Copper death inducer independent of copper ion carrier and application thereof

Abstract

The application provides a copper death inducer independent of copper ion carriers and application thereof. The novel copper death inducer does not need to additionally add copper ions in an extracellular environment. Experimental results show that, under the simultaneous presence of ferroptosis inducers and ferroptosis inhibitors, combined with glutathione inhibitors, cells can be induced to significantly die. The above cell death can be inhibited by a specific inhibitor TTM of copper death, and has typical morphology and biochemical characteristics of copper death, i.e. oligomerization of DLAT protein. Since copper death and copper death drugs have basic and clinical application prospects, the new inducer combination provided by the application will provide a new small molecule induction strategy for copper death related research and application direction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a copper death inducer independent of copper ionophore and application thereof. BACKGROUND

[0002] The research on cell death is a hotspot in the field of biological medicine. According to the classification of cell death by the International Cell Death Nomenclature Committee, cell death can be divided into accidental cell death (ACD) and programmed cell death (PCD). Accidental cell death is caused by uncontrolled accidental injury, while programmed cell death is defined as a cell death process finely regulated by genes, proteins and metabolites. Programmed cell death is widely studied for its potential role in cancer resistance and physiological function in development. At present, according to the different mechanisms of occurrence and development, researchers have defined a variety of programmed cell death modes, such as apoptosis, necroptosis, pyroptosis, alkali death, NETosis, entosis, ferroptosis, cuproptosis and bisulfide death. In March 2022, researchers proposed a new form of cell death and named it cuproptosis.

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

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

[0005] In recent years, copper-induced cell death has become a research hotspot in the field of cell death, as it provides new insights and ways to regulate cell death and new directions and opportunities for the treatment of various diseases.

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

[0007] The relationship between ferroptosis and copper death is not clear at present. There is no report that ferroptosis cells can change cell fate and turn to copper death. As a potential new generation of cancer treatment, it is unknown whether ferroptosis can turn to copper death under the condition of being inhibited (including cells insensitive to ferroptosis, such as human breast cancer cells MCF-7). Any cancer treatment of a compound may exist insensitive cells, and the combination of two different ways of inducing cell programmed death is believed to provide a better choice 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 extra copper ions outside the cells, which greatly limits the basic and applied research of copper death. A new copper death induction method independent of copper ion carriers and extracellular copper ion overload will have a significant impact on the basic and applied research of copper death. In addition, the current induction method of copper death is limited, and the development of new copper death induction agents is of great significance to further understand the mechanism of copper death and design disease treatment strategies based on copper death. SUMMARY

[0008] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a copper death inducer independent of copper ion carriers.

[0009] Another purpose of the present application is to provide the use of the above-mentioned copper death inducer independent of copper ion carriers.

[0010] The purpose of the present application is achieved by the following technical solutions:

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

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

[0013] The ferroptosis inducer comprises at least one of RSL3, ML210 or CMH (Cumene hydroperoxide).

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

[0015] The glutathione inhibitor includes at least one of BSO (buthionine-sulfoximine, an inhibitor of gamma-glutamyl cysteine 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 DLAT, a key protein of copper death.

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

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

[0019] Under the iron death induction condition, if the iron death inhibition condition and the low content of cell glutathione occur at the same time, the cell will change from the iron death induction fate to the copper death, that is, the combination of the iron death and the copper death inhibitor can better prevent and treat the occurrence of cell death under the iron death induction condition.

[0020] The application of the copper death inducer not dependent on copper ion carriers in the preparation of a tumor treatment drug.

[0021] The application of the copper death inducer not dependent on copper ion carriers in the preparation of a treatment drug for iron death-insensitive tumors.

[0022] A tumor treatment drug includes the copper death inducer not dependent on copper ion carriers.

[0023] A treatment drug for iron death-insensitive tumors includes the copper death inducer not dependent on copper ion carriers.

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

[0025] The iron death-insensitive tumor is a tumor insensitive to an iron death inducer.

[0026] The present application has the following advantages and effects relative to the prior art:

[0027] (1) The present application provides a copper death inducer not dependent on copper ion carriers and its application, which is composed of an iron death inducer, an iron death inhibition condition (an iron death inhibitor, which can also be a cell insensitive to iron death), and a glutathione inhibitor. The cell death induced by the inducer is consistent with the cell morphology of copper death induced by the classic copper death condition, can cause the oligomerization of DLAT, a key protein of copper death, and can be rescued by the classic inhibitor TTM of copper death.

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

[0029] (3) At present, the research on copper death is in its infancy, and mainly focuses on bioinformatics analysis, and there are few related experimental findings. The method of inducing copper death is limited to the overloading of copper ions in cells caused by the exogenous addition of copper ion carriers and copper ions. At present, there is no drug inducer that does not depend on copper ion carriers and directly targets the copper death protein pathway to make cells efficiently undergo copper death under the condition that the copper ion concentration outside the cells is normal. Therefore, new copper death induction conditions need to be developed, and new copper death induction conditions will provide a scientific basis for in-depth study of the detailed mechanism of copper death, and provide new ideas for designing disease treatment strategies based on copper death in the future. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a 24h drug toxicity test result graph of BSO in HT-1080 cell lines.

[0031] Figure 2 is a result graph that cell death caused by the inducer combination (BSO combined with RSL3 and NSC668394 / 9-phenanthrol) can be rescued by the copper death inhibitor TTM.

[0032] Figure 3 is a result graph 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 is a result graph of 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 is a result graph of the oligomerization of the copper death marker DLAT protein after the inducer combination (BSO combined with RSL3 and NSC668394 / 9-phenanthrol / Fer-1 / DFO) is treated HT-1080 cells for 8h.

[0035] Figure 6 is a result graph 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 7Figure is the result graph that cell death caused by the combination of inducers (BSO combined with CMH and 9-phenanthrol / Fer-1) can be rescued by the copper death inhibitor TTM.

[0037] Figure 8 Figure is the result graph that 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.

[0038] Figure 9 Figure is the result graph that cell death caused by the combination of inducers (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 application will be further described with reference to the following examples and drawings, but the embodiments of the present application are not limited thereto.

[0040] Unless otherwise specified, the following examples were carried out under conventional experimental conditions or under experimental conditions recommended by the reagent companies. The materials, reagents, etc. used were commercially available reagents and materials unless otherwise specified.

[0041] Experimental materials:

[0042] BSO: purchased from MCE, item number HY-106376A. Dissolved with ultrapure water for 10 min, 250 mM stock solution, the use concentration is 250 μM;

[0043] Tetrathiomolybdate (TTM): purchased from Macklin, item number A828261. Dissolved with DMSO for 10 min, 20 mM stock solution, the use concentration is 20 μM;

[0044] CuCl2: purchased from Macklin, item number C804817. Dissolved with DMSO, 5 mM stock solution, the use concentration is 5 μM;

[0045] Elesclomol: purchased from Macklin, item number E864529. Dissolved with DMSO, 10 mM stock solution, the use concentration is 10 μM;

[0046] RSL3: purchased from MCE, item number HY-100218A. Dissolved with DMSO, 2 mM stock solution, the use concentration is 2 μM;

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

[0048] 9-phenanthrol: purchased from MCE, Cat# HY-122697. DMSO dissolution, 1 mM stock solution;

[0049] EIPA: purchased from MCE, Cat# HY-101840. DMSO dissolution, 50 mM stock solution;

[0050] Fer-1: purchased from MCE, Cat# HY-100579. DMSO dissolution, 10 mM stock solution;

[0051] ML210: purchased from Tocris, Cat# T8375. DMSO dissolution, 10 mM stock solution;

[0052] DFO: purchased from MCE, Cat# HY-B0988. Ultrasonic dissolution in ultrapure water for 10 min, 100 mM stock solution;

[0053] CMH: purchased from Sigma, Cat# 247502. Ultrasonic dissolution in anhydrous ethanol for 10 min, 50 mM stock solution;

[0054] Z-VAD-FMK: purchased from GlpBio, Cat# GC12861. DMSO dissolution, 20 mM stock solution;

[0055] Necrostatin 2 racemate (Nec-1s): purchased from Selleck, Cat# S8641. DMSO dissolution, 10 mM stock solution;

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

[0057] Iodinated propidium dye PI (Propidium Iodide): purchased from Biyun Tian Biological Company, Cat# 40755ES64;

[0058] Hoechst 33342 dye: purchased from Yixing Biological Technology Company, Cat# 40732ES03;

[0059] High-efficiency RIPA tissue / cell rapid lysis solution: purchased from Yaenzyme Biological Company, Cat# PC101;

[0060] BCA protein concentration determination kit: purchased from Thermo Scientific, Cat# 23227;

[0061] BSA: purchased from Yaenzyme Biological Company, Cat# ZJ201;

[0062] DLAT antibody: purchased from Zhengneng Biological Company, Cat# R27216;

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

[0064] Developer: purchased from Biyun Tian Biological Company, item number P0020;

[0065] HT-1080: from the China Academy of Sciences Culture Collection Cell Library, plated one day before the experiment, and the cell density was about 50%-60% on the day of the experiment;

[0066] MCF-7: from Sunbeige Biological Company;

[0067] CO2 incubator: ESCO Company;

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

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

[0070] General optical microscope: Olympus Company;

[0071] Super-clean workbench: Suzhou Purification Instrument Factory;

[0072] Biological safety cabinet: ESCO Company;

[0073] Ultrapure water instrument: MILLIPORE Company;

[0074] CytoFLEX Platform: Beckman Company;

[0075] Cytation 5 cell imaging multifunctional microplate detection system: Agilent Company.

[0076] Example 1

[0077] 24h drug toxicity test experiment 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 3 times with pre-cooled PBS, and after 1 min of trypsin digestion, 1 ml of DMEM complete culture medium was added to terminate the digestion, and the cells were blown down with a pipette. 100 μL of cells, about 5000, were added to each well of a 96-well plate. Since the liquid at the edge of the 96-well plate is easy to evaporate, a circle around it does not add cells, but adds 100 μL of PBS buffer solution. Place the cells in the cell culture incubator at 37℃, 5% CO2 incubation to make the cells adhere.

[0079] (2) The next morning, remove the old culture solution from the culture plate, and add complete culture medium containing 0, 250, 500, 1000, 2000 μΜ BSO to the cells, respectively, and set 3 replicate wells for each group.

[0080] (3) After adding the drug, return the cells to the cell culture incubator for culture, and after 24 h, add 1 μg / ml of iodinated propylidium dye PI (Propidium Iodide) and 1 μg / ml of Hoechst dye to the cells, and stain for 15 min in the dark at 37°C, and then detect the cell death rate by Cytation5 imaging.

[0081] The results are shown in Table 1. Figure 1 As shown in Table 1, BSO alone does not induce HT-1080 cell death, and even at a concentration as high as 2000 μΜ, it has no significant toxicity to the cells within 24 h.

[0082] Example 2

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

[0084] (1) The day before, wash the HT-1080 cells in the cell bottle in logarithmic growth phase with pre-cooled PBS for 3 times, add trypsin to digest the cells for 1 min, then add 1 ml of DMEM complete culture medium to terminate the digestion, and blow the cells down with a pipette. Add 100 μL of cells to each well of the 96-well plate, about 5000 cells. Since the liquid at the edge of the 96-well plate is prone to evaporation, a circle of PBS buffer solution is added instead of cells around the edge. Place the cells in the cell culture incubator at 37°C, 5% CO2 for incubation to allow the cells to adhere.

[0085] (2) The next morning, remove the old culture solution from the culture plate, and add complete culture medium containing 0, 250, 500, 1000, 2000 μΜ BSO to the cells, respectively, and set 3 replicate wells for each group.

[0086] (3) Before adding the drug, prepare fresh culture medium containing each group of drug treatment. Remove the old culture solution, and add the drug to each group, 100 μL per well, and set 3 replicate wells for each group:

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

[0088] b. TTM treatment group: add DMEM complete culture medium containing 20 μΜ TTM;

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

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

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

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

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

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

[0095] i. Single drug or 2-drug treatment group of the copper death-inducing combination described in this case:

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

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

[0098] The results, as shown in Figure 2 Table 1, showed that the cell death rate of the BSO combined with RSL3 and NSC668394 treatment group and the BSO combined with RSL3 and 9-phenanthrol treatment group reached ~ 100%, which was consistent with the copper death induced by the classic extracellular copper ion overload (elesclomol + CuCl2) treatment group and could be rescued by the copper death inhibitor TTM. Among the two copper death-inducing combinations described in this example, only the BSO combined with RSL3 treatment group and the RSL3 alone treatment group induced cell death, but could not be rescued by the copper death inhibitor TTM. It was proved that the BSO combined with RSL3 and NSC668394 / 9-phenanthrol treatment group could induce cell death and could be rescued by the copper death inhibitor TTM.

[0099] Example 3

[0100] Experiment on cell death induced by the combination of inducers (BSO combined with RSL3 and Fer-1 / DFO) which can be rescued by the copper death inhibitor TTM

[0101] (1) One day in advance, the HT-1080 cells in logarithmic growth phase in the cell bottle were washed with pre-cooled PBS for 3 times, trypsin was added to digest the cells for 1 min, then 1 ml of DMEM complete culture medium was added to terminate the digestion, and the cells were blown down with a pipette. 100 μL of cells, about 5000, were added to each well of the 96-well plate. Since the liquid at the edge of the 96-well plate is easy to evaporate, a circle of PBS buffer solution was added instead of cells. The cells were incubated in a cell culture incubator at 37°C, 5% CO2 to adhere to the wall.

[0102] (2) The next morning, the culture plate was taken out, and the BSO group was added. The old culture solution was removed, and the complete culture medium containing a final concentration of 250 μΜ BSO was added to pretreat the cells for 12 h, and 3 replicate wells were set for each group.

[0103] (3) Before adding drugs, fresh culture medium containing various drug treatments was prepared. The old culture solution was removed, and the drugs were added to each group, 100 μL per well, and 3 replicate wells were set for each group:

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

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

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

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

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

[0109] f. The single drug or 2 drug treatment group of the copper death inducing combination according to the present application:

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

[0111] (4) After adding drugs, return to the cell culture box for culture, add propidium iodide (PI) staining solution at a final concentration of 1 μg / ml and Hoechst staining solution at a final concentration of 1 μg / ml after 12 h, stain in the dark at 37°C for 15 min, and detect cell mortality by Cytation5 imaging.

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

[0113] Example 4

[0114] Cell death and survival detection experiment after adding different cell programmed death inhibitors to the inducer combination (BSO combined with RSL3 and NSC668394 / 9-phenanthrol / Fer-1 / DFO)

[0115] (1) Plate in advance, wash the HT-1080 cells in the logarithmic growth phase in the cell bottle with pre-cooled PBS for 3 times, add trypsin to digest the cells for 1 min, then add 1 ml of DMEM complete medium to terminate the digestion, and blow the cells down with a pipette. Add 100 μL of cells to each well of the 96-well plate, about 5000. Since the liquid at the edge of the 96-well plate is easy to evaporate, a circle around it is not added with cells, but 100 μL of PBS buffer solution is added instead. Place the cells in the cell culture box at 37°C, 5% CO2 incubation to make the cells adhere.

[0116] (2) The next morning, take out the culture plate, for the groups with BSO, aspirate the old culture medium, and add complete medium containing BSO at a final concentration of 250 μΜ to pretreat the cells for 12 h, with 3 replicate wells for each group.

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

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

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

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

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

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

[0123] f. The above 5 inducers are respectively added with copper death inhibitor TTM at a final concentration of 20 μΜ, iron death inhibitor Fer-1 at a final concentration of 5 μΜ, apoptosis inhibitor Z-VAD-FMK at a final concentration of 40 μΜ, and necroptosis inhibitor Nec-1s at a final concentration of 10 μΜ.

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

[0125] The results are shown in Table 1. Figure 4 As shown in Table 1, the cell death induced by the above 4 inducers can be rescued by TTM, while other types of cell programmed death inhibitors cannot, proving that the cell death induced by the above 4 new inducer combinations is copper death.

[0126] Example 5

[0127] Detection of oligomerization of DLAT protein, a marker of copper death, after treatment of HT-1080 cells with the combination of inducers (BSO in combination with RSL3 and NSC668394 / 9-phenanthrol / Fer-1 / DFO) for 8h

[0128] 1. Cell sample preparation

[0129] (1) The day before, the HT-1080 cells in logarithmic growth phase in cell bottles were washed 3 times with pre-cooled PBS, trypsin was added to digest the cells for 1 min, 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 300 g for 5 min, the supernatant was poured off, and DMEM was used for resuspension, and the cells were counted with a cell counter. Seed in a 6-well plate, add 2 mL of cell suspension to each well of the 6-well plate, about 200,000 cells per well. Place the cells in an incubator at 37°C, 5% CO2 for 12 h or more to allow the cells to adhere.

[0130] (2) The next morning, the culture plates were removed, and for the groups with BSO, the old culture medium was aspirated, and complete medium containing a final concentration of 500 μΜ BSO was added to pretreat the cells for 12 h, and 3 replicate wells were set up for each group.

[0131] (3) Before adding the drugs, fresh culture medium containing the drug treatment of each group was prepared. The old culture medium was aspirated, and the drugs were added in groups, 2 mL per well, and 3 replicate wells were set up for each group:

[0132] a. Copper death positive control group: add DMEM complete medium containing a final concentration of 1.5 μΜ elesclomol and 1.5 μΜ CuCl2, and treat with drugs for 8 h;

[0133] b. BSO in combination with RSL3 and NSC668394 treatment group: add DMEM complete medium containing a final concentration of 250 μΜ BSO, 2 μΜ RSL3 and 5 μΜ NSC668394, and treat with drugs for 8 h;

[0134] c. BSO in combination with RSL3 and 9-phenanthrol treatment group: add DMEM complete medium containing a final concentration of 250 μΜ BSO, 2 μΜ RSL3 and 5 μΜ 9-phenanthrol, and treat with drugs for 8 h;

[0135] d. BSO in combination with RSL3 and Fer-1 treatment group: add DMEM complete medium containing a final concentration of 250 μΜ BSO, 2 μΜ RSL3 and 5 μΜ Fer-1, and treat with drugs for 8 h;

[0136] e. BSO combined with RSL3 and DFO treatment group: add DMEM complete medium containing final concentration of 250 μΜ BSO, 2 μΜ RSL3 and 100 μΜ DFO, and drug treatment for 8 h;

[0137] f. Ferroptosis control group: add DMEM complete medium containing final concentration of 2 μΜ RSL3, and drug treatment for 3 h;

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

[0139] (4) Continue to place the cells in the incubator after drug addition.

[0140] 2. Protein extraction and Western blotting experiment

[0141] (1) Discard the culture medium in the 6-well plate, and gently wash the cells with cold PBS for 2 times.

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

[0143] (3) Transfer the lysed cells to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 10 min at 4°C, and take the supernatant to a new centrifuge tube.

[0144] (4) The prepared protein sample is measured for protein concentration by BCA method.

[0145] (5) Mix 5x non-reducing protein loading buffer (DLAT protein oligomers are connected by disulfide bonds, so non-reducing protein loading buffer is used) with protein sample at a ratio of 1:4, and boil at 95°C for 5 min. The prepared sample is aliquoted and stored at -80°C.

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

[0147] (7) After protein separation, use 0.45 μm PVDF membrane for 220 mA ice bath wet transfer for 150 min.

[0148] (8) Block with 5% BSA on a shaking bed 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, 1xTBST was used to wash 3 times, 10 min each time. After the corresponding secondary antibody was added and incubated at 4°C for 3h, 1xTBST was used to wash 3 times, 10 min each time, and then developed.

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

[0152] Example 6

[0153] Experiment of cell death induced by inducer combination (BSO combined with ML210 and NSC668394 / 9-phenanthrol) can be rescued by 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 3 times with pre-cooled PBS, and then trypsin was added to digest the cells for 1 min, and 1 ml of DMEM complete medium was added to terminate the digestion. The cells were blown off with a pipette. 100 μL of cells, about 5000, were added to each well of a 96-well plate. Since the liquid at the edge of the 96-well plate is easy to evaporate, a circle around it does not add cells, but adds 100 μL of PBS buffer solution. Place the cells in the cell culture incubator at 37°C, 5% CO2, and incubate to allow the cells to adhere.

[0155] (2) The next morning, the culture plate was taken out, and the BSO group was removed from the old culture medium, and the complete culture medium containing a final concentration of 250 μΜ BSO was added to pretreat the cells for 12h, and 3 replicate wells were set for each group.

[0156] (3) Before adding drugs, prepare fresh culture medium containing various drug treatments. Remove the old culture medium and add drugs in groups, 100 μL per well, 3 replicate wells per group:

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

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

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

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

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

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

[0163] k. The single drug or 2-drug treatment group of the copper death-inducing combination described in the present case:

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

[0165] (4) After adding the drug, return to the cell culture box for culture, 24 h later, add iodine propyl dye PI (Propidium Iodide) with a final concentration of 1 μg / ml and Hoechst dye with a final concentration of 1 μg / ml, avoid light, 37°C staining for 15 min, and Cytation5 imaging detects cell death rate.

[0166] The results are as follows Figure 6As shown, cell death rate of BSO combined with ML210 and NSC668394 / 9-phenanthrol treatment groups reached ~100%, and could be rescued by copper death inhibitor TTM. Among the individual drug or 2-drug combination treatment groups of the induced copper death combination described in this example, only the BSO plus ML210 treatment group and the ML210 alone treatment group induced cell death, but could not be rescued by copper death inhibitor TTM.

[0167] Example 7

[0168] Experiment of cell death induced by the combination of inducers (BSO combined with CMH and 9-phenanthrol / Fer-1) which can be rescued by 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 3 times with pre-cooled PBS, and after trypsin digestion of the cells for 1 min, 1 ml of DMEM complete medium was added to terminate the digestion, and the cells were blown down with a pipette. 100 μL of cells, about 5000, were added to each well of a 96-well plate. Since the liquid at the edge of the 96-well plate is easy to evaporate, a circle around it is not added with cells, but 100 μL of PBS buffer solution is added instead. Place the cells in the incubator at 37°C, 5% CO2, and incubate to allow the cells to adhere.

[0170] (2) The next morning, the cells were taken out, and for the groups with BSO, the old culture medium was removed and the complete culture medium containing a final concentration of 250 μΜ BSO was added to pretreat the cells for 12 h, and 3 replicate wells were set for each group.

[0171] (3) Before adding drugs, fresh culture medium containing drug treatment of each group was prepared. The old culture medium was removed, and the drugs were added in groups, 100 μL per well, and 3 replicate wells were set for each group:

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

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

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

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

[0176] e. Single drug or 2-drug combination treatment group of the copper death-inducing combination described in this case:

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

[0178] (4) After adding the drug, return the cells to the cell culture incubator for culture, and after 13 h, add propidium iodide (PI) with a final concentration of 1 μg / ml and Hoechst dye with a final concentration of 1 μg / ml, stain in the dark at 37 °C for 15 min, and detect the cell death rate by Cytation5 imaging.

[0179] The results are shown in Figure 7 BSO combined with CMH and 9-phenanthrol / Fer-1 treatment groups reached a cell death rate of ~ 100%, and were also partially rescued by the copper death inhibitor TTM. Among the single drug or 2-drug combination treatment groups of the copper death-inducing combination described in this embodiment, only the BSO + CMH treatment group and the CMH alone treatment group induced cell death, but could not be rescued by the copper death inhibitor TTM.

[0180] Example 8

[0181] Experiment on rescue of cell death caused by the inducer combination (EIPA combined with RSL3 and NSC668394 / 9-phenanthrol / Fer-1) by the copper death inhibitor TTM

[0182] (1) One day in advance, the HT-1080 cells in logarithmic growth phase in the cell bottle were washed with pre-cooled PBS for 3 times, and then the cells were digested with trypsin for 1 min, and 1 ml of DMEM complete medium was added to terminate the digestion, and the cells were blown down with a pipette. 100 μL of cells, about 5000, were added to each well of the 96-well plate. Since the liquid at the edge of the 96-well plate is easy to evaporate, a circle of PBS buffer solution was added instead of cells. The cells were incubated in a cell culture incubator at 37°C, 5% CO2 to adhere to the wall.

[0183] (2) The next morning, the culture plate was taken out, and for the BSO group, the old culture solution was removed, and the complete medium containing a final concentration of 250 μΜ BSO was added to pretreat the cells for 12 h, and 3 replicate wells were set for each group.

[0184] (3) Before adding drugs, fresh culture medium containing various drug treatments was prepared. The old culture solution was removed, and the drugs were added to each group, 100 μL per well, and 3 replicate wells were set for each group:

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

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

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

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

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

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

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

[0192] h. BSO combined with RSL3 and NSC668394 treatment group: add DMEM complete medium containing final concentration of 250 μΜ BSO, 2 μΜ RSL3 and 5 μΜ NSC668394;

[0193] i. EIPA combined with RSL3 and NSC668394 treatment group: add DMEM complete medium containing final concentration of 35 μΜ EIPA, 2 μΜ RSL3 and 5 μΜ NSC668394;

[0194] j. EIPA+RSL3+NSC668394+TTM treatment group: add DMEM complete medium containing final concentration of 35 μΜ EIPA, 2 μΜ RSL3, 5 μΜ NSC668394 and 20 μΜ TTM;

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

[0196] l. EIPA combined with RSL3 and 9-phenanthrol treatment group: add DMEM complete medium containing final concentration of 35 μΜ EIPA, 2 μΜ RSL3 and 5 μΜ 9-phenanthrol;

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

[0198] (4) After adding drugs, return to the cell culture box for culture, add propidium iodide (PI) dye with final concentration of 1 μg / ml and Hoechst dye with final concentration of 1 μg / ml after 12 h, stain in dark at 37℃ for 15 min, and detect cell death rate 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%, the cell death rate of the treatment groups of EIPA combined with RSL3 and NSC668394 reached more than 50%, and was consistent with the copper death treatment group induced by classic extracellular copper overload (elesclomol + CuCl2), which could be rescued by copper death inhibitor TTM. It was proved that the treatment groups of EIPA combined with RSL3 and Fer-1 / NSC668394 / 9-phenanthrol could induce cell death, and could be rescued by copper death inhibitor TTM.

[0200] Example 9

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

[0202] (1) One day in advance, the logarithmic growth phase MCF-7 cells in the cell bottle were washed 3 times with pre-cooled PBS, and after trypsin digestion, 1 ml of DMEM complete medium was added to terminate digestion, and the cells were blown down with a pipette. 100 μL of cells, about 5000, were added to each well of a 96-well plate. Since the liquid at the edge of the 96-well plate is easy to evaporate, a circle around it is not added with cells, but 100 μL of PBS buffer solution is added. Place the cells in the incubator at 37°C, 5% CO2, and incubate to allow the cells to adhere.

[0203] (2) The next morning, take out the culture plate, and for the group with BSO, remove the old culture medium and add complete culture medium containing a final concentration of 250 μΜ BSO to pretreat the cells for 12 h, with 3 replicate wells for each group.

[0204] (3) Before adding drugs, prepare fresh culture medium containing various drug treatments. Remove the old culture medium and add drugs in groups, 100 μL per well, with 3 replicate wells for each group:

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

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

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

[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 250 μM BSO and 10 μM RSL3 was added.

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

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

[0213] i. BSO+RSL3+Fer-1+TTM treatment group: Add DMEM complete medium containing 250 μM BSO, 10 μM RSL3, 5 μM Fer-1 and 20 μM TTM.

[0214] (4) After adding the drug, the cells were placed back into the cell culture incubator. After 24 hours, Propidium Iodide (PI) staining solution and Hoechst staining solution with a final concentration of 1 μg / ml were added. The cells were stained at 37°C for 15 minutes in the dark. Cytation 5 imaging was used to detect cell death rate.

[0215] MCF-7 cells are a type of cancer cell that is relatively insensitive to ferroptosis. The results are as follows... Figure 9 As shown, MCF-7 cells are insensitive to ferroptosis; the cell death rate after 24 hours of treatment with the ferroptosis inducer RSL3 is only 30%-40%, while the cell death rate after treatment with BSO combined with RSL3 reaches 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 indicates that BSO combined with RSL3 treatment can induce copper death in ferroptosis-resistant cells, eliminating the need for additional 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) combined, can all induce significant copper death of cells independent of copper ion carriers. That is, the combination of these drugs can be used as a new copper death inducer. And in the cell lines that are not sensitive 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 of cells, which proves that this combination has therapeutic potential for tumors resistant to ferroptosis.

[0217] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be within the scope of protection of the present application.

Claims

1.A tumor therapeutic drug, characterized in that: comprising an iron death inducer, an iron death inhibitor and a glutathione inhibitor; the iron death inducer comprises at least one of RSL3, ML210 or CMH; the iron death inhibitor comprises at least one of Fer-1, DFO, NSC668394 or 9-phenanthrol; the glutathione inhibitor comprises at least one of BSO or EIPA; the tumor comprises at least one of fibrosarcoma or breast cancer; and the tumor is a tumor resistant to the iron death inducer. ​ ​ ​ ​ ​ ​

Images