Introduction Autophagic cell death in cancer cells can be mediated by inhibition of deacetylases. Although extensive studies have focused on the autophagic process in cancer, little is known about the role of autophagy in degrading cytosolic and nuclear components of pancreatic neuroendocrine neoplastic (pNEN) cells leading to cell death, thus improving the therapy of patients affected by pNEN. Methods 2D and 3D human pNEN and pancreatic stellate cells were treated with panobinostat and bafilomycin. Autophagy markers were detected by RT-qPCR, immunofluorescence and western blot. Autophagosomes were detected by electron microscopy and their maturation by real time fluorescence of LC3B stable transfected cells. ChIP was performed at the cAMP responsive element. Immunofluorescence was performed in murine pancreatic tissue. Results We observed that pan-deacetylase inhibitor panobinostat treatment causes autophagic cell death in pNEN cells. We also found that although AMPK-α phosphorylation is counterbalanced by phosphorylated AKT, it is not capable to inhibiting autophagic cell death. Whereas the binding activity of cAMP responsive element is prompted by panobinostat. Although autophagy inhibition prevented autophagosomes synthesis, maturation and cell death, panobinostat treatment induced the accumulation of mature autophagosomes in the cytosol, and the nucleus, leading to disruption of the organelles, cellular digestion and decay. Observation of autophagosome membrane proteins Beclin1 and LC3B aggregation in murine pancreatic islets indicates that autophagy restoration may also lead to autophagosome aggregation in murine insulinoma cells. A basal low expression of autophagy markers was detectable in patients affected by pNEN and, interestingly, the expression of these markers was significantly lower in metastatic pNEN. Discussion/Conclusion Our study highlights that the autophagy functional restoration and prolongation of this catabolic process, mediated by inhibition of deacetylase, is responsible for the reduction of pNEN cells. Prompting of autophagy cell death could be a promising strategy for the therapy of pNEN.


中文翻译：

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自噬介导的死亡对胰腺神经内分泌肿瘤细胞命运的调节

简介 癌细胞中的自噬性细胞死亡可通过抑制脱乙酰酶介导。尽管广泛的研究集中在癌症的自噬过程上，但人们对自噬在降解胰腺神经内分泌肿瘤 (pNEN) 细胞的胞质和核成分导致细胞死亡中的作用知之甚少，从而改善了受 pNEN 影响的患者的治疗。方法 用帕比司他和巴弗洛霉素处理 2D 和 3D 人 pNEN 和胰腺星状细胞。通过 RT-qPCR、免疫荧光和蛋白质印迹检测自噬标记物。通过电子显微镜检测自噬体，并通过 LC3B 稳定转染细胞的实时荧光检测其成熟。ChIP 在 cAMP 反应元件上进行。在小鼠胰腺组织中进行免疫荧光。结果我们观察到泛脱乙酰酶抑制剂帕比司他治疗会导致 pNEN 细胞发生自噬性细胞死亡。我们还发现，尽管 AMPK-α 磷酸化被磷酸化的 AKT 所抵消，但它不能抑制自噬性细胞死亡。而 cAMP 反应元件的结合活性是由 panobinostat 提示的。尽管自噬抑制阻止了自噬体的合成、成熟和细胞死亡，但帕比司他治疗诱导了成熟的自噬体在细胞质和细胞核中的积累，从而导致细胞器的破坏、细胞消化和衰变。小鼠胰岛自噬体膜蛋白 Beclin1 和 LC3B 聚集的观察表明自噬恢复也可能导致小鼠胰岛素瘤细胞自噬体聚集。在受 pNEN 影响的患者中可检测到自噬标志物的基础低表达，有趣的是，这些标志物的表达在转移性 pNEN 中显着降低。讨论/结论 我们的研究强调，通过抑制脱乙酰酶介导的自噬功能恢复和这种分解代谢过程的延长是导致 pNEN 细胞减少的原因。促进自噬细胞死亡可能是治疗 pNEN 的一种有前途的策略。负责减少 pNEN 细胞。促进自噬细胞死亡可能是治疗 pNEN 的一种有前途的策略。负责减少 pNEN 细胞。促进自噬细胞死亡可能是治疗 pNEN 的一种有前途的策略。