Background and PurposeThe 2020 Global Cancer Statistics show that breast cancer is the most prevalent cancer.Trastuzumab,a humanization antibody targeting human epidermal growth factor receptor 2(HER2),plays an important role in the immunotherapy of breast cancer and can significantly improve the life expectancy of patients,but is prone to induce cardiotoxicity.Ferroptosis is an iron-dependent mode of cell death in which ion accumulation and lipid peroxidation are essential features of iron death.Ferroptosis is involved in the development and progression of various cardiovascular diseases such as atherosclerosis,ischemia-reperfusion injury and heart failure.Adriamycin cardiotoxicity is closely related to lipid peroxide accumulation and mitochondrial iron overload,whether trastuzumab-induced cardiomyocyte injury is related to iron death has not been reported.In this study,we investigated the molecular mechanism of trastuzumab-associated cardiotoxicity in mouse cardiomyocytes,taking ferroptosis as the starting point,and provided new ideas for the prevention and treatment of onco-cardiology.Methods1.HL-1 cardiomyocytes treated with the ferroptosis inhibitor Fer-1 or DFO intervention Trastuzumab were assayed for cell viability by Cell Counting Kit-8(CCK-8);the expression of ferroptosis-associated proteins was examined at the transcriptional and translational levels using quantitative real-time polymerase RT-q PCR and Western Blot to detect the expression of ferroptosis-related proteins at the transcriptional and translational levels;2-7-Dichlorouorescin diacetate(DCFH-DA)staining to detect reactive oxygen species(ROS)in cells;the intracellular lipid ROS levels were detected by flow cytometry;the malonaldehyde(MDA)assay kit was used to detect changes in intracellular MDA levels;reduced glutathione(L-GSH)and oxidized glutathione(GSSG)were detected by disulfide(GSSG)kit to measure the GSH/GSSG ratio;Fe2+kit to determine the intracellular Fe2+level.2.Ferroptosis inhibitor,PI3K agonist and inhibitor intervention in Trastuzumab-treated HL-1 cardiomyocytes,Western Blot for phosphorylation levels of PI3K and AKT,and protein expression of Nrf2;immunofluorescence for nuclear translocation of Nrf2.3.Ferroptosis inhibitor Fer-1 or DFO intervention in Trastuzumab-treated HL-1cardiomyocytes,Western Blot assay detects expression of GPx4,4-HNE in mitochondria;Mito Sox detects ROS changes in mitochondria;the Mitochondrial Permeability Transition pore(MPTP)assay kit detects changes in mitochondrial membrane permeability;JC-1 assay detects changes in mitochondrial membrane potential;ATP assay kit detects intracellular ATP levels;assay kit detects the NAD+/NADH and NADP+/NADPH ratio;Co-Immunoprecipitation(Co-IP)detects the interaction of VDAC1 with 4-HNE.Results1.Trastuzumab treatment of HL-1 cells resulted in decreased cell survival,decreased intracellular Erb B2 and GPx4 protein and m RNA expression,increased4-HNE protein expression,increased cellular ROS as detected by DCFH-DA staining,increased intracellular lipid ROS levels as detected by flow cytometry,increased MDA content,decreased GSH/GSSG ratio,elevated intracellular Fe2+levels;intervention with ferroptosis inhibitor Fer-1 or DFO,reversed the above effects of Trastuzumab on HL-1 cells.2.Trastuzumab treatment of HL-1 cells,Western blot showed that Trastuzumab decreased phosphorylation levels of PI3K and AKT,decreased protein expression of Nrf2.PI3K agonist intervention increased phosphorylation levels of PI3K and AKT and protein expression of Nrf2 and increased Nrf2 nuclear translocation;no effect on expression of Erb B2.3.Trastuzumab-treated HL-1 cells showed Trastuzumab decreased GPx4expression in mitochondria,increased 4-HNE expression,increased ROS in mitochondria,increased mitochondrial membrane permeability,decreased mitochondrial membrane potential,decreased intracellular ATP levels,and decreased NAD+/NADH and NADP+/NADPH ratios;ferroptosis inhibitor Fer-1 or DFO reverse the above effects of Trastuzumab on HL-1 cells.Co-IP suggested an interaction between VDAC1 and 4-HNE.Conclusions1.Trastuzumab induces ferroptosis in HL-1 cardiomyocytes by inhibiting the PI3K/AKT signaling pathway,decreasing Nrf2 nuclear translocation,and causing lipid ROS accumulation.2.Trastuzumab causes mitochondrial lipid peroxidation by increasing mitochondrial VDAC1 expression,leading to mitochondrial dysfunction in HL-1cardiomyocytes. |