title: 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.
authors.affiliation: Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Gu
authors.affiliation: Department of Hepatobiliary Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
authors.forename: Daiwei
authors.initials: D
authors.lastname: Wan
authors.affiliation: Department of Ultrasonography, Changshu No. 2 People's Hospital, Changshu, China.
authors.forename: Qinyi
authors.initials: Q
authors.lastname: Qian
authors.affiliation: Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yi
authors.affiliation: Department of Gastroenterology, The First Affiliated Hospital of SooChow University, Suzhou, China.
authors.forename: Zhilong
authors.initials: Z
authors.lastname: He
authors.affiliation: Department of Operating Rooms, The First Affiliated Hospital of SooChow University, Suzhou, China.
authors.forename: Yilin
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Songbing
authors.initials: S
authors.lastname: He
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D000071186:Beclin-1; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000473:pathology*; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C553334:AMBRA1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D005047:Etoposide; D019311:Staurosporine