Upon activation, the 5'-adenosine monophosphate-activated protein kinase (AMPK) increases catabolism, while inhibiting anabolism. The anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), activates AMPK in multiple tumor cell-types (Biochim. Biophys Acta 2016;1863:2916-2933). This acts as an initial cell "rescue response" after iron-depletion mediated by Dp44mT. Considering Dp44mT-mediated AMPK activation, the role of AMPK on Dp44mT cytotoxicity was examined. Dp44mT increased the p-AMPK/AMPK ratio in multiple tumor cell-types over short (24 h) and longer (72 h) incubations. Notably, Dp44mT was more effective in inhibiting tumor cell proliferation after AMPK silencing, potentially due to the loss of AMPK-mediated metabolic plasticity that protects cells against Dp44mT cytotoxicity. The silencing of AMPK-increased cellular cholesterol and stabilized lysosomes against Dp44mT-mediated lysosomal membrane permeabilization. This was substantiated by studies demonstrating that the cholesterol-depleting agent, methyl-β-cyclodextrin (MβCD), restores Dp44mT-mediated lysosomal membrane permeabilization in AMPK silenced cells. The increased levels of cholesterol after AMPK silencing were independent of the ability of AMPK to inhibit the rate-limiting step of cholesterol synthesis via the inactivating phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) at Ser872. In fact, Dp44mT did not increase phosphorylation of HMGCR at (Ser872), but decreased total HMGCR expression similarly in both the presence or absence of AMPK silencing. Dp44mT was demonstrated to increase autophagic initiation after AMPK silencing via an AMPK- and Beclin-1-independent mechanism. Further, there was increased cleaved caspase 3 and cleaved PARP after incubation of AMPK silenced cells with Dp44mT. Overall, AMPK silencing promotes Dp44mT anti-proliferative activity, suggesting a role for AMPK in rescuing its cytotoxicity by inhibiting autophagy and also apoptosis.

中文翻译：

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Dp44mT介导的AMPK在胰腺癌细胞中的活化，调节自噬和凋亡。

激活后，5'-腺苷单磷酸激活的蛋白激酶（AMPK）增加分解代谢，同时抑制合成代谢。抗癌剂二-2-吡啶基酮4,4-二甲基-3-硫代半碳酰胺（Dp44mT）可激活多种肿瘤细胞类型的AMPK（Biochim.Biophys Acta 2016; 1863：2916-2933）。在Dp44mT介导的铁耗竭之后，这充当了初始细胞“救援反应”。考虑到Dp44mT介导的AMPK激活，检查了AMPK对Dp44mT细胞毒性的作用。Dp44mT在短时间（24 h）和较长时间（72 h）孵育中增加了多种肿瘤细胞类型的p-AMPK / AMPK比。值得注意的是，Dp44mT在AMPK沉默后更能有效抑制肿瘤细胞的增殖，这可能是由于AMPK介导的代谢可塑性丧失所致，从而保护了细胞免受Dp44mT细胞毒性。AMPK增加的细胞胆固醇和稳定的溶酶体对Dp44mT介导的溶酶体膜通透性的沉默。这通过研究证实了胆固醇消耗剂甲基-β-环糊精（MβCD）在AMPK沉默的细胞中恢复Dp44mT介导的溶酶体膜通透性的研究。AMPK沉默后，胆固醇水平升高与AMPK通过在Ser872处使3-羟基-3-甲基戊二酰辅酶A还原酶（HMGCR）失活磷酸化来抑制胆固醇合成的限速步骤的能力无关。实际上，Dp44mT不会在（Ser872）处增加HMGCR的磷酸化，但在存在或不存在AMPK沉默的情况下，总HMGCR的表达均会类似地降低。Dp44mT被证明可以通过不依赖AMPK和Beclin-1的机制增加AMPK沉默后的自噬启动。此外，将AMPK沉默的细胞与Dp44mT一起孵育后，裂解的半胱天冬酶3和PARP的裂解增加。总体而言，AMPK沉默促进Dp44mT的抗增殖活性，表明AMPK通过抑制自噬和凋亡而在挽救其细胞毒性中发挥作用。