Copper death inducer independent of copper ion carrier and application of copper death inducer

Abstract

The invention provides a copper death inducer independent of a copper ion carrier and application of the copper death inducer. According to the novel copper death inducer, copper ions do not need to be additionally added in an extracellular environment. Experimental results show that under the coexistence of the ferroptosis inducer and the ferroptosis inhibitor, the treatment of the glutathione inhibitor can induce the cells to generate obvious death. The cell death can be inhibited by a specific inhibitor TTM of copper death, and has typical forms and biochemical characteristics of copper death, namely oligomerization of DLAT protein. Because copper death and copper death drugs have basic and clinical application prospects, the provided new inducer combination will provide a new small molecule induction strategy and research and application directions for copper death related research.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a copper death inducing agent independent of copper ion carriers and application thereof. Background Art

[0002] The study of cell death is a hot topic in the field of biomedicine. According to the classification of cell death by the International Committee on Nomenclature of Cell Death, cell death can be divided into accidental cell death (Accidental Cell Death, ACD) and programmed cell death (Programmed Cell Death, PCD). Accidental cell death is caused by uncontrollable accidental damage, while programmed cell death is defined as a cell death process that is finely regulated by genes, proteins, and metabolites. Programmed cell death has been widely studied for its potential anti-cancer effects and physiological functions during development. At present, according to the different mechanisms of their occurrence and development, researchers have defined a variety of programmed cell death modes, such as apoptosis, necroptosis, pyroptosis, alkaline death, NETosis, Entosis, ferroptosis, copper death, and disulfide death. In March 2022, researchers proposed a new form of cell death and named it Cuproptosis.

[0003] Copper death is a new form of programmed cell death that depends on copper ions. It cannot be suppressed by other known inhibitors of programmed cell death and is also regulated by mitochondrial respiration. Copper ions are essential cofactors for organisms and play an important role in maintaining the functions of certain proteases. Copper ions are usually maintained at low levels in cells. Researchers have found that copper ionophores combined with copper ion overload (i.e., increasing the extracellular copper ion concentration) can significantly cause cytotoxicity and ultimately lead to cellular copper death.

[0004] Copper ion carriers (such as Elesclomol) can transport excess copper ions into cells and release them in the cytoplasm, thereby causing an overload of intracellular copper ions. The overloaded copper ions directly bind to the acylase of the mitochondrial tricarboxylic acid cycle (TCA), promoting the aggregation of acylase (especially dihydrolipoamide S-acetyltransferase, DLAT) and the loss of iron-sulfur cluster protein, thereby inducing protein toxic stress and ultimately causing copper death in cells. Copper ion chelators (Tetrathiomolybdate, TTM) can alleviate the cell-killing effect of elesclomol and copper ion overload. The use of buthionine sulfoximine (BSO) to inhibit the synthesis of glutathione (GSH), a natural chelator of intracellular copper ions, can enhance the cell-killing toxicity of elesclomol and copper ion overload.

[0005] In recent years, copper death has become a research hotspot with increasing attention in the field of cell death because it provides new insights and methods for the regulation of cell death and offers new directions and opportunities for the treatment of a variety of diseases.

[0006] Ferroptosis is a type of programmed cell death named in 2012, characterized by the accumulation of toxic lipid peroxidation and changes in mitochondrial morphology. Unlike apoptosis, ferroptosis does not involve the activation of caspases. Ferroptosis is associated with a variety of diseases, including neurodegenerative diseases, organ and tissue ischemia-reperfusion injury, cancer, and alcoholic liver disease. Ferroptosis is complexly regulated by multiple signaling pathways, and some types of cancer cells show varying degrees of tolerance to ferroptosis. Researchers are actively working to better understand the potential occurrence and inhibition mechanisms of ferroptosis and to develop new therapies for diseases involving this type of cell death.

[0007] At present, the relationship between ferroptosis and copper death is not clear. There are no reports on whether ferroptotic cells can change their cell fate and turn to copper death. As a potential new generation of cancer treatment, ferroptosis is inhibited (including cells insensitive to ferroptosis, such as human breast cancer cells MCF-7). Under what conditions can it turn to copper death? It is also unknown. There may be insensitive cells in any cancer treatment of a compound. The combined use of two different cell programmed death induction methods is believed to provide a better option for cancer treatment. In addition, the current induction method of copper death is only through the use of copper ion carriers (which do not exist under normal physiological conditions) combined with the addition of additional copper ions outside the cell. This induction method of copper death greatly limits the basic and applied research of copper death. A new copper death induction method that does not rely on copper ion carriers and copper ion overload in the extracellular environment will have a significant impact on the basic and applied research of copper death. In addition, the current induction methods of copper death are limited. The development of new agents that induce copper death is of great significance for a deeper understanding of the mechanism of copper death and the design of disease treatment strategies based on copper death. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a copper death inducing agent that does not rely on copper ion carriers.

[0009] Another object of the present invention is to provide the application of the copper death inducing agent that is independent of copper ion carriers.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A copper death inducer that is independent of copper ion carriers, comprising a ferroptosis inducer, a ferroptosis inhibitor, and a glutathione inhibitor.

[0012] A copper death inducer for ferroptosis-insensitive cells, including a glutathione inhibitor and a ferroptosis inducer.

[0013] The ferroptosis inducing agent includes at least one of RSL3, ML210 or CMH (Cumene hydroperoxide).

[0014] The ferroptosis inhibitor includes at least one of Fer-1 (Ferrostatin-1), DFO (Deferoxaminemesylate), NSC668394, and 9-phenanthrol.

[0015] The glutathione inhibitor includes at least one of BSO (buthionine-sulfoximine, an inhibitor of γ-glutamylcysteine ​​synthetase, which can reduce the synthesis of intracellular glutathione) or EIPA (5-(N-Ethyl-N-isopropyl)-Amiloride, a macropinocytosis inhibitor, which can reduce the synthesis of intracellular glutathione).

[0016] The copper death inducer can cause the oligomerization of the copper death key characteristic protein DLAT.

[0017] The copper death is a cell death mode induced by copper ions.

[0018] The copper death can be rescued by TTM, a copper death inhibitor.

[0019] Under ferroptosis-inducing conditions, if ferroptosis-inhibiting conditions and low cellular glutathione content occur simultaneously, the cells will switch from ferroptosis-induced fate to copper death. That is, the combination of ferroptosis and copper death inhibitors can better prevent and control the occurrence of cell death under ferroptosis-inducing conditions.

[0020] The copper death inducing agent independent of copper ion carrier is used in the preparation of tumor treatment drugs.

[0021] The copper death inducer that is independent of copper ion carriers is used in the preparation of therapeutic drugs for ferroptosis-insensitive tumors.

[0022] A tumor treatment drug comprises the above copper death inducer which is independent of copper ion carrier.

[0023] A therapeutic drug for tumors insensitive to ferroptosis, comprising the above-mentioned copper death inducer that is independent of copper ion carriers.

[0024] The tumor includes at least one of fibrosarcoma or breast cancer.

[0025] The tumor insensitive to ferroptosis is a tumor insensitive to ferroptosis inducers.

[0026] Compared with the prior art, the present invention has the following advantages and effects:

[0027] (1) The present invention provides a copper death inducer that is independent of copper ion carriers and its application, the inducer is composed of an ferroptosis inducer, ferroptosis inhibitory conditions (ferroptosis inhibitors, or cells that are insensitive to ferroptosis), and a glutathione inhibitor. The cell death induced by the above inducer is consistent with the morphology of copper death cells induced by classical copper death conditions, can cause oligomerization of the key copper death protein DLAT, and can be rescued by the classical copper death inhibitor TTM.

[0028] (2) The inducer provided by the present invention is not limited to one drug combination, but can be a variety of new copper death inducers produced by arranging and combining different ferroptosis inducers, different ferroptosis inhibition conditions and different glutathione inhibitors.

[0029] (3) Currently, research on copper death is in its infancy and mainly focuses on bioinformatics analysis, with relatively few related experimental findings. The method of inducing copper death is limited to exogenous addition of copper ion carriers and copper ions, which ultimately causes intracellular copper ion overload. At present, there is no drug inducer that is independent of copper ion carriers and directly targets the copper death protein pathway to cause cells to efficiently undergo copper death under normal conditions of extracellular copper ion concentration. Therefore, new copper death induction conditions are urgently needed to be developed. New copper death induction conditions will provide a scientific basis for in-depth research on the detailed mechanism of copper death, as well as new ideas for designing disease treatment strategies based on copper death in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the result of the 24h drug toxicity test of BSO in the HT-1080 cell line.

[0031] Figure 2 This figure shows that cell death caused by a combination of inducers (BSO combined with RSL3 and NSC668394 / 9-phenanthrol) can be rescued by the copper death inhibitor TTM.

[0032] Figure 3 This figure shows that cell death caused by the inducer combination (BSO combined with RSL3 and Fer-1 / DFO) can be rescued by the copper death inhibitor TTM.

[0033] Figure 4 This is a graph showing the cell death rate after adding different programmed cell death inhibitors to the inducer combination (BSO combined with RSL3 and NSC668394 / 9-phenanthrol / Fer-1 / DFO).

[0034] Figure 5 This is the result of oligomerization of copper death marker DLAT protein after HT-1080 cells were treated with inducer combination (BSO combined with RSL3 and NSC668394 / 9-phenanthrol / Fer-1 / DFO) for 8 hours.

[0035] Figure 6 This figure shows that cell death caused by the inducer combination (BSO combined with ML210 and NSC668394 / 9-phenanthrol / Fer-1 / DFO) can be rescued by the copper death inhibitor TTM.

[0036] Figure 7This figure shows that cell death caused by a combination of inducers (BSO combined with CMH and 9-phenanthrol / Fer-1) can be rescued by the copper death inhibitor TTM.

[0037] Figure 8 This figure shows that cell death caused by the inducer combination (EIPA combined with RSL3 and NSC668394 / 9-phenanthrol / Fer-1) can be rescued by the copper death inhibitor TTM.

[0038] Fig. 9 The figure shows the result that cell death caused by the inducer combination (BSO combined with RSL3 or BSO combined with RSL3 and Fer-1) in MCF-7 cells can be rescued by the copper death inhibitor TTM. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0040] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0041] Experimental Materials:

[0042] BSO: purchased from MCE, product number HY-106376A. Dissolved in ultrapure water by ultrasonication for 10 min, 250 mM stock solution, used at a concentration of 250 μM;

[0043] Tetrathiomolybdate (TTM): purchased from McLean, catalog number A828261. Dissolved in DMSO with ultrasound for 10 min, 20 mM stock solution, used at a concentration of 20 μM;

[0044] CuCl2: purchased from McLean, catalog number C804817. Dissolved in DMSO, 5 mM stock solution, used at a concentration of 5 μM;

[0045] Elesclomol: purchased from McLean, catalog number E864529. Dissolved in DMSO, 10 mM stock solution, used at a concentration of 10 μM;

[0046] RSL3: purchased from MCE, catalog number HY-100218A. Dissolved in DMSO, 2 mM stock solution, used at a concentration of 2 μM;

[0047] NSC668394: purchased from MCE, catalog number HY-115492. Dissolved in DMSO, 5 mM stock solution;

[0048] 9-phenanthrol: purchased from MCE, catalog number HY-122697. Dissolved in DMSO, 1 mM stock solution;

[0049] EIPA: purchased from MCE, catalog number HY-101840. Dissolved in DMSO, 50 mM stock solution;

[0050] Fer-1: purchased from MCE, catalog number HY-100579. Dissolved in DMSO, 10 mM stock solution;

[0051] ML210: purchased from Taoshu, catalog number T8375. Dissolved in DMSO, 10 mM stock solution;

[0052] DFO: purchased from MCE, product number HY-B0988. Dissolved in ultrapure water by sonication for 10 min, 100 mM stock solution;

[0053] CMH: purchased from Sigma, catalog number 247502. Dissolved in anhydrous ethanol by sonication for 10 min, 50 mM stock solution;

[0054] Z-VAD-FMK: purchased from GlpBio, catalog number GC12861. Dissolved in DMSO, 20 mM stock solution;

[0055] Necrostatin 2racemate (Nec-1s): purchased from Selleck, catalog number S8641. Dissolved in DMSO, 10 mM stock solution;

[0056] PBS (Phosphate-buffered saline): purchased from Biosharp, catalog number BL302A;

[0057] Propidium iodide stain PI: purchased from Bio-Tech Biotechnology Co., Ltd., catalog number 40755ES64;

[0058] Hoechst 33342 stain: purchased from Yisheng Biotechnology Co., Ltd., catalog number 40732ES03;

[0059] High-efficiency RIPA tissue / cell rapid lysis buffer: purchased from Yazyme Biotechnology Co., Ltd., catalog number PC101;

[0060] BCA protein concentration assay kit: purchased from Thermo Scientific, catalog number 23227;

[0061] BSA: purchased from Yazyme Biotechnology Co., Ltd., catalog number ZJ201;

[0062] DLAT antibody: purchased from Zhengneng Biotechnology, catalog number R27216;

[0063] GAPDH antibody: purchased from Proteintech, catalog number 60004-1-1g;

[0064] Developer: purchased from Bio-Tech Biotechnology Co., Ltd., catalog number P0020;

[0065] HT-1080: Sourced from the cell bank of the Chinese Academy of Sciences Typical Culture Collection Committee, the plates were inoculated one day before the experiment, and the cell density on the day of the experiment was about 50%-60%;

[0066] MCF-7: Source: Senbega Biotech;

[0067] CO2 incubator: ESCO;

[0068] Ordinary low-temperature refrigerator: Haier Company;

[0069] -80℃ ultra-low temperature refrigerator: Thermo Scientific;

[0070] Ordinary optical microscope: Olympus;

[0071] Clean bench: Suzhou Purification Instrument Factory;

[0072] Biological safety cabinet: ESCO;

[0073] Ultrapure water instrument: MILLIPORE;

[0074] CytoFLEX Platform: Beckman Co.

[0075] Cytation 5 Cell Imaging Multi-function Microplate Detection System: Agilent.

[0076] Example 1

[0077] 24h drug toxicity test of BSO in HT-1080 cell line

[0078] (1) One day in advance, the HT-1080 cells in the logarithmic growth phase in the cell bottle were washed three times with pre-cooled PBS. After adding trypsin to digest the cells for 1 minute, 1 ml of DMEM complete medium was added to terminate the digestion, and the cells were blown off with a pipette. 100 μL of cells were added to each well of the 96-well plate, about 5,000 cells. Since the liquid at the edge of the 96-well plate is easy to evaporate, no cells were added around the edge, and 100 μL of PBS buffer solution was added instead. Place the cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall.

[0079] (2) The next morning, the culture plates were removed, the old culture medium was aspirated, and complete culture medium containing final concentrations of 0, 250, 500, 1000, and 2000 μM BSO was added to treat the cells, with three replicate wells set up for each group.

[0080] (3) After adding the drugs, the cells were returned to the cell culture incubator for culture. After 24 hours, propidium iodide (PI) and Hoechst (Hoechst) were added at a final concentration of 1 μg / ml. The cells were stained at 37°C for 15 min in the dark and the cell death rate was detected by Cytation5 imaging.

[0081] The results are as follows Figure 1 As shown, BSO treatment alone did not induce cell death in HT-1080 cells, and even concentrations as high as 2000 μM had no significant toxicity to cells within 24 h.

[0082] Example 2

[0083] Cell death caused by the combination of inducers (BSO combined with RSL3 and NSC668394 / 9-phenanthrol) can be rescued by the copper death inhibitor TTM

[0084] (1) One day in advance, the HT-1080 cells in the logarithmic growth phase in the cell bottle were washed three times with pre-cooled PBS. After adding trypsin to digest the cells for 1 minute, 1 ml of DMEM complete medium was added to terminate the digestion, and the cells were blown off with a pipette. 100 μL of cells were added to each well of the 96-well plate, about 5,000 cells. Since the liquid at the edge of the 96-well plate is easy to evaporate, no cells were added around the edge, and 100 μL of PBS buffer solution was added instead. Place the cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall.

[0085] (2) The next morning, the culture plates were removed. For the group with BSO added, the old culture medium was aspirated and complete culture medium containing a final concentration of 250 μM BSO was added to pretreat the cells for 12 h. Three replicate wells were set up for each group.

[0086] (3) Before adding drugs, prepare fresh culture medium containing drug treatment for each group. Aspirate the old culture medium and add drugs in groups, adding 100 μL to each well. Set up 3 replicate wells for each group:

[0087] a. Blank control group: DMEM complete medium + 3 μL DMSO;

[0088] b. TTM treatment group: DMEM complete medium containing TTM at a final concentration of 20 μM was added;

[0089] c. Copper death positive control group: DMEM complete medium containing elesclomol at a final concentration of 10 μM and CuCl2 at 5 μM was added;

[0090] d.elesclomol+CuCl2+TTM treatment group: DMEM complete medium containing elesclomol at a final concentration of 10 μM, CuCl2 at 5 μM, and TTM at 20 μM was added;

[0091] e. BSO combined with RSL3 and NSC668394 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and NSC668394 at 5 μM was added;

[0092] f. BSO+RSL3+NSC668394+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM, NSC668394 at 5 μM, and TTM at 20 μM was added;

[0093] g. BSO combined with RSL3 and 9-phenanthrol treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, 2 μM RSL3 and 5 μM 9-phenanthrol was added;

[0094] h. BSO+RSL3+9-phenanthrol+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM, 9-phenanthrol at 5 μM, and TTM at 20 μM was added;

[0095] i. The single drug or two-drug treatment groups of the copper-induced death combination described in this case:

[0096] DMEM complete medium containing BSO at a final concentration of 250 μM, DMEM complete medium containing NSC668394 at a final concentration of 5 μM, DMEM complete medium containing 9-phenanthrol at a final concentration of 5 μM, DMEM complete medium containing BSO at a final concentration of 250 μM and NSC668394 at a final concentration of 5 μM, DMEM complete medium containing BSO at a final concentration of 250 μM and 9-phenanthrol at a final concentration of 5 μM, DMEM complete medium containing NSC668394 at a final concentration of 2 μM and DMEM complete medium containing RSL3 with a final concentration of 5 μM, DMEM complete medium containing 9-phenanthrol and 2 μM RSL3 with a final concentration of 5 μM, DMEM complete medium containing BSO with a final concentration of 250 μM and 2 μM RSL3, DMEM complete medium containing BSO with a final concentration of 250 μM, 2 μM RSL3 and 20 μM TTM, DMEM complete medium containing RSL3 with a final concentration of 2 μM, and DMEM complete medium containing RSL3 and 20 μM TTM with a final concentration of 2 μM.

[0097] (4) After adding the drugs, the cells were returned to the cell culture incubator for culture. After 12 h, propidium iodide (PI) and Hoechst (Hoechst) were added at a final concentration of 1 μg / ml. The cells were stained at 37°C for 15 min in the dark and the cell death rate was detected by Cytation5 imaging.

[0098] The results are as follows Figure 2 As shown, the cell death rate of the treatment groups of BSO combined with RSL3 and NSC668394 and BSO combined with RSL3 and 9-phenanthrol reached ~100%, which is consistent with the copper death treatment group induced by classic extracellular copper ion overload (elesclomol+CuCl2), and can be rescued by the copper death inhibitor TTM. Among the single drug or two drug combination treatment groups of the two copper death inducing combinations described in this example, only the BSO plus RSL3 treatment group and the RSL3 treatment group alone induced cell death, but could not be rescued by the copper death inhibitor TTM. It is proved that the treatment group of BSO combined with RSL3 and NSC668394 / 9-phenanthrol can induce cell death and can be rescued by the copper death inhibitor TTM.

[0099] Example 3

[0100] Experiments showing that cell death caused by a combination of inducers (BSO combined with RSL3 and Fer-1 / DFO) can be rescued by the copper death inhibitor TTM

[0101] (1) One day in advance, the HT-1080 cells in the logarithmic growth phase in the cell bottle were washed three times with pre-cooled PBS. After adding trypsin to digest the cells for 1 minute, 1 ml of DMEM complete medium was added to terminate the digestion, and the cells were blown off with a pipette. 100 μL of cells were added to each well of the 96-well plate, about 5,000 cells. Since the liquid at the edge of the 96-well plate is easy to evaporate, no cells were added around the edge, and 100 μL of PBS buffer solution was added instead. Place the cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall.

[0102] (2) The next morning, the culture plates were removed. For the group with BSO added, the old culture medium was removed and complete culture medium containing a final concentration of 250 μM BSO was added to pretreat the cells for 12 h. Three replicate wells were set up for each group.

[0103] (3) Before adding drugs, prepare fresh culture medium containing drug treatment for each group. Aspirate the old culture medium and add drugs in groups, adding 100 μL to each well. Set up 3 replicate wells for each group:

[0104] a. Blank control group: DMEM complete medium + 3 μL DMSO;

[0105] b. BSO combined with RSL3 and Fer-1 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, 2 μM RSL3 and 5 μM Fer-1 was added;

[0106] c. BSO+RSL3+Fer-1+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM, Fer-1 at 5 μM, and TTM at 20 μM was added;

[0107] d. BSO combined with RSL3 and DFO treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and DFO at 100 μM was added;

[0108] e. BSO+RSL3+DFO+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM, DFO at 100 μM, and TTM at 20 μM was added;

[0109] f. A single drug or two drug treatment groups of the copper death inducing combination of the present invention:

[0110] DMEM complete medium containing Fer-1 at a final concentration of 5 μM, DMEM complete medium containing DFO at a final concentration of 100 μM, DMEM complete medium containing BSO at a final concentration of 250 μM and 5 μM Fer-1, DMEM complete medium containing BSO at a final concentration of 250 μM and 100 μM DFO, DMEM complete medium containing Fer-1 at a final concentration of 5 μM and 2 μM RSL3, DMEM complete medium containing DFO at a final concentration of 100 μM and 2 μM RSL3. For other single drug or two drug treatment groups, see Example 2.

[0111] (4) After adding the drugs, the cells were returned to the cell culture incubator for culture. After 12 h, propidium iodide (PI) and Hoechst (Hoechst) were added at a final concentration of 1 μg / ml. The cells were stained at 37°C for 15 min in the dark and the cell death rate was detected by Cytation5 imaging.

[0112] Fer-1 and DFO are two classic and most commonly used inhibitors in the field of ferroptosis. Figure 3 As shown, the cell death rate after 12 h of treatment with BSO combined with RSL3 and Fer-1 and BSO combined with RSL3 and DFO was close to 100%, but it could be rescued by the copper death inhibitor TTM.

[0113] Example 4

[0114] Cell death and viability assays after adding different programmed cell death inhibitors to inducer combinations (BSO combined with RSL3 and NSC668394 / 9-phenanthrol / Fer-1 / DFO)

[0115] (1) One day in advance, the HT-1080 cells in the logarithmic growth phase in the cell bottle were washed three times with pre-cooled PBS. After adding trypsin to digest the cells for 1 minute, 1 ml of DMEM complete medium was added to terminate the digestion, and the cells were blown off with a pipette. 100 μL of cells were added to each well of the 96-well plate, about 5,000 cells. Since the liquid at the edge of the 96-well plate is easy to evaporate, no cells were added around the edge, and 100 μL of PBS buffer solution was added instead. Place the cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall.

[0116] (2) The next morning, the culture plates were removed. For the group with BSO added, the old culture medium was removed and complete culture medium containing a final concentration of 250 μM BSO was added to pretreat the cells for 12 h. Three replicate wells were set up for each group.

[0117] (3) Before adding drugs, prepare fresh culture medium containing drug treatment for each group. Aspirate the old culture medium and add drugs in groups, adding 100 μL to each well. Set up 3 replicate wells for each group:

[0118] a. Copper death positive control group: DMEM complete medium containing elesclomol at a final concentration of 10 μM and CuCl2 at 5 μM was added;

[0119] b. BSO combined with RSL3 and Fer-1 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, 2 μM RSL3 and 5 μM Fer-1 was added;

[0120] c. BSO combined with RSL3 and DFO treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and DFO at 100 μM was added;

[0121] d. BSO combined with RSL3 and NSC668394 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and NSC668394 at 5 μM was added;

[0122] e. BSO combined with RSL3 and 9-phenanthrol treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and 9-phenanthrol at 5 μM was added;

[0123] f. The above five inducers were further added with copper death inhibitor TTM at a final concentration of 20 μM / ferroptosis inhibitor Fer-1 at a final concentration of 5 μM / apoptosis inhibitor Z-VAD-FMK at a final concentration of 40 μM / necroptosis inhibitor Nec-1s at a final concentration of 10 μM.

[0124] (4) After adding the drugs, the cells were returned to the cell culture incubator for culture. After 12 h, propidium iodide (PI) and Hoechst (Hoechst) were added at a final concentration of 1 μg / ml. The cells were stained at 37°C for 15 min in the dark and the cell death rate was detected by Cytation5 imaging.

[0125] The results are as follows Figure 4 As shown, the cell death induced by the above four inducers can be rescued by TTM, just like the copper death treatment group induced by classic extracellular copper ion overload (elesclomol+CuCl2), while other types of inhibitors of programmed cell death cannot. This proves that the cell death induced by the above four new induction combinations is copper death.

[0126] Example 5

[0127] Oligomerization detection experiment of copper death marker DLAT protein after HT-1080 cells were treated with inducer combination (BSO combined with RSL3 and NSC668394 / 9-phenanthrol / Fer-1 / DFO) for 8 hours

[0128] 1. Cell Sample Preparation

[0129] (1) One day in advance, the HT-1080 cells in the logarithmic growth phase in the cell bottle were washed three times with pre-cooled PBS, and the cells were digested with trypsin for 1 minute, and then 1 ml of DMEM complete medium was added to terminate the digestion. The cells were blown off with a pipette, and the cell suspension was transferred to a centrifuge tube. Centrifuged at 300g for 5 minutes, the supernatant was poured out, and the cells were resuspended in DMEM and counted with a cell counter. Planted in a 6-well plate, 2 mL of cell suspension was added to each well of the 6-well plate, and about 200,000 cells were added to each well. Placed in a cell culture incubator at 37°C and 5% CO2 for more than 12 hours to allow the cells to adhere to the wall.

[0130] (2) The next morning, the culture plates were removed. For the group with BSO added, the old culture medium was aspirated and complete culture medium containing a final concentration of 500 μM BSO was added to pretreat the cells for 12 h. Three replicate wells were set up for each group.

[0131] (3) Before adding drugs, prepare fresh culture medium containing drug treatment for each group. Aspirate the old culture medium, add drugs in groups, add 2 mL to each well, and set up 3 replicate wells for each group:

[0132] a. Copper death positive control group: DMEM complete medium containing elesclomol at a final concentration of 1.5 μM and CuCl2 at 1.5 μM was added and treated for 8 h;

[0133] b. BSO combined with RSL3 and NSC668394 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and NSC668394 at 5 μM was added and treated for 8 h;

[0134] c. BSO combined with RSL3 and 9-phenanthrol treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and 9-phenanthrol at 5 μM was added and treated for 8 h;

[0135] d. BSO combined with RSL3 and Fer-1 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and Fer-1 at 5 μM was added and treated for 8 h;

[0136] e. BSO combined with RSL3 and DFO treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and DFO at 100 μM was added and treated for 8 h;

[0137] f. Ferroptosis control group: DMEM complete medium was added with RSL3 at a final concentration of 2 μM and treated for 3 h;

[0138] g. Blank control group: DMEM complete medium + 2 μL DMSO.

[0139] (4) After adding the drug, continue to place the cell incubator for incubation.

[0140] 2. Protein Extraction and Western Blotting

[0141] (1) Discard the culture medium in the 6-well plate and slowly add cold PBS to wash the cells twice.

[0142] (2) RIPA lysis buffer and PMSF were mixed in a ratio of 100:1. 100 μL of the prepared lysis buffer was added to each well of a 6-well plate and lysed on ice for 30 min.

[0143] (3) Transfer the lysed cells to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 10 min at 4°C. Transfer the supernatant to a new centrifuge tube.

[0144] (4) The protein concentration of the prepared protein samples was measured using the BCA method.

[0145] (5) Mix 5x non-reducing protein loading buffer (DLAT protein oligomers are linked by disulfide bonds, so non-reducing protein loading buffer should be used) with the protein sample at a ratio of 1:4 and cook at 95°C for 5 min. Aliquot the prepared samples and store at -80°C.

[0146] (6) Use 4%-8% SDS-PAGE gel electrophoresis, the electrophoresis system is Tris-Gly, the electrophoresis condition is a constant voltage of 120V, and the protein loading amount is 30μg.

[0147] (7) After protein separation, transfer the protein using a 0.45 μm PVDF membrane in an ice bath at 220 mA for 150 min.

[0148] (8) Block with 5% BSA on a shaker at room temperature for 2 h.

[0149] (9) Incubate with DLAT antibody (1:1000 dilution) and internal reference GAPDH antibody (1:5000 dilution) at 4°C overnight.

[0150] (10) After the antibody was recovered, the membrane was washed three times with 1xTBST for 10 min each time. The corresponding secondary antibody was added and incubated at 4°C for 3 h, then washed three times with 1xTBST for 10 min each time before development.

[0151] The results are as follows Figure 5 As shown, BSO combined with RSL3 and NSC668394 / 9-phenanthrol / Fer-1 / DFO treatment, consistent with the classic copper death induced by extracellular copper ion overload (elesclomol+CuCl2), can lead to oligomerization of DLAT protein. The DLAT protein in the blank control group and the iron death inducer treatment group did not oligomerize. DLAT protein oligomerization is believed to cause cytotoxic stress and is a key factor in copper death.

[0152] Example 6

[0153] Experiments showing that cell death caused by a combination of inducers (BSO combined with ML210 and NSC668394 / 9-phenanthrol) can be rescued by the copper death inhibitor TTM

[0154] (1) One day in advance, the HT-1080 cells in the logarithmic growth phase in the cell bottle were washed three times with pre-cooled PBS. After adding trypsin to digest the cells for 1 minute, 1 ml of DMEM complete medium was added to terminate the digestion, and the cells were blown off with a pipette. 100 μL of cells were added to each well of the 96-well plate, about 5,000 cells. Since the liquid at the edge of the 96-well plate is easy to evaporate, no cells were added around the edge, and 100 μL of PBS buffer solution was added instead. Place the cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall.

[0155] (2) The next morning, the culture plates were removed. For the group with BSO added, the old culture medium was removed and complete culture medium containing a final concentration of 250 μM BSO was added to pretreat the cells for 12 h. Three replicate wells were set up for each group.

[0156] (3) Before adding drugs, prepare fresh culture medium containing drug treatment for each group. Aspirate the old culture medium and add drugs in groups, adding 100 μL to each well. Set up 3 replicate wells for each group:

[0157] a. Blank control group: DMEM complete medium + 3 μL DMSO;

[0158] b. TTM treatment group: DMEM complete medium containing TTM at a final concentration of 20 μM was added;

[0159] c. BSO combined with ML210 and NSC668394 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, ML210 at 10 μM and NSC668394 at 5 μM was added;

[0160] d. BSO+ML210+NSC668394+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, ML210 at 10 μM, NSC668394 at 5 μM, and TTM at 20 μM was added;

[0161] e. BSO combined with ML210 and 9-phenanthrol treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, 10 μM ML210 and 10 μM 9-phenanthrol was added;

[0162] f. BSO+ML210+9-phenanthrol+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, ML210 at 10 μM, 9-phenanthrol at 10 μM, and TTM at 20 μM was added;

[0163] k. Single drug or two drug treatment groups in the copper death induction combination described in this case:

[0164] DMEM complete medium containing 9-phenanthrol at a final concentration of 10 μM, DMEM complete medium containing BSO at a final concentration of 250 μM and 10 μM 9-phenanthrol, DMEM complete medium containing NSC668394 at a final concentration of 5 μM and 10 μM ML210, DMEM complete medium containing 9-phenanthrol at a final concentration of 10 μM and 10 μM ML210, DMEM complete medium containing BSO at a final concentration of 250 μM and 10 μM ML210, DMEM complete medium containing BSO at a final concentration of 250 μM and 10 μM ML210 and 20 μM TTM, DMEM complete medium containing ML210 at a final concentration of 10 μM, DMEM complete medium containing ML210 at a final concentration of 10 μM and 20 μM TTM.

[0165] (4) After adding the drugs, the cells were returned to the cell culture incubator for culture. After 24 hours, propidium iodide (PI) and Hoechst (Hoechst) were added at a final concentration of 1 μg / ml. The cells were stained at 37°C for 15 min in the dark and the cell death rate was detected by Cytation5 imaging.

[0166] The results are as follows Figure 6As shown, the cell death rate of the BSO combined with ML210 and NSC668394 / 9-phenanthrol treatment group reached 100%, and could be rescued by the copper death inhibitor TTM. Among the single drug or two-drug combination treatment groups of the copper death inducing combination described in this example, only the BSO plus ML210 treatment group and the ML210 treatment group alone induced cell death, but could not be rescued by the copper death inhibitor TTM.

[0167] Example 7

[0168] Experiments showing that cell death caused by a combination of inducers (BSO combined with CMH and 9-phenanthrol / Fer-1) can be rescued by the copper death inhibitor TTM

[0169] (1) One day in advance, the HT-1080 cells in the logarithmic growth phase in the cell bottle were washed three times with pre-cooled PBS. After adding trypsin to digest the cells for 1 minute, 1 ml of DMEM complete medium was added to terminate the digestion, and the cells were blown off with a pipette. 100 μL of cells were added to each well of the 96-well plate, about 5,000 cells. Since the liquid at the edge of the 96-well plate is easy to evaporate, no cells were added around the edge, and 100 μL of PBS buffer solution was added instead. Place the cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall.

[0170] (2) The next morning, the cells were removed. For the group with BSO added, the old culture medium was aspirated and complete culture medium containing a final concentration of 250 μM BSO was added to pretreat the cells for 12 h. Three replicate wells were set up for each group.

[0171] (3) Before adding drugs, prepare fresh culture medium containing drug treatment for each group. Aspirate the old culture medium and add drugs in groups, adding 100 μL to each well. Set up 3 replicate wells for each group:

[0172] a. BSO combined with CMH and 9-phenanthrol treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, CMH at 50 μM and 9-phenanthrol at 2 μM was added;

[0173] b. BSO+CMH+9-phenanthrol+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, CMH at 50 μM, 9-phenanthrol at 2 μM, and TTM at 20 μM was added;

[0174] c. BSO combined with CMH and Fer-1 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, CMH at 100 μM and Fer-1 at 10 μM was added;

[0175] d. BSO+CMH+Fer-1+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, CMH at 100 μM, Fer-1 at 10 μM, and TTM at 20 μM was added;

[0176] e. Single drug or two drug treatment groups in the copper death induction combination described in this case:

[0177] DMEM complete medium containing 9-phenanthrol at a final concentration of 2 μM, DMEM complete medium containing Fer-1 at a final concentration of 10 μM, DMEM complete medium containing BSO at a final concentration of 250 μM and 2 μM 9-phenanthrol, DMEM complete medium containing BSO at a final concentration of 250 μM and 10 μM Fer-1, DMEM complete medium containing 9-phenanthrol at a final concentration of 2 μM and 50 μM CMH, DMEM complete medium containing Fer-1 at a final concentration of 10 μM and 100 μM CMH, DMEM complete medium containing CMH at a final concentration of 50 μM, DMEM complete medium containing CMH at a final concentration of 50 μM and 20 μM TTM, DMEM complete medium containing CMH at a final concentration of 100 μM, DMEM complete medium containing CMH at a final concentration of 100 μM and 20 μM TTM.

[0178] (4) After adding the drugs, the cells were returned to the cell culture incubator for culture. After 13 h, propidium iodide (PI) and Hoechst (1 μg / ml) were added. The cells were stained at 37°C for 15 min in the dark. Cytation5 imaging was used to detect cell death rate.

[0179] The results are as follows Figure 7 As shown, the cell death rate in the BSO combined with CMH and 9-phenanthrol / Fer-1 treatment groups reached 100%, which could also be partially rescued by the copper death inhibitor TTM. Among the single drug or two-drug combination treatment groups of the copper death inducing combination described in this example, only the BSO plus CMH treatment group and the CMH treatment group alone induced cell death, but could not be rescued by the copper death inhibitor TTM.

[0180] Example 8

[0181] Cell death caused by the combination of inducers (EIPA combined with RSL3 and NSC668394 / 9-phenanthrol / Fer-1) can be rescued by the copper death inhibitor TTM

[0182] (1) One day in advance, the HT-1080 cells in the logarithmic growth phase in the cell bottle were washed three times with pre-cooled PBS. After adding trypsin to digest the cells for 1 minute, 1 ml of DMEM complete medium was added to terminate the digestion, and the cells were blown off with a pipette. 100 μL of cells were added to each well of the 96-well plate, about 5,000 cells. Since the liquid at the edge of the 96-well plate is easy to evaporate, no cells were added around the edge, and 100 μL of PBS buffer solution was added instead. Place the cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall.

[0183] (2) The next morning, the culture plates were removed. For the group with BSO added, the old culture medium was aspirated and complete culture medium containing a final concentration of 250 μM BSO was added to pretreat the cells for 12 h. Three replicate wells were set up for each group.

[0184] (3) Before adding drugs, prepare fresh culture medium containing drug treatment for each group. Aspirate the old culture medium and add drugs in groups, adding 100 μL to each well. Set up 3 replicate wells for each group:

[0185] a. Blank control group: DMEM complete medium + 3 μL DMSO;

[0186] b. Ferroptosis positive control group: DMEM complete medium containing RSL-3 at a final concentration of 2 μM was added;

[0187] c. Copper death positive control group: DMEM complete medium containing elesclomol at a final concentration of 1.5 μM and CuCl2 at 1.5 μM was added;

[0188] d.elesclomol+CuCl2+TTM treatment group: DMEM complete medium containing elesclomol at a final concentration of 1.5 μM, CuCl2 at 1.5 μM, and TTM at 20 μM was added;

[0189] e. BSO combined with RSL3 and Fer-1 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and Fer-1 at 5 μM was added;

[0190] f. EIPA combined with RSL3 and Fer-1 treatment group: DMEM complete medium containing EIPA at a final concentration of 35 μM, RSL3 at 2 μM and Fer-1 at 5 μM was added;

[0191] g. EIPA+RSL3+Fer-1+TTM treatment group: DMEM complete medium containing 35 μM EIPA, 2 μM RSL3, 5 μM Fer-1 and 20 μM TTM was added;

[0192] h. BSO combined with RSL3 and NSC668394 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and NSC668394 at 5 μM was added;

[0193] i. EIPA combined with RSL3 and NSC668394 treatment group: DMEM complete medium containing EIPA at a final concentration of 35 μM, RSL3 at 2 μM and NSC668394 at 5 μM was added;

[0194] j. EIPA+RSL3+NSC668394+TTM treatment group: DMEM complete medium containing 35 μM EIPA, 2 μM RSL3, 5 μM NSC668394 and 20 μM TTM was added;

[0195] k. BSO combined with RSL3 and 9-phenanthrol treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM and 9-phenanthrol at 5 μM was added;

[0196] 1. EIPA combined with RSL3 and 9-phenanthrol treatment group: DMEM complete medium containing EIPA at a final concentration of 35 μM, RSL3 at 2 μM and 9-phenanthrol at 5 μM was added;

[0197] m. EIPA+RSL3+9-phenanthrol+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 2 μM, 9-phenanthrol at 5 μM, and TTM at 20 μM was added;

[0198] (4) After adding the drugs, the cells were returned to the cell culture incubator for culture. After 12 h, propidium iodide (PI) and Hoechst (Hoechst) were added at a final concentration of 1 μg / ml. The cells were stained at 37°C for 15 min in the dark and the cell death rate was detected by Cytation5 imaging.

[0199] The results are as follows Figure 8As shown, the cell death rate of the treatment groups of EIPA combined with RSL3 and Fer-1, and EIPA combined with RSL3 and 9-phenanthrol reached more than 80%, and the cell death rate of the treatment group of EIPA combined with RSL3 and NSC668394 reached more than 50%, which is consistent with the copper death treatment group induced by classic extracellular copper ion overload (elesclomol+CuCl2), and can be rescued by the copper death inhibitor TTM. It proves that the treatment group of EIPA combined with RSL3 and Fer-1 / NSC668394 / 9-phenanthrol can induce cell death, and can be rescued by the copper death inhibitor TTM.

[0200] Example 9

[0201] Experiments in which cell death caused by the inducer combination (BSO combined with RSL3 or BSO combined with RSL3 and Fer-1) in MCF-7 cells could be rescued by the copper death inhibitor TTM.

[0202] (1) One day in advance, the MCF-7 cells in the logarithmic growth phase in the cell bottle were washed three times with pre-cooled PBS. After adding trypsin to digest the cells, 1 ml of DMEM complete medium was added to terminate the digestion, and the cells were blown off with a pipette. 100 μL of cells were added to each well of the 96-well plate, about 5,000 cells. Since the liquid at the edge of the 96-well plate is easy to evaporate, no cells were added around the edge, and 100 μL of PBS buffer solution was added instead. Place the cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall.

[0203] (2) The next morning, the culture plates were removed. For the group with BSO added, the old culture medium was removed and complete culture medium containing a final concentration of 250 μM BSO was added to pretreat the cells for 12 h. Three replicate wells were set up for each group.

[0204] (3) Before adding drugs, prepare fresh culture medium containing drug treatment for each group. Aspirate the old culture medium and add drugs in groups, adding 100 μL to each well. Set up 3 replicate wells for each group:

[0205] a. Blank control group: DMEM complete medium + 2 μL DMSO;

[0206] b. Copper death positive control group: DMEM complete medium containing elesclomol at a final concentration of 10 μM and CuCl2 at 5 μM was added;

[0207] c.elesclomol+CuCl2+TTM treatment group: DMEM complete medium containing elesclomol at a final concentration of 10 μM, CuCl2 at 5 μM, and TTM at 20 μM was added

[0208] d. BSO treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM was added;

[0209] e. RSL3 treatment group: DMEM complete medium containing RSL3 at a final concentration of 10 μM was added;

[0210] f. BSO combined with RSL3 group: DMEM complete medium containing BSO at a final concentration of 250 μM and RSL3 at 10 μM was added;

[0211] g. BSO+RSL3+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 10 μM, and TTM at 20 μM was added;

[0212] h. BSO+RSL3+Fer-1 treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 10 μM, and Fer-1 at 5 μM was added;

[0213] i. BSO+RSL3+Fer-1+TTM treatment group: DMEM complete medium containing BSO at a final concentration of 250 μM, RSL3 at 10 μM, Fer-1 at 5 μM, and TTM at 20 μM was added;

[0214] (4) After adding the drugs, the cells were returned to the cell culture incubator for culture. After 24 hours, propidium iodide (PI) and Hoechst (Hoechst) were added at a final concentration of 1 μg / ml. The cells were stained at 37°C for 15 min in the dark and the cell death rate was detected by Cytation5 imaging.

[0215] MCF-7 cells are a type of cancer cell that is relatively insensitive to ferroptosis. Fig. 9 As shown in the figure, MCF-7 cells are insensitive to ferroptosis. The mortality rate after 24h treatment with the ferroptosis inducer RSL3 is only 30%-40%, while the mortality rate after BSO combined with RSL3 treatment can reach 80%-90%, and can be rescued by the copper death inhibitor TTM, but not by the ferroptosis inhibitor Fer-1. Cell death induced by BSO combined with RSL3 and Fer-1 can also be rescued by the copper death inhibitor TTM. This shows that BSO combined with RSL3 treatment can cause copper death in cells resistant to ferroptosis, and there is no need to add ferroptosis inhibitors.

[0216] In summary, different ferroptosis inducers (RSL3 / ML210 / CMH), different ferroptosis inhibitors (Fer-1 / DFO / NSC668394 / 9-phenanthrol) and different glutathione inhibitors (BSO / EIPA) can all induce significant copper death in cells that is independent of copper ion carriers. That is, the combined treatment of these drugs can be used as a new type of copper death inducer. And in cell lines that are insensitive to ferroptosis (ferroptosis inducers can only cause partial cell death), only the combination of ferroptosis inducer RSL3 and glutathione inhibitor BSO can cause significant copper death in cells, proving that this combination has therapeutic potential for tumors that resist ferroptosis.

[0217] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A copper death inducing agent that does not rely on copper ion carriers, characterized in that: Including ferroptosis inducers, ferroptosis inhibitors and glutathione inhibitors.

2. A copper death inducer for cells insensitive to ferroptosis, comprising a ferroptosis inducer and a glutathione inhibitor.

3. The copper death inducing agent according to claim 1 or 2, characterized in that: The ferroptosis inducing agent includes at least one of RSL3, ML210 or CMH.

4. The copper death inducing agent according to claim 1, characterized in that: The ferroptosis inhibitor includes at least one of Fer-1, DFO, NSC668394, and 9-phenanthrol.

5. The copper death inducing agent according to claim 1 or 2, characterized in that: The glutathione inhibitor includes at least one of BSO and EIPA.

6. The copper death inducing agent according to claim 1 or 2, characterized in that: The copper death inducer can cause the oligomerization of the copper death key characteristic protein DLAT.

7. The copper death inducing agent according to claim 1 or 2, characterized in that: The copper death inducer does not require the addition of additional copper ions in the extracellular environment.

8. Use of the copper death inducer independent of copper ion carriers as claimed in claim 1 in the preparation of tumor therapeutic drugs.

9. Use of the copper death inducer independent of copper ion carriers as claimed in claim 2 in the preparation of tumor therapeutic drugs that are insensitive to ferroptosis.

10. The use according to claim 8 or 9, characterized in that: The tumor includes at least one of fibrosarcoma or breast cancer.

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