Upregulation and activation of p53 by erastin-induced reactive oxygen species contribute to cytotoxic and cytostatic effects in A549 lung cancer cells
Upregulation and activation of p53 by erastin-induced reactive oxygen species contribute to cytotoxic and cytostatic effects in A549 lung cancer cells
复制标题
DOI：
10.3892/or.2018.6585
复制
发表时间：
2018-10-01
期刊：
影响因子：
4.2
通讯作者：
Jiang, Rong
中科院分区：
文献类型：
--
作者：
Huang, Chaoli;Yang, Mengchang;Jiang, Rong
肿瘤抑制蛋白p53是多种细胞过程的关键调控因子，通过诱导凋亡细胞死亡发挥其肿瘤抑制功能。然而，新出现的证据表明p53也参与诱导铁凋亡，这是一种独特的铁依赖性非凋亡细胞死亡形式，由ras选择性致死性小分子erastin触发。先前的研究表明，暴露于erastin会导致ROS积累和氧化应激增加。在本研究中，我们用erastin培养A549细胞，检测ROS积累。半定量western blotting分析诱导的ROS对p53活性的影响。为了确定ROS如何激活p53，将ROS清除剂NAC和ATM激酶抑制剂KU-55933与erastin共孵育，然后进行western blot分析。将p53或SLC7A11 siRNA导入A549细胞沉默靶基因表达，然后进行ROS检测，阐明ROS激活的p53对其靶基因SLC7A11的调控作用。采用Annexin V-FITC/PI染色检测erastin暴露对凋亡细胞的诱导作用。为了进一步评估erastin处理对细胞增殖的影响，我们进行了EdU染色和细胞周期流式细胞术分析。在依赖于Erastin诱导的ROS的肺癌A549细胞中，Erastin暴露上调并激活p53，从而转录激活其下游靶基因，包括p21和Bax。随后，通过erastin处理激活p53，抑制SLC7A11并诱导ROS积累，表明p53与erastin诱导的ROS之间存在潜在的反馈回路。通过使用caspase抑制剂Z-VAD-FMK，我们发现，erastin诱导的p53可导致铁致凋亡和凋亡细胞死亡，并通过将细胞周期阻滞在G1期来抑制细胞增殖。综上所述，这些结果表明p53可能参与细胞毒性和细胞抑制作用，并与erastin诱导的ROS建立反馈回路有关。
The tumour-suppressor protein p53 is a key regulator of multiple cellular processes and exerts its tumour-suppressor function by inducing apoptotic cell death. However, emerging evidence indicates that p53 is also involved in inducing ferroptosis, which is a unique iron-dependent form of non-apoptotic cell death triggered by the RAS-selective lethal small molecule erastin. Previous studies have shown that erastin exposure induces increased ROS accumulation and oxidative stress. In the present study, we incubated A549 cells with erastin and detected ROS accumulation. Semi-quantitative western blotting was performed to analyse the effect of the induced ROS on p53 activity. To determine how ROS activate p53, NAC, an ROS scavenger, and KU-55933, an ATM kinase inhibitor, were employed to co-incubate with erastin, followed by western blot analysis. Either p53 or SLC7A11 siRNA was introduced into A549 cells to silence the target-gene expression, followed by ROS detection to illustrate the regulatory role of ROS -activated p53 on its target gene SLC7A11. Annexin V-FITC/PI staining was performed to detect the induction of apoptotic cell death by erastin exposure. To further assess the effects of erastin treatment on cellular proliferation, EdU staining and cell cycle flow cytometric analysis were performed. Erastin exposure upregulated and activated p53 and thus, transcriptionally activated its downstream target genes, including p21 and Bax, in lung cancer A549 cells dependent on erastin-induced ROS. Subsequently, activated p53 by erastin treatment suppressed SLC7A11 and induced ROS accumulation, indicating the potential feedback loop between p53 and erastin-induced ROS. By employing the caspase inhibitor Z-VAD-FMK, it was revealed that erastin-induced p53 contributed to both ferroptotic and apoptotic cell death and inhibited cell proliferation via arresting the cell cycle at G1 phase. Collectively, these results indicated that p53 may contribute to the cytotoxic and cytostatic effects associated with establishing a feedback loop with ROS induced by erastin.