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.
PLoS One. 2014 Feb 26;9(2):e90151. doi: 10.1371/journal.pone.0090151. eCollection 2014.
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.
最近的研究揭示了自噬相关基因相关（ATG）蛋白 Ambra1 在自噬存活反应中的作用，并且已经表明 Ambra1 调节胚胎干细胞中的 Beclin1 和 Beclin1 依赖性自噬。然而，Ambra1 是否在结直肠癌细胞的自噬途径中发挥重要作用尚不清楚。在这项研究中，我们假设 Ambra1 是 CRC 细胞系中自噬和细胞凋亡的重要调节剂。为了验证这一假设，我们通过评估内源性微管相关蛋白 1 轻链 3（LC3）定位、自噬体（透射电子显微镜）的存在和 LC3 蛋白水平（Western blot）来证实血清饥饿的 SW620 CRC 细胞中的自噬活性。用 staurosporine 或依托泊苷处理 SW620 细胞后，通过 Western blot 检测 Ambra1 的表达。使用钙蛋白酶和半胱天冬酶抑制剂来验证钙蛋白酶和半胱天冬酶是否负责 Ambra1 的切割。为了研究 Ambra1 在细胞凋亡中的作用，用 staurosporine 和依托泊苷处理 Ambra1 敲低细胞。通过 Annexin-V 和 PI 染色和 MTT 测定来测量细胞凋亡和活力。我们确定血清剥夺诱导的自噬与结直肠癌细胞系中 Ambra1 的上调有关。在 staurosporine 或依托泊苷诱导的细胞凋亡过程中，Ambra1 的表达减少。钙蛋白酶和半胱天冬酶可能负责 Ambra1 的降解。当用 siRNA 降低 Ambra1 表达时，SW620 细胞对 staurosporine 或依托泊苷诱导的细胞凋亡更敏感。此外，饥饿诱导的自噬减少。最后，Ambra1 和 Beclin1 的共免疫沉淀表明，在血清饥饿或雷帕霉素处理的 SW620 细胞中，Ambra1 和 Beclin1 相互作用，表明 Ambra1 通过与 Beclin1 相互作用来调节 CRC 细胞中的自噬。总之，Ambra1 是 CRC 细胞中自噬和细胞凋亡的关键调节剂，它维持自噬和细胞凋亡之间的平衡。
