Histone deacetylase SIRT6 regulates chemosensitivity in liver cancer cells via modulation of FOXO3 activity

Abstract

Liver cancer is the leading cause of cancer‑related mortality worldwide and its incidence is increasing. Considerable effort has been made in recent decades to improve the diagnosis and treatment of liver cancer. Advanced liver cancer often exhibits a poor response to chemotherapy and poor prognosis due to acquired chemoresistance and tumor recurrence. Understanding the precise molecular mechanisms that are responsible for chemotherapeutic drug‑induced cell death could potentially identify novel therapeutic targets and improve liver cancer treatment. In the present study, it was demonstrated that in response to doxorubicin, the most frequently used chemical compound for liver cancer treatment, histone deacetylase sirtuin 6 (SIRT6) is specifically downregulated. This enables forkhead box O3 (FOXO3) upregulation, translocation into the nucleus and increased expression of its target genes p27 and Bim, which further induce apoptosis. Overexpression of SIRT6, but not enzyme‑inactivated mutants, prevents FOXO3 translocation into the nucleus and doxorubicin‑induced cell death. SIRT6 interacts with FOXO3 and this interaction increases FOXO3 ubiquitination and decreases its stability. Finally, it was identified that the effect of SIRT6 in preventing doxorubicin‑induced cell death requires FOXO3. Overexpression of SIRT6 could not prevent doxorubicin‑induced cell death in FOXO3‑knockdown cells. Therefore, it was concluded that SIRT6 plays a central role in determining doxorubicin‑induced cell death via modulation of FOXO3 activity. Therapeutic targeting of SIRT6 and/or FOXO3 may offer novel strategies for treatment of liver cancer.

Figure 1.

Doxorubicin (DOX) treatment specifically decreases SIRT6. (A) HepG2 cells were treated with 1 µM DOX for 36 h. Cells were harvested at various times as indicated. SIRT1, SIRT4 and SIRT6 mRNA levels were evaluated by reverse transcription-quantitative polymerase chain reaction. (B) HepG2 and Huh7 cells were treated with 1 µM DOX for various times as indicated. Protein levels of SIRT1, SIRT4 and SIRT6 were evaluated by western blot analysis. GAPDH was used as a loading control. (C) Immunofluorescence staining of HepG2 cells that were either untreated (Control) or treated with DOX for various times as indicated by using SIRT6 antibody (green) and DAPI staining (blue). Data are presented as the mean ± standard error of the mean of three independent experiments with technical duplicates. *P<0.05, **P<0.01 vs. control; one-way ANOVA. SIRT, sirtuin.

Figure 2.

Doxorubicin (DOX)-induced SIRT6 downregulation is required for apoptosis. (A) HepG2 cells were treated with varies dose of DOX and caspase-3/-7 activity was examined at 36 h after treatment. (B and C) HepG2 cells were treated with 1 µM DOX for 36 h. (B) Protein levels of cleaved PARP (C-PARP) and cleaved caspase-3 (C-CASP3) were evaluated by western blot analysis. GAPDH was used as a loading control. (C) Cell death was evaluated by TUNEL assay. (D) Huh7 cells were transfected with Flag-tagged SIRT6 or SIRT6 H133Y for 24 h and treated with DOX (1 µM) for 36 h. Protein expression of SIRT6, FOXO3, cleaved PARP and caspase-3 was evaluated by western blot analysis. (E) HepG2 cells were transfected with Flag-tagged SIRT6 or SIRT6 H133Y for 24 h and treated with DOX (1 µM) for 36 h. Cell death was evaluated by TUNEL assay. Data are presented as the mean ± standard error of the mean of three independent experiments with technical duplicates. **P<0.01, ***P<0.001 vs. control; one- way ANOVA. PARP, poly(ADP ribose) polymerase; SIRT, sirtuin; FOXO3, forkhead box O3.

Figure 3.

Doxorubicin (DOX) increases FOXO3 activation. HepG2 cells were treated with 1 µM DOX for 36 h, then cells were harvested at various times as indicated. (A) Protein levels of FOXO3, FOXO1, p53 and Bax were evaluated by western blot analysis. GAPDH was used as a loading control. (B) Immunofluorescence staining of HepG2 and Huh7 cells that were either untreated (Control) or treated with DOX for various times as indicated by using FOXO3 antibody (green) and DAPI staining (blue). Scale bar, 50 µm. (C-E) HepG2 cells were either untreated (Control) or treated with DOX for 24 h. (C) Promoter binding of FOXO3 was evaluated by chromatin immunoprecipitation assay. Data are presented as the mean ± standard error of the mean of three independent experiments with technical duplicates. *P<0.05, **P<0.01 vs. control, two-tailed unpaired Student's t-test. (D) p27 and Bim mRNA levels were evaluated by reverse transcription-quantitative polymerase chain reaction. (E) Protein levels were evaluated by western blot analysis. Graphs show mean ± SEM of three independent experiments with technical duplicates. Data are presented as the mean ± standard error of the mean of three independent experiments with technical duplicates. *P<0.05, **P<0.01 vs. control; one-way ANOVA. FOXO3, forkhead box O3; FOXO1, forkhead box O1.

Figure 4.

SIRT6 destabilizes FOXO3 by promoting FOXO3 ubiquitination. (A) Interaction between SIRT6 and FOXO3. HeLa cells were transfected with Flag-tagged SIRT6 for 24 h. Flag-SIRT6 or FOXO3 were immunoprecipitated and the input and immune complexes were assessed for the presence of FOXO3 or SIRT6 by western blot analysis. (B) FOXO3 was immunoprecipitated from cell lysis as in (A) Acetylated, phosphorylated and ubiquitinated FOXO3 and total amount of FOXO3 were assessed by western blot analysis. (C) HepG2 cells were transfected with Flag-SIRT6 or SIRT6 H133Y for 24 h. Protein level of total FOXO3 and p-FOXO3 S253 were evaluated by western blot analysis. (D) HepG2 cells were transfected with Flag-SIRT6 for 24 h and cells were treated with CHX (100 µM) for various times as indicated. Protein levels of FOXO3 and SIRT6 were evaluated by western blot analysis. FOXO3, forkhead box O3; SIRT, sirtuin; CHX, cycloheximide.

Figure 5.

SIRT6 directs doxorubicin (DOX)-induced cell death through FOXO3. (A) HepG2 cells were transduced with shRNA specific for FOXO3 (shFOXO3) or non-targeting shRNA (shCON) for 72 h. FOXO3 and SIRT6 protein levels were evaluated by western blot analysis. (B) Cells in A were treated with DOX for 36 h and cell death was evaluated by TUNEL assay. (C and D) HA-FOXO3 and/or Flag-SIRT6 were transfected into HepG2 cells followed by treatment with DOX for 24 h. Immunofluorescence staining for FOXO3 and SIRT6 was performed using FOXO3 (green) and Flag (red) antibodies. Scale bar, 20 µm. (E and F) Cells in A were transfected with Flag-SIRT6 or SIRT6 H133Y for 24 h and treated with DOX for 36 h. (E) Protein levels were evaluated by western blot analysis. (F) Cell death was evaluated by TUNEL assay. Data are presented as the mean ± standard error of the mean of three independent experiments. *P<0.05 vs. shCON; one-way ANOVA.