CYP2J2 产生的环氧二十碳三烯酸以依赖 PPAR 的方式促进胰腺导管腺癌的铁死亡抵抗。
CYP2J2produced epoxyeicosatrienoic acids contribute to the ferroptosis resistance of pancreatic ductal adenocarcinoma in a PPARdependent manner.
机构信息
Department of Laboratory Medicine, Third Xiangya Hospital, Central South University, Changsha 410013.
Department of Laboratory Medicine, Xiangya School of Medicine, Central South University, Changsha 410013.
出版信息
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2021;46(9):932-941. doi: 10.11817/j.issn.1672-7347.2021.210413.
OBJECTIVES
Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant digestive tract tumors with a poor prognosis and high recurrence rate. Recently, ferroptosis resistance has been found in PDAC. However, the underlying mechanism of ferroptosis resistance has not been fully elucidated. Cytochrome P450 2J2 (CYP2J2) is the main enzyme which mediates arachidonic acid to produce epoxyeicosatrienoic acids (EETs) in human tissues. It has been reported that EETs involve in the development of cancer, while the roles of EETs in PDAC and ferroptosis remain unclear.This study aims to explore the effect of CYP2J2/EETs on ferroptosis of human pancreatic ductal adenocarcinoma cells PANC-1 cells and the underlying mechanisms.
METHODS
The tumor tissues and para-carcinoma tissues of 9 patients with PDAC were collected and the expression of CYP2J2 was detected with real-time PCR and Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of 8,9-dihydroxyeicosatrienoic acid (8,9-DHET), and the degradation product of 8,9-epoxyeicosa-trienoic acid (8,9-EET). PANC-1 cells were used in this study. The ferroptosis inducer erastin was used to induce ferroptosis. The intracellular long-chain acyl-CoA synthetase 4 (ACSL4) protein level, lactate dehydrogenase (LDH) activity, malondialdehyde (MDA) content, Fe concentration, and cell survival were detected. The 8,9-EET was pretreated to observe its effect on erastin-induced ferroptosis in PANC-1 cells. Lentivirus was used to construct a CYP2J2 knockdown cell line to observe its effect on the ferroptosis of PANC-1 cells induced by erastin. A peroxisome proliferation-activated receptor γ (PPARγ) blocker was used to observe the effect of 8,9-EET on erastin-induced glutathione peroxidase 4 (GPX4) and MDA content in PANC-1 cells.
RESULTS
High expression of CYP2J2 was found in PDAC, accompanied by an increased level of 8,9-DHET. The 8,9-EET pretreatment significantly attenuated the PANC-1 cell death induced by erastin. The 8,9-EET reduced the Fe concentration, LDH activity and MDA content, and ACSL4 protein expression in erastin-treated PANC-1 cells. The 8,9-EET also restored the ferroportin (FPN) and ferroptosis suppressor protein 1 (FSP1) mRNA expressions in erastin-treated PANC-1 cells. But CYP2J2 knockdown exacerbated the erastin-induced ferroptosis in PANC-1 cells. Besides, CYP2J2 knockdown furtherly down-regulated the gene expression of FPN and FSP1. The 8,9-EET increased the expression of GPX4 in the erastin-treated PANC-1 cells, which was eliminated by a PPARγ blocker GW9662. And GW9662 abolished the anti-ferroptosis effects of 8,9-EET.
CONCLUSIONS
CYP2J2/EETs are highly expressed in PDAC tissues. EETs inhibit the ferroptosis via up-regulation of GPX4 in a PPARγ-dependent manner, which contributes to the ferroptosis resistance of PDAC.
目的
胰腺导管腺癌（PDAC）是最恶性的消化道肿瘤之一，预后差，复发率高。最近，在 PDAC 中发现了铁死亡抵抗。然而，铁死亡抵抗的潜在机制尚未完全阐明。细胞色素 P450 2J2（CYP2J2）是在人类组织中介导花生四烯酸生成环氧二十碳三烯酸（EETs）的主要酶。据报道，EETs 参与癌症的发展，而 EETs 在 PDAC 和铁死亡中的作用尚不清楚。本研究旨在探讨 CYP2J2/EETs 对人胰腺导管腺癌 PANC-1 细胞铁死亡的影响及其潜在机制。
方法
收集 9 例 PDAC 患者的肿瘤组织和癌旁组织，采用实时 PCR 和 Western blot 检测 CYP2J2 的表达。酶联免疫吸附试验（ELISA）检测 8,9-二羟二十碳三烯酸（8,9-DHET）和 8,9-环氧二十碳三烯酸（8,9-EET）降解产物的水平。本研究采用 PANC-1 细胞。用铁死亡诱导剂 erastin 诱导铁死亡。检测细胞内长链酰基辅酶 A 合成酶 4（ACSL4）蛋白水平、乳酸脱氢酶（LDH）活性、丙二醛（MDA）含量、Fe 浓度和细胞存活率。用 8,9-EET 预处理观察其对 erastin 诱导的 PANC-1 细胞铁死亡的影响。用慢病毒构建 CYP2J2 敲低细胞系，观察其对 erastin 诱导的 PANC-1 细胞铁死亡的影响。用过氧化物酶体增殖物激活受体γ（PPARγ）抑制剂观察 8,9-EET 对 erastin 诱导的 PANC-1 细胞谷胱甘肽过氧化物酶 4（GPX4）和 MDA 含量的影响。
结果
PDAC 中 CYP2J2 高表达，伴随 8,9-DHET 水平升高。8,9-EET 预处理显著减轻 erastin 诱导的 PANC-1 细胞死亡。8,9-EET 降低了 erastin 处理的 PANC-1 细胞中的 Fe 浓度、LDH 活性和 MDA 含量以及 ACSL4 蛋白表达。8,9-EET 还恢复了 erastin 处理的 PANC-1 细胞中的铁转运蛋白（FPN）和铁死亡抑制蛋白 1（FSP1）mRNA 表达。但 CYP2J2 敲低加剧了 erastin 诱导的 PANC-1 细胞铁死亡。此外，CYP2J2 敲低进一步下调了 erastin 处理的 PANC-1 细胞中 FPN 和 FSP1 的基因表达。8,9-EET 增加了 erastin 处理的 PANC-1 细胞中 GPX4 的表达，该表达被过氧化物酶体增殖物激活受体γ（PPARγ）抑制剂 GW9662 消除。而 GW9662 消除了 8,9-EET 的抗铁死亡作用。
结论
CYP2J2/EETs 在 PDAC 组织中高表达。EETs 通过 PPARγ 依赖性方式上调 GPX4 抑制铁死亡，有助于 PDAC 的铁死亡抵抗。
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