AIMS Inhibitor for the apoptosis-stimulating protein of p53 (iASPP) has been reported to be correlated with 5-fluorouracil (5-Fu) resistance in renal cell carcinoma. Here, we uncover mechanisms of iASPP-Nrf2-ROS regulation of 5-Fu resistance which are important for the development of alternative treatment strategies for gastric adenocarcinoma treatment. METHODS We analyzed iASPP and Nrf2 through TCGA RNA-seq data, UALCAN analysis, and cBioPortal datasets. Intracellular ROS generation was determined by 2',7'-dichloro-fluorescin diacetate staining. Transwell was used to evaluate the invasion. The expression of iASPP, Nrf2, HO-1, and GSTP1 was tested using western blot. RESULTS We found that iASPP KD led to an apparent 5-Fu-induced ROS accumulation in MGC803 and SCG790 cells. Accompanied by iASPP KD, Nrf2 was markedly decreased. iASPP-induced ROS inhibition relies on Nrf2, and due to both knocked down iASPP and Nrf2, the level of ROS did not show an obvious difference with Nrf2 KD solely. Similarly, iASPP KD failed to enhance the Nrf2 KD-mediated ROS accumulation after 5-Fu treatment, suggesting that iASPP-induced antioxidative effects related to 5-Fu resistance are partially dependent on Nrf2. Also, the combination of iASPP KD and Nrf2 KD did not show any synergistic effect on apoptosis after 5-Fu treatment in MGC803 and SCG790 cells. Further studies revealed that iASPP KD or Nrf2 KD could decrease the expression of HO-1 and GSTP1. CONCLUSIONS Our data highlight that iASPP plays a crucial role in the inhibition of 5-Fu-induced apoptosis resistance by removing ROS accumulation in gastric adenocarcinoma, and that the removal of ROS induced by iASPP is Nrf2 signaling dependent.

中文翻译：

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iASPP介导的ROS抑制作用取决于胃腺癌中Nrf2抗氧化信号通路的5-Fu耐药性。

据报道，在肾细胞癌中，p53凋亡刺激蛋白的AIMS抑制剂（iASPP）与5-氟尿嘧啶（5-Fu）耐药性相关。在这里，我们揭示了iASPP-Nrf2-ROS调节5-Fu耐药性的机制，这些机制对于制定胃腺癌替代治疗策略至关重要。方法我们通过TCGA RNA序列数据，UALCAN分析和cBioPortal数据集分析了iASPP和Nrf2。通过2'，7'-二氯荧光素二乙酸酯染色确定细胞内ROS的产生。Transwell用于评估入侵。使用蛋白质印迹测试了iASPP，Nrf2，HO-1和GSTP1的表达。结果我们发现iASPP KD在MGC803和SCG790细胞中导致了明显的5-Fu诱导的ROS积累。伴随iASPP KD，Nrf2明显减少。iASPP诱导的ROS抑制依赖于Nrf2，并且由于同时击倒了iASPP和Nrf2，因此ROS的水平与单独使用Nrf2 KD并没有显示明显的差异。同样，iASPP KD在5-Fu处理后未能增强Nrf2 KD介导的ROS积累，表明iASPP诱导的与5-Fu抗性相关的抗氧化作用部分取决于Nrf2。同样，iASPP KD和Nrf2 KD的组合在MGC803和SCG790细胞中经5-Fu处理后对细胞凋亡也没有显示任何协同作用。进一步的研究表明，iASPP KD或Nrf2 KD可以降低HO-1和GSTP1的表达。结论我们的数据强调，iASPP通过去除胃腺癌中的ROS积累在抑制5-Fu诱导的细胞凋亡抗性中起着至关重要的作用，