白花丹素调控Nrf-2/Keap1信号通路诱导膀胱癌细胞铁死亡

Plumbagin Induces Ferroptosis Through Nrf-2/Keap1 Signaling Pathway in Bladder Cancer Cells

目的:探讨白花丹素作为一种新型的铁死亡诱导剂在膀胱癌抑制中的作用机制。方法:本研究中使用了膀胱癌细胞T24。采用细胞增殖与活性检测-8(CCK-8)法检测白花丹素(0.1、1、2、3、6、12、24、48μmol·L^(-1))对T24细胞活力的影响。采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V FITC/PI)凋亡试剂盒检测白花丹素(1.5、3、6μmol·L^(-1))对T24细胞凋亡的影响。采用不同的抑制剂(铁死亡抑制剂Fer-1,凋亡抑制剂VAD,坏死性凋亡抑制剂Nec-1)与白花丹素(6μmol·L^(-1))联合使用。采用活性氧荧光探针(DCFH-DA),丙二醛(MDA)和谷胱甘肽(GSH)试剂盒分别检测不同浓度的白花丹素(1.5、3、6μmol·L^(-1))对T24细胞内活性氧水平,MDA和GSH的含量,脂质过氧化荧光探针(C11-BODIPY)荧光探针检测白花丹素(1.5、3、6μmol·L^(-1))对T24细胞中过氧化物水平的影响。蛋白免疫印迹法(Western blot)检测白花丹素(1.5、3、6μmol·L^(-1))细胞中溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化酶(GPX4)、核因子E2相关因子-2(Nrf-2)和Kelch样ECH关联蛋白1(Keap1)的蛋白表达的影响。结果:与空白组比较,白花丹素组T24细胞的活性明显降低(P<0.05),IC50为3.52μmol·L^(-1)。与空白组比较,白花丹素组(1.5、3、6μmol·L^(-1))T24细胞凋亡率明显升高(P<0.05);与单独使用6μmol·L^(-1)的白花丹素组比较,铁死亡抑制剂和凋亡抑制剂组能够逆转6μmol·L^(-1)的白花丹素对T24细胞增殖抑制作用(P<0.05)。与空白组比较,白花丹素组(1.5、3、6μmol·L^(-1)),T24细胞ROS、MDA及脂质过氧化物的含量明显升高,GSH水平明显降低,铁死亡相关蛋白SLC7A11、GPX4以及Nrf-2/Keap1明显降低(P<0.05)。结论:白花丹素能诱导细胞铁死亡,其机制与Nrf-2/Keap1信号通路有关。

Objective:To explore the mechanism of plumbagin as a novel ferroptosis inducer in bladder cancer inhibition.Method:Bladder cancer T24 cells were used in this study.The effect of different concentrations of plumbagin(0.1,1,2,3,6,12,24,48μmol·L^(-1))on the viability of T24 cells was detected by cell counting kit-8(CCK-8).The effect of different concentrations of plumbagin(1.5,3,6μmol·L^(-1))on the apoptosis of T24 cells was detected by annexin V-fluorescein isothiocyanate(Annexin V FITC)/PI apoptosis kit.Different inhibitors(ferroptosis inhibitor Fer-1,apoptosis inhibitor VAD,and necroptosis inhibitor Nec-1)were used in combination with plumbagin(6μmol·L^(-1)).Reactive oxygen species(ROS)fluorescent probe(DCFH-DA),malonaldehyde(MDA),and glutathione(GSH)kits were used to detect the effects of different concentrations of plumbagin(1.5,3,6μmol·L^(-1))on the level of ROS and the content of MDA and GSH in T24 cells,respectively.The effect of different concentrations of plumbagin(1.5,3,6μmol·L^(-1))on peroxide levels in T24 cells was detected by C11-BODIPY fluorescent probe.Western blot was used to detect the effect of different concentrations of plumbagin(1.5,3,6μmol·L^(-1))on the protein expression of solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4),nuclear factor E2-related factor-2(Nrf-2),and Kelch-like ECHassociated protein 1(Keap1).Result:Compared with the blank group,plumbagin could inhibit the activity of T24 cells(P<0.05)with IC50 of 3.52μmol·L^(-1).At the concentrations of 1.5,3,6μmol·L^(-1),plumbagin significantly promoted the apoptosis of T24 cells(P<0.05)as compared with the blank group.Compared with the plumbagin group at 6μmol·L^(-1),the ferroptosis inhibitor and apoptosis inhibitor groups could reverse the inhibitory effect of 6μmol·L^(-1) plumbagin on the proliferation of T24 cells(P<0.05).Compared with the blank group,the plumbagin groups at 1.5,3,6μmol·L^(-1) showed increased content of ROS,MDA,and lipid peroxides in T24 cells,decreased GSH level,and reduced SLC7A11,GPX4,and Nrf-2/Keap1(P<0.05).Conclusion:plumbagin can induce ferroptosis,and its mechanism is related to the Nrf-2/Keap1 signaling pathway.