鸦胆子苦醇调节Nrf2/SLC7A11/GPX4信号通路对皮肤鳞癌细胞铁死亡的影响

Influence of brusatol on ferroptosis in cutaneous squamous cell carcinoma cells by regulating Nrf2/SLC7A11/GPX4 signaling pathway

摘要:

目的 探讨鸦胆子苦醇调节核因子E2相关因子2（nuclear factor erythroid-2 related factor 2，Nrf2）/溶质载体家族7成员11（solute carrier family 7 member 11，SLC7A11）/谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）信号通路对皮肤鳞癌（cutaneous squamous cell carcinoma，cSCC）细胞铁死亡的影响。方法 CCK-8法检测0、100、250、500、700、900 nmol/L鸦胆子苦醇处理后人cSCC细胞系A431细胞增殖率，筛选出合适的鸦胆子苦醇作用浓度。体外培养的A431细胞随机分为对照组、鸦胆子苦醇低剂量组、鸦胆子苦醇高剂量组、特丁基对苯二酚（tert-butylhydroquinone，TBHQ；Nrf2激活剂）组、鸦胆子苦醇高剂量+TBHQ组，以鸦胆子苦醇和TBHQ分组处理后，采用CCK-8法、Hoechst 33342染色法检测各组A431细胞增殖与凋亡；采用试剂盒检测各组A431细胞抗氧化因子［谷胱甘肽（glutathione，GSH）、过氧化氢酶（catalase，CAT）、超氧化物歧化酶（superoxide dismutase，SOD）］水平及铁死亡指标［铁含量、活性氧（reactive oxygen species，ROS）及丙二醛（malondialdehyde，MDA）］水平；采用免疫印迹实验检测各组A431细胞增殖、凋亡及Nrf2/SLC7A11/GPX4信号通路相关蛋白表达。结果 与对照组相比，鸦胆子苦醇低、高剂量组细胞增殖率、GSH及CAT、SOD水平，以及增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）及Bcl-2、Nrf2、SLC7A11、GPX4蛋白表达均降低（P<0.05），凋亡率、铁含量、ROS及MDA水平和Bax蛋白表达均升高（P<0.05）；鸦胆子苦醇高剂量组细胞增殖率、GSH及CAT、SOD水平，以及PCNA及Bcl-2、Nrf2、SLC7A11、GPX4蛋白表达相比鸦胆子苦醇低剂量组进一步降低（P<0.05），凋亡率、铁含量、ROS及MDA水平和Bax蛋白表达进一步升高（P<0.05）；TBHQ组细胞增殖率、GSH及CAT、SOD水平，以及PCNA及Bcl-2、Nrf2、SLC7A11、GPX4蛋白表达升高（P<0.05），凋亡率、铁含量、ROS及MDA水平和Bax蛋白表达降低（P<0.05）。与鸦胆子苦醇高剂量组相比，鸦胆子苦醇高剂量+TBHQ组细胞增殖率、GSH及CAT、SOD水平，以及PCNA及Bcl-2、Nrf2、SLC7A11、GPX4蛋白表达升高（P<0.05），凋亡率、铁含量、ROS及MDA水平和Bax蛋白表达降低（P<0.05）。结论 鸦胆子苦醇通过下调Nrf2/SLC7A11/GPX4信号通路而促进铁死亡，诱导cSCC细胞凋亡，并抑制其增殖。

Abstract:

Objective To investigate the influence of brusatol on ferroptosis in cutaneous squamous cell carcinoma（cSCC） by regulating nuclear factor erythroid-2 related factor 2（Nrf2）/solute carrier family 7 member 11（SLC7A11）/glutathione peroxidase 4（GPX4） signaling pathway.Methods The proliferation rate of human cSCC cell line A431 cells after treatment with 0，100，250，500，700，900 nmol/L of brusatol was detected by CCK-8 method，and the appropriate concentration of brusatol was screened out. The A431 cells cultured in vitro were randomly separated into control group，low-dose brusatol group， high-dose brusatol group，tert-butylhydroquinone（TBHQ；Nrf2 activator） group，and high-dose brusatol+TBHQ group. After treatment with brusatol and TBHQ，respectively，the proliferation and apoptosis of A431 cells in each group were detected by CCK-8 method and Hoechst 33342 staining method；Kits were applied to detect the levels of antioxidant factors［glutathione（GSH），catalase（CAT），and superoxide dismutase（SOD）］ and ferroptosis indicators［iron content，reactive oxygen species（ROS） and malondialdehyde（MDA）］ in A431 cells in each group；the expressions of proliferation，apoptosis and Nrf2/SLC7A11/GPX4 signaling pathway-related proteins of A431 cells in each group were detected by Western blot.Results Compared with the control group，the cell proliferation rate，GSH，CAT，SOD levels，PCNA，Bcl-2，Nrf2，SLC7A11，and GPX4 protein expressions in the low and high dose brusatol groups were all decreased（P<0.05），and the apoptosis rate，iron content，ROS and MDA levels，and Bax protein expression were all increased（P<0.05）；the cell proliferation rate，GSH，CAT，SOD levels，proliferating cell nuclear antigen（PCNA），Bcl-2，Nrf2，SLC7A11，and GPX4 protein expressions in the high-dose brusatol group were further decreased than the low-dose brusatol group（P<0.05），and the apoptosis rate，iron content，ROS and MDA levels，and Bax protein expression were further increased（P<0.05）；the cell proliferation rate，GSH，CAT，SOD levels，PCNA，Bcl-2， Nrf2，SLC7A11，and GPX4 protein expressions in TBHQ group were increased（P<0.05），and the apoptosis rate，iron content，ROS and MDA levels，and Bax protein expression were decreased（P<0.05）. Compared with the high-dose brusatol group，the cell proliferation rate，GSH，CAT，SOD levels，PCNA，Bcl-2，Nrf2，SLC7A11，and GPX4 protein expressions in the high-dose brusatol alcohol+TBHQ group were increased （P<0.05），and the apoptosis rate，iron content，ROS and MDA levels，and Bax protein expression were decreased（P<0.05）.Conclusion Brusatol promotes ferroptosis，induces apoptosis of cSCC cells，and inhibits its proliferation by down-regulating Nrf2/SLC7A11/GPX4 signaling pathway.