SIRT1 and AROS suppress doxorubicin-induced apoptosis via inhibition of GSK3β activity in neuroblastoma cells

Abstract

SIRT1, the best-characterized member of the sirtuin family of deacetylases, is involved in cancer, apoptosis, inflammation, and metabolism. Active regulator of SIRT1 (AROS) was the first identified direct regulator of SIRT1. An increasing number of reports have indicated that SIRT1 plays an important role in controlling brain tumors. Here, we demonstrated that depletion of SIRT1 and AROS increases doxorubicin-mediated apoptosis in human neuroblastoma SH-SY5Y cells. Glycogen synthase kinase 3β (GSK3β) promoted doxorubicin-mediated apoptosis, but this effect was abolished by overexpression of SIRT1 and AROS. Interestingly, SIRT1 and AROS interacted with GSK3β and increased inhibitory phosphorylation of GSK3β on Ser9. Finally, we determined that AROS cooperates with SIRT1 to suppress GSK3β acetylation. Taken together, our results suggest that SIRT1 and AROS inhibit GSK3β activity and provide additional insight into drug resistance in the treatment of neuroblastoma.

Keywords: GSK3β; SIRT1; apoptosis; neuroblastoma.

Figure 1.

SIRT1 and AROS deficiency increase doxorubicin (DOX)-induced cytotoxicity. SH-SY5Y cells were transfected with sh-Luciferase (sh-Luc), sh-SIRT1, and sh-AROS. At 48 h after transfection, cells were treated with 0.1 μM DOX for 12 h. (A) Cleaved PARP-1 was detected by western blotting (WB; arrow). Knockdown efficiency was measured using RT-PCR with SIRT1 or AROS primers. GAPDH was used as an internal control. (B) SH-SY5Y Cells were fixed in 70% ethanol and stained with propidium iodide, and the Sub-G1 fraction was analyzed by flow cytometry. Quantification of the Sub-G1 population in response to DOX. Each value represents the mean ± standard deviation (SD) of three independent experiments (**P < 0.01). (C) Chromatin condensation and nuclear fragmentation as detected by Hoechst staining. (D) Quantification of apoptotic cell death in response to DOX. The percentage of apoptotic cells was calculated. All data are expressed as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01).

Figure 2.

SIRT1 and AROS abolish the effects of GSK3β on DOX-induced apoptosis. (A) SH-SY5Y cells were exposed to 0.1 μM DOX for 12 h. Cells were treated with 10 μM SB216763 2 h before DOX treatment. PARP-1 cleavage was detected by WB (arrow). (B) SH-SY5Y cells were co-transfected with GFP-GSK3β and Flag-AROS or Flag-SIRT1. At 24 h after transfection, cells were treated with 0.1 μM DOX for 12 h. Cleaved PARP-1 was observed by WB (arrow).

Figure 3.

SIRT1 and AROS associate with GSK3β. For immunoprecipitation (IP) assay, HEK293 cells were co-transfected with GFP-GSK3β and empty vector or Flag-SIRT1. Interaction was determined by IP with anti-Flag antibody and subsequent WB with anti-GFP antibody. (B) HEK293 cells were transfected with GFP-GSK3β and empty vector or Flag-AROS. IP was performed with anti-Flag antibody and bound protein was examined with anti-GFP antibody. (C) HEK293 cells were transfected with GFP-GSK3β, GFP-SIRT1, and empty vector or Flag-AROS. Cell lysates were immunoprecipitated with anti-Flag antibody and bound protein was examined with anti-GFP antibody. (D) SH-SY5Y cells were transfected with GFP-GSK3β, Flag-SIRT1, and Myc-AROS. Cells were then incubated with rabbit polyclonal anti-SIRT1 antibody and mouse monoclonal anti-Myc antibody. Cellular location was observed by rhodamine-conjugated anti-rabbit IgG and DyLight 405-conjugated anti-mouse IgG using confocal microscopy

Figure 4.

Effects of SIRT1 and AROS on the phosphorylation of GSK3β. (A) HEK293 cells were transiently transfected with Flag-SIRT1 and/or Flag-AROS. Phosphorylation of GSK3β at Ser9 was monitored by anti-pS9-GSK3β antibody. (B) SH-SY5Y cells were transfected with sh-Luc, sh-SIRT1, or sh-AROS. Lysates were subjected to WB with anti-pS9-GSK3β antibody. Knockdown efficiency was measured using RT-PCR with SIRT1 or AROS primers. GAPDH was used as an internal control.

Figure 5.

Effects of SIRT1 and AROS on deacetylation of GSK3β. (A) HEK293 cells were co-transfected with Flag-GSK3β and GFP-SIRT1 or Myc-AROS. Cell lysates were subjected to IP with anti-Flag antibody and analyzed by WB with anti-acetyl lysine antibody. (B) HEK293 cells were transfected with sh-Luc, sh-SIRT1, or sh-AROS. Cell lysates were subjected to IP with anti-Flag antibody and analyzed by WB with anti-acetyl lysine antibody.