The Role Of Bcl-2in Regulating Autophagy Activity And Cell Survival During Starvation

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.