD.
Results: Autophagy was induced by serum starvation and Rapamycin inneuroblastoma SH-SY5Y cells. Western Blotting detected elevations of autophagy relatedproteins, including Beclin1, LC3and lysosomal enzyme Cathepsin D, and autophagicsubstrate p62was detected to be down-regulated, indicting autophagy was induced in thiscell line. Anti-apoptotic protein Bcl-2was also up-regulated accompanying autophagyinduction. When knockdown of Bcl-2by siRNA duplex or blockade of Bcl-2function withthe small molecular inhibitors, HA14-1and ABT-737, serum starvation induced apoptoticcell death, as evidenced by the reduction in cell viability (MTT), the eversion ofphosphatidylserine/increased membrane permeability (Annexin V/PI) and the blockage ofcell death by pan-caspase inhibitor, Z-VAD. Knockdown of Bcl-2did not increase LC3 turnover induced by serum starvation, but enhanced down-regulation of autophagicsubstrate p62, and LC3-II accumulated in the present of lysosome inhibitor, NH4CL, thesedata indicted that autophagic flux was further increased. Using different stages of inhibitorsof autophagy, E64D or Bafilomycin A1, to inhibit autophagy, MTT results showed thatstarvation-induced cell death under serum starvation could be partially rescued, indictingautophagy is involved in the process of cell death.
Conclusions: In normal conditions, serum starvation-induced autophagy wasprotective, however, when Bcl-2was down-regulated, and the balance betweenanti-autophagic and pro-autophagic was broken, in such case, enhance autophagic activitycould lead to cell death under starvation condition, suggesting Bcl-2plays an importantrole in preventing autophagy overactivation and promot cell survival by inhibitinginitiation of apoptosis.
引文
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