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.

Ambra1 in autophagy and apoptosis: Implications for cell survival and chemotherapy resistance

“…Ambra1 may primarily play a pro-survival role due to the positive induction of autophagy, which has been previously demonstrated in in vivo (mouse embryos) and in vitro (2FTG cells, SW620 cells and neural stem cells) studies (3,4,73,74). According to these results, the accumulation of Ambra1 in cells will promote autophagy occurrence and suppress apoptosis execution, which may decrease the effectiveness of chemotherapy.…”

“…Although certain studies indicate that autophagy participates in chemotherapy resistance, the mechanism is not yet clear (1). A recent series of studies suggest that activating molecule in Beclin 1-regulated autophagy protein 1 (Ambra1) is an important factor in regards to the association between autophagy and apoptosis, and may control the reciprocal conversion between the two processes to decide the resulting cell death or survival (2)(3)(4). Therefore, Ambra1 may be an important factor of autophagy involved in cancer treatment and chemotherapy resistance.…”

Conformational Flexibility Enables the Function of a BECN1 Region Essential for Starvation-Mediated Autophagy

“…To our knowledge, this is the first BECN1 region to be directly implicated in up-regulating autophagy in response to starvation, rather than impacting general autophagy levels. Both AMBRA1 and UVRAG have previously been shown to also be important for the up-regulation of autophagy (8, 72, 74). This may suggest that mutation of conserved BECN1 FHD residues abolishes the interaction with either AMBRA1 or UVRAG, or perhaps yet some other uncharacterized interaction, thereby abrogating the starvation-induced up-regulation of autophagy.…”

Downregulation of Enhancer of Zeste Homolog 2 (EZH2) is essential for the Induction of Autophagy and Apoptosis in Colorectal Cancer Cells

“…In our study, we found increased expression of LC3-II and Ambra1 in CRC cell lines after transfection with EZH2-shRNA or addition of exogenous DZNep. LC3-II and Ambra1 are the marker proteins of occurrence of autophagy [30,31,32]. Our previous study evidenced that the increased expression of Ambra1 corresponded to the induction of autophagy [33].…”

“…There are multiple epigenetic mechanisms, such as chromatin modulation, histone modification, and microRNAs (miRNAs) that regulate the autophagy. For example, the activity of histone deacetylase SIRT1 [32] or histone mark H3K4me [34] regulates autophagy. As an epigenetic gene, EZH2 has only been reported to regulate in HCC cells [35] and glioblastoma multiforme [36].…”