敲低铁蛋白导致依马替尼诱导神经母细胞瘤细胞发生铁死亡。
Knockdown of ferroportin accelerates erastin-induced ferroptosis in neuroblastoma cells.
机构信息
Department of Pediatric Surgery, Second Hospital, Hebei Medical University, Hebei, Shijiazhuang, China.
出版信息
Eur Rev Med Pharmacol Sci. 2018 Jun;22(12):3826-3836. doi: 10.26355/eurrev_201806_15267.
OBJECTIVE
Ferroptosis is a new-found iron-dependent form of non-apoptotic regulated cell death (RCD), which is activated on therapy with several antitumor agents, but the potential mechanism remains unclear. Erastin, exhibiting selectivity for RAS-mutated cancer cells, induces ferroptosis by increasing iron and lipid reactive oxygen species (ROS) levels in cell. Ferroportin (Fpn), the sole iron export protein, participates in the regulation of intracellular iron concentration. In this study, we investigated the role of Fpn on ferroptosis induced by erastin in SH-SY5Y cells.
MATERIALS AND METHODS
The cell viability was determined by CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit. The activity of caspase-3 was measured by ELISA kit. qRT-PCR was performed to examine the mRNA expression of Fpn. Western blot assay was conducted to examine the expression level of marker proteins. Specific commercial kits were used to examine the levels of MDA, ROS and iron in cells, respectively.
RESULTS
Ferroptosis was evaluated by intracellular lipid ROS level and iron concentration. Hepcidin could prevent erastin-induced ferroptosis by degrading Fpn. Erastin (5 μg/mL) was observed to induce ferroptosis in neuroblastoma cells at 6 hours, which was promoted by knockdown of Fpn. The expression of Fpn gene and protein was decreased in SH-SY5Y cells treated with erastin. After treatment with erastin, Fpn siRNA transfection in SH-SY5Y cells was able to accelerate ferroptosis-associated phenotypic changes. Fpn acted as a negative regulator of ferroptosis by reducing intracellular iron concentration. Knockdown of Fpn enhanced anticancer activity of erastin.
CONCLUSIONS
These results suggested that knockdown of Fpn accelerated erastin-induced ferroptosis by increasing iron-dependent lipid ROS accumulation, highlighting Fpn as a potential therapeutic target site for neuroblastoma. Thus, Fpn inhibitors may provide new access for chemosensitization of neuroblastoma.
目的
铁死亡是一种新发现的铁依赖性非凋亡调控细胞死亡（RCD）形式，它在几种抗肿瘤药物治疗时被激活，但潜在机制尚不清楚。恩拉霉素（erastin）对 RAS 突变型癌细胞具有选择性，通过增加细胞内铁和脂质活性氧（ROS）水平诱导铁死亡。铁蛋白（Fpn）是唯一的铁输出蛋白，参与细胞内铁浓度的调节。在这项研究中，我们研究了 Fpn 在恩拉霉素诱导的 SH-SY5Y 细胞铁死亡中的作用。
材料和方法
通过 CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay 试剂盒测定细胞活力。通过 ELISA 试剂盒测定 caspase-3 的活性。qRT-PCR 用于检测 Fpn 的 mRNA 表达。Western blot 检测用于检测标记蛋白的表达水平。分别使用特定的商业试剂盒来检测细胞内 MDA、ROS 和铁的水平。
结果
通过细胞内脂质 ROS 水平和铁浓度来评估铁死亡。铁调素（hepcidin）可通过降解 Fpn 来预防恩拉霉素诱导的铁死亡。在 6 小时时，观察到 5μg/mL 的恩拉霉素诱导神经母细胞瘤细胞发生铁死亡，而 Fpn 的敲低可促进这种作用。在用恩拉霉素处理的 SH-SY5Y 细胞中，Fpn 基因和蛋白的表达均降低。在用恩拉霉素处理后，SH-SY5Y 细胞中 Fpn siRNA 的转染能够加速与铁死亡相关的表型变化。Fpn 通过降低细胞内铁浓度作为铁死亡的负调节因子。Fpn 的敲低增强了恩拉霉素的抗癌活性。
结论
这些结果表明，敲低 Fpn 通过增加铁依赖性脂质 ROS 积累加速恩拉霉素诱导的铁死亡，突出了 Fpn 作为神经母细胞瘤的潜在治疗靶点。因此，Fpn 抑制剂可能为神经母细胞瘤的化疗增敏提供新的途径。
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