Ambra1 is an essential regulator of autophagy and apoptosis in SW620 cells: pro-survival role of Ambra1
Authors: Bin Yi
Liang Wang
Zhilong He
Songbing He
Qinyi Qian
Yilin Gu
Daiwei Wan
Wen Gu
Journal: PLoS ONE (2014)
Discipline: Multidisciplinary
Keywords: Ambra1; autophagy; apoptosis; colorectal cancer cells; Beclin1
Affiliations: Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, China., Department of Hepatobiliary Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China., Department of Ultrasonography, Changshu No. 2 People's Hospital, Changshu, China., Department of Gastroenterology, The First Affiliated Hospital of SooChow University, Suzhou, China., Department of Operating Rooms, The First Affiliated Hospital of SooChow University, Suzhou, China., Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, China ; Institute of Health Science and Shanghai Institute of Immunology, Shanghai Institute for Biological Science, Chinese Academy of Science and Shanghai Jiao Tong University School of Medicine, Shanghai, China ; Washington University School of Medicine, St Louis, Missouri, United States of America.

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. 

Retracted: No

PubPeer comments:
 Figure 1F. * Magenta boxes: The Starvation 24h -BafA1 panel appears to overlap with the Ctrl +BafA1 panel. Reported to the journal in October 2015, but no update as of today. ![file] , 
 Figure 4A. * Blue boxes: The VPS34 and ATG5 panels look unexpectedly similar, even though they represent proteins with a very different molecular weight. Reported to the journal in October 2015, but no update as of today. ![file] , 
 A new finding, not part of the concerns reported in 2015, found with the aid of ImageTwin.io. * Yellow boxes: The Starvation 12h -BafA1 panel in Figure 1 appears to overlap with the Starvation 24h siAmbra1#3 panel in Figure 7. * Magenta boxes: The Starvation 24h -BafA1 panel in Figure 1 appears to overlap with the Ctrl +BafA1 panel in Figure 1 (same concern as reported in comment #1) ![file] ,