CYP2J2介导生成的EETs通过PPARγ促进胰腺导管腺癌铁死亡抵抗

CYP2J2-produced epoxyeicosatrienoic acids contribute to the ferroptosis resistance of pancreatic ductal adenocarcinoma in a PPARγ-dependent manner

目的:胰腺导管腺癌是消化系统的恶性肿瘤,预后差,复发率高。最新研究表明铁死亡抵抗是胰腺导管腺癌的病理机制之一,但发生铁死亡抵抗的潜在机制尚未阐明。细胞色素P4502J2(CYP2J2)是人体组织细胞中介导花生四烯酸生成环氧二十碳三烯酸(epoxyeicosatrienoic acids,EETs)的关键酶。研究显示EETs参与肿瘤的发生发展,然而EETs在胰腺导管腺癌中的作用及其对铁死亡的影响尚不清楚。本研究探讨CYP2J2及EETs对铁死亡诱导剂erastin诱导的胰腺导管腺癌铁死亡的影响及其机制。方法:收集9例胰腺导管癌患者的肿瘤组织及相应的癌旁组织,采用real-time PCR和蛋白质印迹法检测CYP2J2表达,酶联免疫吸附实验(ELISA)法检测8,9-EET分解产物8,9-碳三烯酸(8,9-dihydroxyeicosatrienoic acids,8,9-DHET)的含量。体外实验以人胰腺导管腺癌细胞系PANC-1为研究对象,采用erastin诱导铁死亡,检测细胞内长链脂酰辅酶A合成酶4(acyl-CoA synthetase 4,ACSL4)的蛋白质水平、乳酸脱氢酶(lactate dehydrogenase,LDH)活性、丙二醛(malondialdehyde,MDA)含量、Fe^(2+)浓度及细胞存活状况。给予8,9-EET预处理,观察其对erastin诱导的PANC-1细胞铁死亡的影响。采用慢病毒构建CYP2J2敲低的细胞系,观察其对erastin诱导的PANC-1细胞铁死亡的影响。采用过氧化物酶体增殖激活受体γ(peroxisome proliferation-activated receptorγ,PPARγ)阻断剂处理,观察其对8,9-EET调节erastin诱导的PANC-1细胞谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)和MDA含量的影响。结果:CYP2J2在胰腺导管腺癌组织中高表达,其下游代谢产物8,9-DHET水平亦显著升高。8,9-EET预处理显著减少erastin诱导的PANC-1细胞铁死亡,同时降低PANC-1细胞中Fe^(2+)浓度、LDH活性、MDA含量及ACSL4蛋白质表达。此外,8,9-EET可部分恢复膜铁转运蛋白(ferroportin,FPN)和铁死亡抑制蛋白(ferroptosis suppressor protein 1,FSP1)的基因表达。shCYP2J2能够加剧erastin诱导的PANC-1细胞铁死亡,下调FPN和FSP1的基因表达。GPX4的表达,该效应可被PPARγ阻断剂GW9662消除。结论:CYP2J2/EETs高表达于胰腺导管腺癌组织,EETs通过激活PPARγ上调GPX4的水平,抑制铁死亡,从而促进胰腺导管腺癌铁死亡抵抗。

Objective: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 P4502J2(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-epoxyeicosatrienoic 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^(2+)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 receptorγ(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^(2+)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 downregulated 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 PPARγblocker GW9662.And GW9662 abolished the anti-ferroptosis effects of 8,9-EET.Conclusion: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.