Cryptotanshinone Suppresses Triple Negative Breast Cancer Via Ferroptosis

Breast cancer is the cancer with the highest morbidity and mortality among women in the world.Triple-negative breast cancer（TNBC）is the most malignant subtype of breast cancer.Due to its high heterogeneity and lack of specific biomarkers,chemotherapy is still the main treatment option,which leads to poor efficacy,side effects,drug resistance and other problems.In recent years,immune checkpoint inhibitors have achieved encouraging therapeutic effects,but only for a few PD-L1 positive patients.Traditional Chinese medicine Salvia miltiorrhiza Bunge has been used for thousands of years in the treatment of"breast carbuncle".Its fat-soluble diterpene quinone compound cryptotanshinone has been widely proved to have significant anti-tumor activity,but the mechanism is unclear and not deeply studied.Therefore,the purpose of this study is to systematically evaluate the efficacy of cryptotanshinone in TNBC treatment,and to explore its possible anti-tumor immunomodulatory mechanism,so as to provide important preliminary data for systematically explaining the multi-level and multi-target anti-tumor mechanism of cryptotanshinone.Firstly,the anti-tumor activity of cryptotanshinone in vitro was investigated by CCK-8assay,founding that cryptotanshinone could effectively inhibit triple negative breast cancer cell lines MDA-MB-231 and 4T1-Luc.The mechanism of cryptotanshinone on 4T1-Luc cells was explored by proteomic technology,founding that cryptotanshinone could regulate ferroptosis.Phenotypic detection showed that cryptotanshinone could significantly increase the contents of total iron and ROS in cells,and the corresponding morphological characteristics of ferroptosis were also observed by transmission electron microscope.It was also found that the promoting effect of cryptotanshinone on ferroptosis could be significantly neutralized by ferroptosis inhibitor Fer-1,indicating that cryptotanshinone could directly promote ferroptosis of tumor cells in vitro.The syngeneic triple negative breast cancer model was established by in situ inoculation of 4T1-Luc cells in female BALB/c mice.The tumor volume and body weight of tumor-bearing mice were dynamically detected within 22 days after intraperitoneal injection of cryptotanshinone.The main organs and in situ tumor tissues were stripped,photographed and pathologically examined after treatment.It was found that cryptotanshinone effectively inhibited tumor growth without causing obvious side effects.Immunohistochemical results of tumor tissues showed that in vivo administration of cryptotanshinone could increase the lipid peroxidation products of tumor tissue in tumor-bearing mice,suggesting the clue related to ferroptosis in vivo.It is worth noting that,cryptotanshinone could significantly increase the proportion of lymphocytes and decrease the proportion of neutrophils in the peripheral blood of tumor-bearing mice by blood routine analysis,suggesting that the anti-tumor mechanism of cryptotanshinone is also related to immune cells.In order to further investigate the anti-tumor activity of cryptotanshinone,the survival time of BALB/c and NOD/SCID tumor-bearing mice was investigated.It was found that cryptotanshinone could significantly prolong the survival time of immune tumor-bearing mice,but decreased the curative effect in immunodeficient NOD/SCID mice.It is suggested that in addition to directly inducing ferroptosis in TNBC cells,the anti-tumor effect of cryptotanshinone is also closely related to immune system.In order to explore the immune cells regulated by cryptotanshinone,the tumor immune microenvironment map of syngeneic orthotopic triple negative breast cancer model mice was drawn by single cell transcriptome sequencing and bioinformatics analysis for the first time,and 7 cell clusters with the largest number were identified.It was found that cluster 1～4 were neutrophils,cluster 5 was macrophages,cluster 6 was monocytes and cluster 7 was T cells.The proportion of all kinds of immune cells changed in varying degrees after administration of cryptotanshinone in vivo,and the immunomodulatory effect of cryptotanshinone was explained from the transcriptome level of single cell for the first time.By consulting the literature and mining of single cell sequencing data,it is preliminarily determined that the four clusters of neutrophils are tumor-promoting N2 neutrophils,anti-tumor N1 neutrophils,granulocyte-like myelogenic suppressor cells and immature neutrophils.Based on the higher abundance of single-cell transcriptome sequencing data,the differential expression genes were sequenced,and the heat maps of the top 200 genes were drawn.The characteristic gene expression map of neutrophils in situ tumor of syngeneic orthotopic triple negative breast cancer model mice was obtained.Pseudotime analysis of neutrophils showed that because cryptotanshinone inhibited immunosuppressive neutrophils,neutrophils in the administration group were mainly in the relatively early stage with anti-tumor function compared with the model group.The most famous and the highest proportion of immune cells were further studied,and discovered the target cells directly affected by cryptotanshinone,granulocyte-like myeloid derived suppressor cells.Through proteomic study and enrichment analysis,it was found that cryptotanshinone regulated ferroptosis.Through enrichment analysis of the sequencing data of immunosuppressive neutrophil to investigate the changes in transcription level,it was also found that cryptotanshinone can induce ferroptosis.Phenotypic detection showed that cryptotanshinone significantly increased intracellular total iron,Fe²⁺,ROS,lipid peroxidation and its product 4-HNE.The ultrastructural changes observed by transmission electron microscope also explained the induced effect of cryptotanshinone on granulocyte-like myeloid derived suppressor cell ferroptosis from the morphological point of view.The mechanism of cryptotanshinone was also verified by ferroptosis inhibitor Fer-1,iron chelator DFO,and apoptosis inhibitor Z-VAD.It was found that DFO could most significantly neutralize the ferroptosis promoting effect of cryptotanshinone,followed by Fer-1 and Z-VAD,which were significantly different from those of cryptotanshinone alone,indicating that cryptotanshinone mainly promoted primary granulocyte-like myeloid derived suppressor cell death through ferroptosis,especially iron metabolism.Finally,based on the fact that cryptotanshinone has a stronger promoting effect on ferroptosis to granulocyte-like myeloid derived suppressor cell than triple negative breast cancer cell line 4T1-Luc,the targets of cryptotanshinone on granulocyte-like myeloid derived suppressor cell were screened by unlabeled DARTS（Drug affinity responsive target stability）/proteomic technology.Enrichment analysis of different proteins showed that cryptotanshinone could affect mitochondria,plasma membrane,antioxidant activity and peroxidase,which were closely related to ferroptosis.Furthermore,conducted literature research on candidate proteins,and HMGB2（High mobility group protein B2）and FTH（Ferritin heavy chain）were selected as potential targets for follow-up study.The analysis of the interaction between target protein and cryptotanshinone by molecular docking and microscale thermophoresis assay showed that the binding effect of cryptotanshinone to FTH was better than HMGB2,and it was a classic protein involved in ferroptosis.Therefore,only the FTH was verified by Westen Blot.The results showed that the direct binding of cryptotanshinone to target protein FTH made it easier to be hydrolyzed by pronase,and decreased its stability.In conclusion,this research systematically explored the mechanism of cryptotanshinone inhibiting tumor growth by promoting ferroptosis of granulocyte-like myeloid derived suppressor cells and triple-negative breast cancer cells by proteomics,single cell transcriptome sequencing,ferroptosis phenotype detection,DARTS and molecular docking,providing data support for the development of cryptotanshinone as a new drug against triple negative breast cancer,and was of great significance to prove the"multi-level,multi-target"pharmacological effects of natural products.