Ambra1 is an essential regulator of autophagy and apoptosis in SW620 cells: pro-survival role of Ambra1

Abstract

Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells. However, whether Ambra1 plays an important role in the autophagy pathway in colorectal cancer cells is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in CRC cell lines. To test this hypothesis, we confirmed autophagic activity in serum-starved SW620 CRC cells by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes (transmission electron microscopy) and LC3 protein levels (Western blotting). Ambra1 expression was detected by Western blot in SW620 cells treated with staurosporine or etoposide. Calpain and caspase inhibitors were employed to verify whether calpains and caspases were responsible for Ambra1 cleavage. To examine the role of Ambra1 in apoptosis, Ambra1 knockdown cells were treated with staurosporine and etoposide. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in colorectal cancer cell lines. Ambra1 expression decreased during staurosporine- or etoposide-induced apoptosis. Calpains and caspases may be responsible for Ambra1 degradation. When Ambra1 expression was reduced by siRNA, SW620 cells were more sensitive to staurosporine- or etoposide-induced apoptosis. In addition, starvation-induced autophagy decreased. Finally, Co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated SW620 cells, suggesting that Ambra1 regulates autophagy in CRC cells by interacting with Beclin1. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in CRC cells that maintains the balance between autophagy and apoptosis.

Figure 1. Autophagy is induced by starvation in SW620 cells.

A: Western blot analysis of LC3 expression in SW620 cells. B: Quantitation of the optical density of LC3-II/LC3-I at various time points. C: Western blot of Ambra1, Beclin1 and cathepsin D expression after serum starvation at various time points. D: Quantitation of the optical density of Ambra1, cathepsin D and Beclin1. E: The relative Ambra1 expression after serum starvation for the indicated times as determined by real-time PCR. F: Immunofluorescent images of SW620 cells treated with or without 100 nM BafA1 for the indicated times. G: Quantification of autophagy in SW620 cells after serum starvation. The data are presented as the mean ± SD of three independent experiments (p<0.05).

Figure 2. Representative electron micrographs of starvation-induced autophagy in SW620 cells at various time points.

A: Control group. B: Starvation for 6 h. C: Starvation for 12 h. D: Starvation for 24 h.

Figure 3. Ambra1 degradation during staurosporine-induced apoptosis.

A: SW620 cells treated with 1 µM staurosporine. Ambra1, Beclin1, Vps34 and ATG5 expression as well as the appearance of cleaved PARP fragments were analyzed by Western blot at different time points. B: Normalized quantitation of the Ambra1 optical density in SW620 cells treated with staurosporine for the indicated times. C: SW620 cells treated with 1 µM staurosporine for the indicated times and analyzed by Western blot for pro-capsase-3 and cleaved caspase-3. D: Normalized quantitation of the cleaved caspase-3 optical density in SW620 cells treated with staurosporine for the indicated times. E: SW620 cells treated with various doses of staurosporine for 6 h. Ambra1 levels were measured by Western blot. F: Normalized quantitation of the Ambra1 optical density in SW620 cells treated with the indicated doses of staurosporine.

Figure 4. Ambra1 degradation during etoposide-induced apoptosis.

A: SW620 cells treated with 1 µM staurosporine. Ambra1, Beclin1, Vps34 and ATG5 expression was measured by Western blot at various time points. B: Normalized quantitation of the Ambra1 optical density in SW620 cells treated with etoposide for the indicated times. C: SW620 cells treated with 1 µM staurosporine for the indicated times and analyzed by Western blot for pro-capsase-3 and cleaved caspase-3. D: Normalized quantitation of the cleaved caspase-3 optical density in SW620 cells treated with etoposide for the indicated times. E: SW620 cells treated with various doses of etoposide for 6 h. Ambra1 levels were measured by Western blot. F: Normalized quantitation of the Ambra1 optical density in SW620 cells treated with the indicated doses of staurosporine. The data are presented as the mean ± SD of three independent experiments (p<0.05).

Figure 5. Calpains and caspases are responsible for Ambra1 cleavage.

A: SW620 cells treated with 2 µM staurosporine for 6 h in the presence or absence of 20 µM calpain inhibitor (CL) and/or 10 µM caspase inhibitor (z-VAD-fmk). Ambra1 expression was evaluated by Western blot. B: SW620 cells treated with 1 µg/ml etoposide for 6 h in the presence or absence of 20 µM calpain inhibitor (CL) and/or 10 µM caspase inhibitor (z-VAD-fmk). Ambra1 expression was evaluated by Western blot. The data are presented as the mean ± SD of three independent experiments (p<0.05).

Figure 6. The pro-survival function of Ambra1 in SW620 cells.

A: Western blot analysis of Ambra1 expression in SW620 cells after transient transfection with negative control or Ambra1-siRNA oligonucleotides. These cells were analyzed by MTT assay. B: SW620 cells cultured in serum-free medium for 12 or 24 h. Cell viability was determined by MTT assay. C: Western blot analysis of Ambra1 expression in SW620 cells after transient transfection with negative control and Ambra1-siRNA oligonucleotides. These cells were analyzed by flow cytometry. D: SW620 cells serum starved for 24 h. Cell death was measured by flow cytometry. E: Western blot analysis of Ambra1 expression in SW620 cells after transient transfection with negative control or Ambra1-siRNA oligonucleotides. These cells were assayed for staurosporine-induced cell death. F: SW620 cells treated with staurosporine (2 µM for 6 h). Cell death was measured by flow cytometry. G: Western blot analysis of Ambra1 expression in SW620 cells after transient transfection with negative control or Ambra1-siRNA oligonucleotides. These cells were assayed for etoposide-induced cell death. H: SW620 cells treated with etoposide (5 µg/ml for 24 h). Cell death was measured by flow cytometry. The data are presented as the mean ± SD of three independent experiments (p<0.05).

Figure 7. Ambra1 downregulation suppresses autophagy.

A: Western blot analysis of Ambra1 expression in SW620 cells after transient transfection with negative control or Ambra1-siRNA oligonucleotides. B: Quantitation of the optical density of the Ambra1 bands after transfection. C and D: LC3 levels in negative control and si-Ambra1 serum-starved SW620 cells. E: Immunofluorescence images of SW620 cells treated with 100 nM BafA1. SW620 cells were transfected with negative control or Ambra1-siRNA oligonucleotides. F: The quantification of positive dots in the negative control and si-Ambra1 groups. The data are presented as the mean ± SD of three independent experiments (p<0.05).

Figure 8. Ambra1 interacts with Beclin1 in SW620 cells.

A and B: Co-immunoprecipitation assays with SW620 cell lysates. SW620 cells were serum-starved or not (control) for 12 h. Next, the cells were incubated with anti-Ambra1 antisera or control sera (A) or anti-Beclin1 antisera or control sera (B) covalently bound to protein A-sepharose beads. After extensive washes, the eluted proteins were separated and subjected to Beclin1 and Ambra1 immunoblotting. The procedure for (C) and (D) is similar to that for (A) and (B) with the exception that the cells were treated or not with rapamycin (100 nM for 12 h) instead of being serum-starved.