Mechanism Of Sodium Selenite Induced Ferroptosis In Triple Negative Breast Cancer Cells

Breast cancer is the most common malignant tumor in women worldwide,with the highest incidence and mortality of female malignant tumors.Triple negative breast cancer（TNBC）is a typical subtype of breast cancer that can not benefit clinically from traditional targeted therapies.Selenium（Se）is an essential trace element for human body and has the potential to prevent and treat cancers.Selenium can induce apoptosis,necrosis and autophagy of cancer cells.Extensive preclinical evidences showed that selenium had an inhibitory effect on the occurrence of breast cancer.Ferroptosis,a kind of programmed cell death,is different from apoptosis,necrosis,autophagy,etc.,and is an important research hotspot in recent years.There have been some relevant studies on ferroptosis in TNBC,but whether selenium can induce ferroptosis in TNBC and its related molecular mechanism have not been reported.In this study,human TNBC cell MDA-MB-231 were used as the study object,and human non-TNBC cell MCF-7 were used as the control.Sodium selenite（Na₂SeO₃）was used as the treatment object.The effect of Na₂SeO₃ on ferroptosis was determined by western blotting,immunofluorescence,transmission electron microscopy,flow cytometry and other methods.The expression level of ferroptosis related proteins,lipid peroxidation level,Fe²⁺level,GSH content,GPx enzyme activity,mitochondrial morphological changes related to ferroptosis were determined.At the same time,the effect of Na₂SeO₃ on ferroptosis was further verified by using ferroptosis inhibitors DFO and Fer-1.It has been reported that ataxia telangiectasia mutated（ATM）played a potential regulatory role in ferroptosis of some cancer cells.In this study,the effect of Na₂SeO₃on ATM kinase activation was detected by immunofluorescence.Furthermore,ATM was knocked down by transient transfection or kinase activity was inhibited by KU55933 treatment.And then,the relevant indexes of ferroptosis were detected and the role of ATM protein in Na₂SeO₃ induced ferroptosis was clarified,thus providing new research data for the effect of selenium on ferroptosis.The results are as follows:1.Na₂SeO₃ induced ferroptosis in MDA-MB-231 cells.（1）After 24 h treatment with different concentrations of Na₂SeO₃（0,5,6,7,8,9,10μM）,the cell viability of MDA-MB-231 cells was significantly decreased in all treatment groups（P<0.01）.The cell viability was decreased to about 70%,50%,30%when treated with 7,8,9μM Na₂SeO₃,which were selected as the follow-up treatment concentrations.（2）The proportion of L-ROS positive cells,MDA and Fe²⁺content were significantly increased by Na₂SeO₃（P<0.01）.（3）The GSH content and the GPx enzyme activity were significantly decreased by Na₂SeO₃（P<0.01）.（4）GPx4 and FTH1 protein expression levels were significantly decreased（P<0.01）.（5）Na₂SeO₃ treatment can lead to degradation or even disappearance of mitochondrial cristae,increase of mitochondrial membrane density,rupture of outer membrane,etc.The results indicated that Na₂SeO₃ treatment induced ferroptosis in MDA-MB-231 cells.However,there was no significant change in ferroptosis related indexes of MCF-7cells treated with 7,8,and 9μM Na₂SeO₃,indicating that Na₂SeO₃ could not induce ferroptosis of MCF-7 cells.Meanwhile,MDA-MB-231 cells were treated with ferroptosis inhibitors DFO or Fer-1 together with Na₂SeO₃（24 h）.The results showed that compared with Na₂SeO₃alone treatment group,the cell vitality,MDA content,Fe²⁺content and the expressions of ferroptosis related proteins GPx4 and FTH1 were significantly increased in combined treatment group（P<0.05）.It was further demonstrated that Na₂SeO₃ induced ferroptosis in MDA-MB-231 cells.2.Na₂SeO₃ induced ATM protein activation in MDA-MB-231 cells.Immunofluorescence assay showed that the fluorescence focus of p-ATM S1981appeared in the cytoplasmic region in Na₂SeO₃-treated cells,and the fluorescence intensity was significantly enhanced（P<0.01）,indicating that Na₂SeO₃ activated ATM protein.The intracellular p-ATM S1981 expression level was significantly decreased when treated with ATM protein kinase inhibitor KU55933（10μM）for 72 h or si ATM transient transfection for 96 h.The results indicated that both KU55933pretreatment for 72 h and si ATM transient transfection for 96 h could significantly inhibit the ATM protein activation induced by Na₂SeO₃,which could be used for subsequent experiments.3.Na₂SeO₃ induced ferroptosis in MDA-MB-231 cells by activating ATM protein.（1）Compared with Na₂SeO₃ group,the protein expression levels of GPx4 and FTH1 were significantly increased after pretreatment with KU55933 or transfection of si ATM（P<0.05）.（2）the number of dead cells,MDA and Fe²⁺contents were significantly decreased,while GSH content and GPx enzyme activity were significantly increased in KU55933 pretreatment group（P<0.05）;Similarly,in si ATM transient transfection group,Fe²⁺content was significantly decreased,GSH content and GPx enzyme activity were significantly increased（P<0.05）.These results suggest that inhibition of ATM kinase activity attenuate the occurrence of Na₂SeO₃ induced ferroptosis,and ATM kinase play an important regulatory role in Na₂SeO₃ induced ferroptosis in MDA-MB-231 cells.In conclusion,Na₂SeO₃ induced ferroptosis and ATM protein activation in MDA-MB-231 cells.Na₂SeO₃ induced ferroptosis was inhibited by ATM kinase inhibitor or si ATM,suggesting that Na₂SeO₃-induced ferroptosis was mediated by ATM protein in MDA-MB-231 cells.In this thesis,the inhibitory mechanism of Na₂SeO₃ on TNBC was investigated from the angle of ferroptosis,providing a new perspective for the treatment of breast cancer.