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

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.