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

Gu W, Wan D, Qian Q, Yi B, He Z, Gu Y, Wang L, He S - PLoS ONE (2014)

Bottom Line: When Ambra1 expression was reduced by siRNA, SW620 cells were more sensitive to staurosporine- or etoposide-induced apoptosis.In addition, starvation-induced autophagy decreased.In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in CRC cells that maintains the balance between autophagy and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, China.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3936000&req=5

pone-0090151-g002: 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.

Mentions: To further confirm that the autophagosomes were induced by starvation, we examined serum-starved SW620 cells via fluorescence microscopy and transmission electron microscopy (TEM). An antibody against endogenous LC3 was used to detect autophagy by immunofluorescence. Endogenous LC3 was redistributed from a diffuse pattern to visible cytoplasmic puncta (Figure 1F). Figure 1G illustrates the average number of LC3 positive dots in SW620 cells. The TEM images confirmed the induction of autophagy at various time points as indicated by autophagosome visualization (Figure 2). These data demonstrated that autophagy was triggered by starvation and that Ambra1 was associated with autophagy.


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

Gu W, Wan D, Qian Q, Yi B, He Z, Gu Y, Wang L, He S - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3936000&req=5

pone-0090151-g002: 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.
Mentions: To further confirm that the autophagosomes were induced by starvation, we examined serum-starved SW620 cells via fluorescence microscopy and transmission electron microscopy (TEM). An antibody against endogenous LC3 was used to detect autophagy by immunofluorescence. Endogenous LC3 was redistributed from a diffuse pattern to visible cytoplasmic puncta (Figure 1F). Figure 1G illustrates the average number of LC3 positive dots in SW620 cells. The TEM images confirmed the induction of autophagy at various time points as indicated by autophagosome visualization (Figure 2). These data demonstrated that autophagy was triggered by starvation and that Ambra1 was associated with autophagy.

Bottom Line: When Ambra1 expression was reduced by siRNA, SW620 cells were more sensitive to staurosporine- or etoposide-induced apoptosis.In addition, starvation-induced autophagy decreased.In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in CRC cells that maintains the balance between autophagy and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, China.

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.

Show MeSH
Related in: MedlinePlus