Use of (+)-strebloside for preparing an iron death inducer for non-hodgkin lymphoma cells

Abstract

The application discloses application of (+) -Strebloside in preparation of a non-Hodgkin lymphoma ferroptosis inducer. Researches of the application show that (+) -Strebloside can induce non-Hodgkin lymphoma to occur ferroptosis through STEAP3, and experiments in vitro and in vivo prove that (+) -Strebloside not only has a good antitumor effect on non-Hodgkin lymphoma cells, but also shows high tolerance and low systemic toxicity in a nude mouse in vivo, so that it is indicated that the ferroptosis pathway is one of effective antitumor pathways of (+) -Strebloside, and (+) -Strebloside has the potential as a ferroptosis inducer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to application of a cardiac glycoside compound (+)-Strebloside in preparation of a medicine for treating non-Hodgkin lymphoma, in particular to application of (+)-Strebloside in preparation of a ferroptosis inducer for non-Hodgkin lymphoma cells. BACKGROUND

[0002] Non-Hodgkin lymphoma (NHL) is the largest sub-branch of lymphoma, accounting for about 90% of all lymphomas, and ranks sixth in the incidence of malignant tumors, which poses a significant burden to human health.

[0003] The incidence of non-Hodgkin lymphoma is related to immune deficiency, chronic antigen stimulation, genetics and other factors, and the treatment level is relatively complex.

[0004] The R-CHOP-based chemotherapy regimen is currently the most effective method for treating NHL. However, high-dose chemotherapy can cause bone marrow suppression and even life-threatening complications. Tumor invasion and chemotherapy can also increase the risk of blood clotting, so patients prone to thrombosis have limited opportunities for NHL chemotherapy. In addition, other clinical treatments for non-Hodgkin lymphoma include CAR-T-based cell therapy, bispecific antibody immunotherapy and lenalidomide, and BTK and PI3K inhibitor targeted therapy. The above treatment regimens greatly improve the survival rate and cure rate of NHL patients, but high prices and serious side effects severely limit their clinical use. Therefore, finding new interventions or further exploring anti-tumor mechanisms, discovering new drugs and new targets, can provide more possibilities for NHL treatment.

[0005] Natural products are an important source of anti-tumor active molecules, and can provide skeletal diversity for the discovery of new therapeutic entities for NHL. At the same time, exploring potential natural products as a starting point for developing or optimizing new anti-tumor drugs may provide additional treatment options for NHL.

[0006] Inhibition of cell cycle and apoptosis is a key method of many preclinical and clinical cancer treatment drugs. However, in clinical practice, we can easily find that many tumor cells will produce chemotherapeutic resistance and apoptosis induction defects. Therefore, new drugs that can induce non-apoptotic cell death are of great significance. Ferroptosis is a regulated non-apoptotic form of cell death, which is characterized by cell morphology, ferroptosis can cause mitochondrial to be small, membrane density to be high, mitochondrial cristae to disappear or decrease, and cell components are mainly manifested as iron ion and reactive oxygen species accumulation, mitochondrial membrane potential reduction and reduction of the core enzyme GPX4 of the antioxidant system (glutathione system). Because cancer cells need higher levels of iron and lipid metabolism to grow than normal cells, which theoretically makes them more susceptible to ferroptosis. Therefore, targeting ferroptosis has been identified as a promising approach to developing cancer therapies. Recent studies have shown that artemether can synergistically promote solafenib-induced apoptosis and ferroptosis in non-Hodgkin's lymphoma by inhibiting the STAT3 pathway. Therefore, it is of great significance to further screen drugs and explore the specific mechanism of drugs in non-Hodgkin's lymphoma ferroptosis, and it is also helpful to provide more precise and timely treatment for non-Hodgkin's lymphoma patients. SUMMARY

[0007] (+)-Strebloside (Formula I) is a strong cardiac glycoside compound isolated from the roots of Streblus asper Lour of Moraceae.

[0008]

[0009] The inventors found that (+)-Strebloside has significant growth inhibition activity on non-Hodgkin's lymphoma cells in in vivo and in vitro experiments, and animal acute toxicity tests show that (+)-Strebloside exhibits systemic low toxicity in mice. In addition, (+)-Strebloside can induce the up-regulation of sub-iron ion concentration, ROS, lipid peroxide and cytochrome C in NHL cells through STEAP3, while GPX4, ATP and mitochondrial membrane potential are reduced in a concentration-dependent manner, suggesting that (+)-Strebloside can induce NHL ferroptosis through STEAP3, and the ferroptosis pathway is one of the effective pathways for its anti-tumor effect. Therefore, (+)-Strebloside can be used as a ferroptosis inducer to inhibit the proliferation of non-Hodgkin's lymphoma cells and play an anti-tumor role. The present application provides a certain theory for the research and development of new drugs for NHL, and also provides a certain idea and theoretical basis for subsequent clinical combination drug and adjuvant chemotherapy drug.

[0010] The purpose of the present application is to provide the use of (+)-Strebloside in the preparation of a drug for treating non-Hodgkin's lymphoma.

[0011] Preferably, the use is the use of (+)-Strebloside in the preparation of a medicament for treating non-Hodgkin lymphoma by at least one of the following actions:

[0012] inhibiting non-Hodgkin lymphoma cell proliferation;

[0013] inhibiting non-Hodgkin lymphoma cell metastasis;

[0014] inducing non-Hodgkin lymphoma cell ferroptosis.

[0015] Another object of the present application is to provide the use of (+)-Strebloside in the preparation of a non-Hodgkin lymphoma cell ferroptosis inducer.

[0016] Preferably, the use is the use of (+)-Strebloside in the preparation of a medicament for inducing non-Hodgkin lymphoma cell ferroptosis by STEAP3.

[0017] The non-Hodgkin lymphoma cells include diffuse large B-cell lymphoma (OCI-LY3), Burkitts lymphoma cells (Raji), and human T lymphocyte leukemia cells (Jurkat). BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Results of in vitro experiments of (+)-Strebloside inhibiting NHL cell proliferation; wherein A is the IC 50 of (+)-Strebloside on Raji cells, B is the IC 50 of (+)-Strebloside on Jurkat and OCI-LY3 cells, respectively.

[0019] Figure 2 Effects of (+)-Strebloside on in vitro NHL cell colony formation; wherein A is a soft agar plate colony experiment to evaluate the proliferation inhibition ability of (+)-Strebloside in NHL cell lines, and B is a quantitative statistical diagram of soft agar plate colony.

[0020] Figure 3 Pictures of tumor tissues in nude mice.

[0021] Figure 4Figure 6 is the results of in vivo inhibition of NHL cell growth and metastasis by (+)-Strebloside; wherein A is the average weight change of the tumors in the control group, the positive group, the high-dose treatment group of (+)-Strebloside, and the low-dose treatment group of (+)-Strebloside; B is the difference in body weight of the nude mice before and after administration in each group; C is the average volume change of the tumors in the control group, the positive group, the high-dose treatment group of (+)-Strebloside, and the low-dose treatment group of (+)-Strebloside; and D is the tumor inhibition rate between the positive group, the high-dose treatment group of (+)-Strebloside, and the low-dose treatment group of (+)-Strebloside.

[0022] Figure 5 Figure 7 is the results of revealing the oxidative stress and ion homeostasis disorder mediated by (+)-Strebloside through proteomics; wherein A is 52 up-regulated proteins and 10 down-regulated proteins; B is the oxidative stress-related up-regulated proteins; C is the mitochondria-related enriched proteins; and D is the oxidative stress-related down-regulated ions.

[0023] Figure 6 Figure 8 is the preliminary toxicity evaluation results of (+)-Strebloside; wherein A is the body weight change of the mice; and B is the H&E staining results of the heart, liver, spleen, lung, kidney, and lymphoid tissue of the mice in the (+)-Strebloside administration group.

[0024] Figure 7 Figure 9 is the results of up-regulating the expression level of STEAP3 protein by (+)-Strebloside in vitro; wherein A is the expression level of STEAP3 protein in the blank group and the Raji cell administration group; and B is the expression level of STEAP3 protein in the blank group and the Jurkat and OCI-LY3 cell administration groups.

[0025] Figure 8 Figure 10 is the expression level of STEAP3 protein in the subcutaneous transplanted tumor tissue of the nude mice after treatment with (+)-Strebloside (4 mg / kg).

[0026] Figure 9 Figure 11 is the results of up-regulating the expression level of STEAP3 gene by (+)-Strebloside in vitro.

[0027] Figure 10 Figure 12 is the results of the Fe 2+The enrichment experiment investigation results; among them, A is the blank control group and the expression level of ferrous ion concentration in Raji cells and Jurkat cells under different concentrations of (+)-Strebloside treatment, B is the quantitative analysis diagram of ferrous ion concentration in Raji cells and Jurkat cells under blank control and (+)-Strebloside (0.5 μM) treatment.

[0028] Figure 11 The results of the experiment of relieving (+)-Strebloside-mediated NHL proliferation inhibition as an iron chelator; among them, A is the change of ferrous ion concentration in Raji cells under the action of (+)-Strebloside and iron ion chelator Deferasirox; B is the quantitative diagram of ferrous ion in Raji cells under the action of (+)-Strebloside and iron ion chelator Deferasirox; C is the survival rate of Raji cells under the action of (+)-Strebloside and iron ion chelator; D is the survival rate of Jurkat cells under the action of (+)-Strebloside and iron ion chelator.

[0029] Figure 12 The results of the experiment of relieving (+)-Strebloside-mediated NHL proliferation inhibition as an iron chelator; among them, A is the change of ferrous ion concentration in Raji cells under the action of (+)-Strebloside and iron ion chelator Deferasirox; B is the quantitative diagram of ferrous ion in Raji cells under the action of (+)-Strebloside and iron ion chelator Deferasirox; C is the survival rate of Raji cells under the action of (+)-Strebloside and iron ion chelator; D is the survival rate of Jurkat cells under the action of (+)-Strebloside and iron ion chelator.

[0030] Figure 13 The results of the experiment of detecting iron death indicators ROS, lipid peroxide and GPX4; among them, A is the ROS level in Raji cells under the action of blank group and different concentrations of (+)-Strebloside, B is the lipid peroxide level in Raji cells under the action of blank group and different concentrations of (+)-Strebloside, C is the expression level of GPX4 in Raji cells under the action of different concentrations of (+)-Strebloside.

[0031] Figure 14The experimental results for the detection of mitochondrial membrane potential (ΔΨm), ATP and cytochrome C are as follows: A is the level of mitochondrial membrane potential in Raji cells treated with different concentrations of (+)-Strebloside, B is the level of ATP in Raji cells treated with different concentrations of (+)-Strebloside, and C is the expression level of cytochrome C protein in Raji cells treated with different concentrations of (+)-Strebloside.

[0032] Figure 15 The experimental results of Steap3-siRNA transfection efficiency screening are as follows.

[0033] Figure 16 The experimental results of Raji cell transfection are as follows: A is the level of GPX4 after Raji cell transfection, B is the quantitative analysis chart of GPX4 after Raji cell transfection, and C is the cell viability after Raji cell transfection.

[0034] Figure 17 The experimental results of Jurkat cell transfection are as follows: A is the level of GPX4 after Jurkat cell transfection, B is the quantitative analysis chart of GPX4 after Jurkat cell transfection, and C is the cell viability after Jurkat cell transfection. DETAILED DESCRIPTION

[0035] The technical solutions of the present application are further described below in conjunction with examples, but the present application is not limited by the examples. In the following examples, the experimental methods are conventional methods unless otherwise specified.

[0036] Statistical analysis was completed by Excel and GraphPad Prism 9.3.0. Unless otherwise specified, all values are shown as the mean ± standard deviation (SD) of three experiments performed in parallel. Multiple comparisons between means were performed using multivariate analysis of variance, and p values < 0.05 were considered statistically significant.

[0037] (+)-Strebloside (provided by the College of Chinese Medicine, China Pharmaceutical University) was dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich) to obtain a (+)-Strebloside stock solution (concentration of 1019.5 μM), which was stored at -20°C and diluted with culture medium, and freshly prepared before each use.

[0038] Cell lines: Human Burkitt's lymphoma cell line (Raji) was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and certified by STR analysis. Human T lymphocyte cell line (Jurkat) and diffuse large B-cell lymphoma cell line (OCI-LY3) were both from Zhejiang University (Zhejiang, China).

[0039] Cell culture: Raji, Jurkat and OCI-LY3 cells were seeded in RPMI-1640 complete medium containing 10% fetal bovine serum, and incubated at 37°C in a 5% CO2 incubator, and passaged every 1-2 days.

[0040] Mice: Female BALB / c nude mice were purchased from Shanghai National Laboratory Animal Center (Shanghai, China) and maintained in a pathogen-free environment, provided with sufficient food and water, and controlled at a relative humidity of 50-60% and a temperature of 22-26°C with a 12-hour light / dark cycle. Mice were euthanized using cervical dislocation. All animal experiments were approved by the Pharmaceutical Animal Experimental Center of China Pharmaceutical University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

[0041] Drugs and reagents: 10% fetal bovine serum (FBS; Gibco, USA); RPMI-1640 high glucose medium (KGM31800S) was purchased from Jiangsu Keygen Biotech Co., Ltd.; sterile PBS (KGB5001) was purchased from Jiangsu Keygen Biotech Co., Ltd.; DMSO (30072418) was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; CCK-8 (C0039) was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.; Matrigel (356235) was purchased from Corning Biological; Doxorubicin (R034347) was purchased from Ruon Biological; physiological saline (ST341) and goat serum blocking solution (C0265) were purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.; 4% paraformaldehyde (G1101) was purchased from Wuhan Sevier Biological Technology Co., Ltd.; Anti-STEAP3 antibody (ab151566) was purchased from Abcom; protease inhibitor (SCP0110) and phosphatase inhibitor (P2850) were purchased from Merck Millipore; Coomassie brilliant blue staining solution (P0017), RIPA lysis solution (P0013B), PMSF (ST505), BCA protein concentration determination kit (P0010), SDS-PACE protein loading buffer (P0015), and skimmed milk powder (P0216) were purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.; high-sensitivity ECL chemiluminescence solution (180-501) was purchased from Tanon; Anti-Cytochrome C antibody (11940) was purchased from Cellsignaling Technology; Anti-GAPDH-antibody (AP0063), Goat anti-Mouse IgG-HRP (BS12478), and Goat anti-Rabbit IgG-HRP (bs13278) were purchased from Nanjing Boster Biological Technology Co., Ltd.; RNA extraction kit (YFXM0001) and SYBY Green PCR kit (YFXM0001) were purchased from Nanjing Wingsnow Biological Technology Co., Ltd.; Deferasirox (HY-17359), Ferrostatin-1 (HY-100579), U0126-EtOH (HY-12031), and SP600125 (HY-12041) were purchased from MCE; FerrofraRed fluorescent probe (MX4558) was purchased from MaokangBiotech; reactive oxygen species detection kit (S0033S) was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.; BODIPY 581 / 591 C11 probe (D3861) was purchased from Thermo Fisher; mitochondrial membrane potential detection kit (C2006) and ATP detection kit (S0026) were purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.

[0042] Instruments: CO2 incubator (briefly incubator), biosafety cabinet (ESCO), optical inverted microscope (Olympus China Co., Ltd.), panoramic scanner (Jinan Danjil Electronics Co., Ltd.), vacuum centrifugal concentrator (USA Thermo Fisher Corporation), ultra-high performance liquid, high-resolution mass spectrometer (Bruker Beijing Technology Co., Ltd.), full-wavelength multifunctional enzyme label instrument (Thermo), oscillation incubator (Shanghai Minquan Instrument Co., Ltd.), inverted biological microscope (Nexcope), fluorescence microscope (Olympus), section scanner (Hamamatsu), flow cytometer (USA BD Medical Instrument Co., Ltd.), PCR amplifier, real-time fluorescent quantitative PCR instrument (Bole Life Medicine Product Shanghai Co., Ltd.).

[0043] Example 1

[0044] In vitro inhibition of NHL cell proliferation experiment

[0045] Various NHL cell lines (including Raji, Jurkat and OCI-LY3 cells) were characterized for (+)-Strebloside-mediated cell proliferation inhibition activity using CCK-8 assay.

[0046] Raji, Jurkat and OCI-LY3 cells were transferred from a large dish to an EP tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cell mass was collected, resuspended with complete culture medium, counted under a microscope with a hemocytometer, transferred and inoculated in a 96-well plate, 200 μL / well, 5000 cells per well. Shake the Chinese character "zizhi" to ensure uniform distribution of cells, place in the incubator for 24 h, set up blank, control and drug groups, replace the drug-containing medium in the drug group ((+)-Strebloside is diluted with serum-free medium, set 8 gradient concentrations, 100 μM, 50 μM, 25 μM, 5 μM, 0.5 μM, 0.1 μM, 0.01 μM, 0.001 μM, the blank group is the same volume of serum-free medium, and the control group is only serum-free medium with cells. Incubate in the incubator for 24 h, 48 h and 72 h, add CCK-8 reagent to each well, 20 μL per well, incubate at 37°C for 2-4 hours. Use the enzyme label instrument to measure the optical density (OD) at 450 nm at different times. The experiment was repeated 3 times.

[0047] In vitro evaluation of NHL cell proliferation inhibition can be expressed by half inhibition concentration (IC 50 ), IC 50 is calculated by GraphPad Prism 9.3.0 software.

[0048] Survival rate % = (OD 给药组 - OD 空白组 ) / (OD对照组 OD0) x 100%

[0049] Results: The survival rates of the three NHL cells were all decreased in a concentration-dependent manner after treatment with (+)-Strebloside. The IC50 values of Raji cells treated with (+)-Strebloside for 24 hours, 48 hours and 72 hours were 0.436 μM, 0.278 μM and 0.098 μM, respectively. 50 Figure 1 A). Both Jurkat and OCI-LY3 cell lines showed similar concentration-dependent cell proliferation inhibition as Raji cells under treatment with different concentrations of (+)-Strebloside. The IC50 values of Jurkat and OCI-LY3 cells treated with (+)-Strebloside for 24 hours were 0.317 μM and 0.186 μM, respectively. 50 Figure 1 B).

[0050] Colony formation experiment

[0051] Raji and Jurkat cells in the logarithmic growth phase were taken, and each cell was set up a control group and a drug administration group (the concentration of (+)-Strebloside in the drug administration group was 0.1 μM). Drug administration group: the cells were transferred to an EP tube and centrifuged at 1000 rpm for 5 min, resuspended with a drug-containing medium ((+)-Strebloside was diluted with serum-free medium to a concentration of 0.1 μM), counted under a microscope by using a hemocytometer, and 1 x 10 6 cells were inoculated in a small dish. After 24 hours of drug administration, the cells were centrifuged at 1000 rpm for 5 min, and the cell mass was collected. The cells were resuspended with fresh complete medium, counted, and the cell density was adjusted to 1 x 10 3 / mL. 0.5 mL of the cell suspension (about 500 cells) was taken, complete medium was added to 9.4 mL to obtain a drug administration group cell suspension, and the cell suspension was placed in an incubator for preheating. Control group: the drug-free serum-free medium was used to replace the drug-containing medium, and the rest was the same as the drug administration group. The control group cell suspension was obtained.

[0052] Preparation of the bottom agar: 5 g of agar powder was dissolved in 100 mL of distilled water, and heated in a boiling water bath until completely dissolved (the concentration of agar was 5%). When 1 mL of 5% agar was cooled to 45°C, 9 mL of complete medium preheated to 37°C was quickly added, mixed well, and 1 mL was added to a 6-well plate. The plate was naturally solidified at room temperature, and 3 samples were repeated for each experiment.

[0053] ​​Preparation of upper layer agar: 0.6 mL of 5% agar at 45°C was added to 9.4 mL of the above-mentioned preheated cell suspension of the administration group, mixed quickly, and then 0.8 mL was quickly added to a 6-well plate, which was allowed to naturally solidify at room temperature. Similarly, 0.6 mL of 5% agar at 45°C was added to 9.4 mL of the above-mentioned preheated cell suspension of the control group, mixed quickly, and then 0.8 mL was quickly added to a 6-well plate, which was allowed to naturally solidify at room temperature. Each experiment was repeated 3 samples.

[0054] After 3 weeks of routine culture, the number of colonies (colonies composed of more than 50 cells) was counted, and the cloning efficiency (%) = number of colonies / inoculated cell number x 100.

[0055] Results: In the soft agar plate cloning experiment, Raji and Jurkat cells treated with non-killing concentration of (+)-Strebloside showed lower colony formation compared with the control group (p<0.05, Figure 2 A and B).

[0056] Example 2

[0057] Tumor growth experiment in mouse xenograft model

[0058] The anti-tumor effect of (+)-Strebloside in BALB / c nude mice was evaluated using tumor xenografts transplanted with Raji cell lines. Specifically, 100 μL of Raji cell suspension (1 x 10 7 cells / mouse) was injected subcutaneously into the right forelimb of BALB / c nude mice, and the tumor volume was measured and calculated every other day. When the volume of the palpable tumor reached about 50 mm 3 The mice were randomly divided into the following groups: control group, positive group, (+)-Strebloside low-dose treatment group, and (+)-Strebloside high-dose treatment group, with 5 mice in each group. The nude mice were administered drugs on days 7, 9, 11, 13, 15, 17, 19, and 21 after tumor inoculation. The positive group, the (+)-Strebloside low-dose treatment group, and the (+)-Strebloside high-dose treatment group were intraperitoneally injected with Doxorubicin 10 mg / kg, (+)-Strebloside 2 mg / kg, and (+)-Strebloside 4 mg / kg, respectively, every two days. All drugs were prepared with normal saline, and the injection volume was 100 μL each time. The control group of mice was injected with 100 μL of normal saline each time. The tumor growth and health status of all mice were monitored every two days. On the 15th day of treatment, the mice were sacrificed, the tumors were removed, and the measurements and photographs were taken.

[0059] Tumor volume formula: V = a2 b / 2, wherein a represents the minimum diameter and b represents the maximum diameter.

[0060] Results are shown in Figure 3 and Figure 4 . The body weight of mice reflects the overall health of the mice and the toxicity of the drug. The body weight of nude mice was monitored during drug treatment, and there was no significant difference in the body weight of each group before drug treatment and at the end of treatment Figure 4 B). In the excised subcutaneously transplanted tumors, the average tumor weight of the control group was 0.391 ± 0.138 g, which was significantly higher than that of the low-dose treatment group and the high-dose treatment group of (+)-Strebloside (0.105 ± 0.096 g and 0.073 ± 0.054 g, respectively, p < 0.01, Figure 4 A). In addition, the average tumor volume of the control group was 631.17 ± 317.16 mm 3 , while the average tumor volume of the low-dose treatment group and the high-dose treatment group of (+)-Strebloside was 281.29 ± 120.34 mm 3 and 183.80 ± 80.03 mm 3 , respectively Figure 4 C), with a tumor inhibition rate of >75%, which was significantly lower than that of the control group Figure 4 D). This means that (+)-Strebloside can significantly inhibit the growth of tumors in nude mice.

[0061] Proteomics analysis

[0062] To further explore the effect of (+)-Strebloside on NHL, the inventors harvested tumors from mice in the high-dose treatment group of (+)-Strebloside and the control group and performed proteomics analysis.

[0063] (1) Protein extraction and enzymolysis

[0064] Harvest the subcutaneous tumor of nude mice, first fix with 4% paraformaldehyde, grind with liquid nitrogen, add 500 μL of lysis buffer, and perform ultrasonic lysis, all steps are carried out at 4°C; after lysis, centrifuge at 12000 rpm for 5 min, take the supernatant for quantification, and use the BCA protein quantification kit to quantify the concentration; then take an equal amount of protein sample for enzymolysis, slowly add 10% TCA and acetic acid, vortex well, precipitate at 4°C for 2 h, centrifuge at 5000 rpm for 5 min, discard the supernatant, wash with pre-cooled acetone for 2-3 times, dry, add TEAB with a final concentration of 200 mM, ultrasonic mixing, add protease according to the mass ratio of protease to protein 1:50, digest at 37°C overnight, then add 10 mM dithiothreitol (DTT) to the protein, incubate at 56°C in the dark for 30 min, then add 40 mM iodoacetamide (IAA), incubate at room temperature in the dark for 30 min.

[0065] (2) Analysis by liquid chromatography-mass spectrometry

[0066] The peptide segment is dissolved in an aqueous solution containing 0.1% formic acid and 2% acetonitrile, and separated by a NanoElute ultra-high performance liquid system. The mobile phase A (0.1% formic acid and 2% acetonitrile aqueous solution), the mobile phase B (0.1% formic acid and 100% acetonitrile solution), the gradient elution is set as 0-70 min, 6%→24% B; 70-82 min, 24%→35% B; 82-86 min, 35%→80% B; 86-90 min, 80% B; the flow rate is 450 nL / min. The peptide segment separated by the ultra-high performance liquid system is injected into the Capillary ion source for ionization, and then enters the timeTOF Pro 2 mass spectrometer for further analysis, and the secondary chromatography scanning range is set as 100-1700 m / z. The data set is collected in parallel accumulation series fragmentation (PASEF) mode, 10 PASEF mode secondary mass spectrometry is collected after each primary mass spectrometry is collected, the secondary mass spectrometry mother electron charge number is controlled within 0-5, and the tandem mass spectrometry scanning dynamic time is set as 30 s to avoid repeated scanning of the mother ion.

[0067] (3) Identification and quantification of proteins

[0068] Maxquant (v1.6.15.0) is used to search the obtained secondary mass spectrometry data. Mus_musculus_10090_SP__20220107.fasta (17097 sequences) is used as the search database, and the reverse library is added to calculate the false positive rate caused by random matching. In addition, common pollution libraries are added to the database to eliminate the influence of pollution proteins on the identification results. The polypeptides with a confidence level higher than 95% are selected for further analysis. The average mass of three biological repeats is used as the protein ratio for bioinformatics analysis.

[0069] (4) GO clustering analysis, differential protein localization, and differential protein functional enrichment analysis

[0070] Differential analysis was performed on the retrieved differentially expressed proteins, and annotation analysis was conducted using eggnog-mapper software (v2.0). This software is based on the EggNOG database. Here, the GO ID of each protein annotation result was extracted, and then the proteins were functionally classified according to cellular components, molecular functions, and biological processes. Enrichment analysis p-values ​​were obtained using a binomial test, and corrected using Bonferroni's multiple comparison method.

[0071] result:

[0072] Proteomics revealed (+)-Strebloside-mediated oxidative stress and ion homeostasis dysregulation in NHL. Specifically, 52 upregulated proteins and 10 downregulated proteins were identified (ploidy change > 1.5). Figure 5 A). Subsequently, enrichment analysis of the biological processes of differentially expressed proteins was performed based on Gene Ontology (GO) annotations (p<0.05), revealing proteins associated with lipid peroxidation and inorganic ion binding processes. Functional enrichment analysis of differentially expressed proteins showed that the expression of eight oxidative stress-related proteins was significantly upregulated in the (+)-Strebloside high-dose treatment group. Figure 5 B), cytochrome c activity and a range of fatty acid metabolism and catalytic activities were also significantly upregulated. Most of the differentially expressed proteins between the two groups were located in mitochondria (29.03%). Figure 5 C) Some mitochondrial proteins are components of the respiratory chain complex. Mitochondria are the primary site of reactive oxygen species (ROS) production; mitochondrial dysfunction leads to increased ROS production, triggering oxidative stress and cell damage. In areas of functional enrichment, calcium binding, metal ion binding, and cation binding activities are downregulated, resulting in decreased intracellular calcium and metal ion homeostasis. Figure 5 D) indicates that cardiac glycoside (+)-Strebloside acts as a Na + / K + The native ligand of α-ATPase (NKA) induces ion pump inhibition and leads to intracellular ion homeostasis imbalance. Furthermore, proteomics enrichment revealed upregulation of STEAP3 protein expression (1.664) and 11 other proteins associated with iron transport, suggesting that iron may also be involved in (+)-Strebloside-mediated NHL cell killing.

[0073] Example 3

[0074] Preliminary toxicity assessment of (+)-Strebloside

[0075] The preliminary toxicity experiment of (+)-Strebloside was carried out by using mouse acute toxicity test, and the specific method was as follows:

[0076] 4-week-old female BALB / c nude mice (18-22 g) were adaptively fed in an SPF environment for one week, randomly and uniformly distributed, and the drug administration groups were set: (+)-Strebloside administration group (20 mg / kg, physiological saline dissolution administration), 5 mice in each group, intraperitoneal injection administration, and the injection volume was 100 μL. The nude mice only received 1 dose of treatment, and 14 d after administration, the surviving mice were euthanized, and the heart, liver, spleen, lung, kidney, and lymph node were harvested for H&E staining.

[0077] Results: After administration of (+)-Strebloside (20 mg / kg), no abnormal behavior was observed in mice. No significant weight loss was detected in mice after two weeks of administration Figure 6 A). Two weeks after administration, no obvious lesions were observed in the heart, liver, spleen, lung, kidney, and lymph node of mice, and H&E staining showed that the integrity of the mouse tissue morphology had no obvious toxic reaction Figure 6 B).

[0078] Example 4

[0079] Effect of (+)-Strebloside on STEAP3 protein expression

[0080] Western blot: The drug-containing medium was obtained by diluting the stock solution of (+)-Strebloside with serum-free medium, and the final concentration of (+)-Strebloside in the drug-containing medium was 0.01, 0.05, 0.1, and 0.5 μM, respectively. The Raji, Jurkat, and OCI-LY3 cells in the logarithmic growth phase were cultured in 6-well plates, respectively. After 24 h of culture, a blank group (the culture medium was replaced with fresh serum-free medium) and a drug administration group (the culture medium was replaced with a drug-containing medium) were set up. The final concentration of (+)-Strebloside in the drug-containing medium used in the drug administration group of Raji cells was 0.01, 0.05, 0.1, and 0.5 μM, respectively, and the final concentration of (+)-Strebloside in the drug-containing medium used in the drug administration group of Jurkat and OCI-LY3 cells was 0.5 μM. After 48 h of culture, the protein was extracted from the cells. The cells were collected in an EP tube and centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Then, the cell lysate was added according to the ratio of 20 mg:(150-250) μL, and the cells were lysed on ice for 30 min. After centrifugation at 12000 rpm for 5 min, the supernatant was collected, and the concentration was determined using the BCA protein assay method. The standard sample was diluted with PBS to 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM, 0.03125 mM, and 0 mM, respectively, and then 20 μL of the diluted standard protein and the extracted protein to be tested were added in turn. BCA color reagent (200 μL / well) was added, and the absorbance was measured at 465 nm to make a standard curve. The protein of each group was divided into aliquots, and the secondary structure of the protein was removed by boiling in water for 5 min. An equal amount of protein was subjected to 10% SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Merck Millipore, USA). The membrane was blocked with 5% skim milk at room temperature for 2-3 hours, and then incubated with diluted primary antibody at 4°C overnight. After the end, TPST was washed quickly for three times, each for 10 min. Then, the goat anti-rabbit or mouse secondary antibody coupled with horseradish peroxidase (Beyotime, Shanghai, China) was incubated at room temperature for 2 hours, and then washed with TPST for three times, each for 10 min. GAPDH, tubulin-α, and β-actin were used as internal controls. The signal was visualized by chemiluminescence, and the image was analyzed using Image J software.

[0081] Conclusion: The results of the Western blot experiment are shown in Figure 7 It can be seen that (+)-Strebloside significantly up-regulates the expression of STEAP3 protein in a dose-dependent manner.

[0082] Immunohistochemistry is a method of determining the expression of a target protein in cells of a tissue by developing a color reagent for a labeled antibody through a chemical reaction. The method is as follows: First, a tumor is transplanted subcutaneously into a nude mouse, and the tumor-bearing method is the same as in Example 2. When the tumor is developed to a volume of about 50 mm 3 Four-week-old female BALB / c nude mice (18-22 g) were randomly divided into two groups, five in each group: a Control group (normal saline) and a (+)-Strebloside administration group (4 mg / kg, administered in normal saline), administered intraperitoneally, and the nude mice received only one dose of treatment. After 14 days of administration, the nude mice were sacrificed, the tumor mass was weighed, and then quickly preserved with 4% paraformaldehyde. The embedded tumor tissue block was then sliced by a paraffin microtome, the wax slices were attached to a glass slide, and the glass slide was dried at 45°C. Antigen repair: The paraffin sections were deparaffinated at room temperature, hydrated, and subjected to heat antigen repair to unfold the proteins in the tissue. After repair, 3% hydrogen peroxide was added, and the endogenous enzymes were inactivated by incubation at room temperature for 10 min. Blocking: The sections were blocked with goat serum blocking solution at room temperature for 10 min. Incubation of primary antibody: Then, the primary antibody was incubated according to the antibody dilution ratio, and the incubation was performed at 4°C overnight. Incubation of secondary antibody: After incubation of the primary antibody, the glass slide was warmed at room temperature for 30 min, washed with PBS for 3 min, washed with distilled water for 3 min, and then the secondary antibody was diluted and incubated according to the instructions. Staining: 50 μL of streptomyces antibiotic-peroxidase solution was added, and the glass slide was incubated at room temperature for 10 min; washed with PBS for 10 min, and then washed with distilled water for 3 min. 100 μL of DAB solution was added, and the glass slide was observed under a microscope. Eosin was added for 1-2 min, 1% hydrochloric acid alcohol was added for 3 s, and then the glass slide was washed with tap water, dehydrated, and sealed with neutral balsam. Finally, the images were collected and analyzed.

[0083] Conclusion: Immunohistochemical staining showed that Figure 8 Compared with the Control group, the administration group showed obvious brown color, indicating that STEAP3 was significantly up-regulated in the tumor of the mouse treated with (+)-Strebloside.

[0084] Effect of (+)-Strebloside on the expression of STEAP3 gene

[0085] The effect of (+)-Strebloside on the expression of STEAP3 gene was investigated by qRT-PCR.

[0086] Primer selection and synthesis: The primer premier 5 was used to design the primer, and the primer sequence with no abnormal structure, no dimer, G, C content of 40-60%, and high specificity was selected. The primers required in this experiment were synthesized by Wuhan Seville Biotechnology Co., Ltd., and the purity was PAGE level. Sterile ultrapure water was used for dissolution, and the concentration was 10 μM.

[0087] Total RNA extraction in cells: The Raji cells in logarithmic growth phase were cultured in a 6-well plate and incubated for 24 h. The blank group and the drug group were set. The culture medium of the blank group was replaced with fresh serum-free culture medium, and the drug group was replaced with drug-containing culture medium ((+)-Strebloside final concentration 0.5 μM). Incubate for 48 h. After incubation, collect the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, take the precipitate (i.e. cells), and extract the total RNA in the cells according to the instructions of the RNA extraction kit. Dissolve the total RNA in DEPC water for reverse transcription.

[0088] Complementary DNA synthesis: NANO analysis quantifier is needed before reverse transcription to quantify the total RNA, then according to the manufacturer's instructions RNA reverse transcriptase, target gene, internal standard primer, reverse the RNA to cDNA.

[0089] Real-time fluorescence quantitative PCR: 2x SYBR Green Fastq PCR premix (YiFeiXue biotechnology, Nanjing, China) was used according to the manufacturer's instructions on the BIO-RADS1000 device (BIO-RAD, USA). Different programs were used to analyze the total RNA and cDNA synthesis of RT. The mRNA level was normalized to β-actin, and the standard 2 -ΔΔCt method was calculated.

[0090] Conclusion: The results of qRT-PCR experiment are shown in Figure 9 , which shows that (+)-Strebloside up-regulates the expression of SREAP3 gene in Raji cells.

[0091] (+)-Strebloside mediates Fe 2+ overload

[0092] STEAP3 is a metal reductase with physiological activity of reducing Fe 3+ to ferrous iron (Fe 2+ ) in vivo. Therefore, the cell was treated with ferrous ion fluorescent probe (FerroFarRed) to evaluate the intracellular ferrous ion concentration.

[0093] Take 50 nmol Ferrofar Red fluorescent probe, prepare Ferrofar Red stock solution with DMSO to get the concentration of 1 mM.

[0094] Take the log phase of Raji, Jurkat cells cultured in 6-well plates for 24 h, centrifuged at 1000 rpm, and the supernatant was washed with PBS once. At this time, set the Ctrl group (Ctrl group added with serum-free medium), drug administration group (drug administration group added with 0.05, 0.1, 0.5 μM Strebloside), and the drug preparation process was diluted with serum-free medium. Incubate for 48 h, centrifuge at 1000 rpm for 5 min, and wash the precipitate (i.e. cells) with PBS once; then dilute the Ferrofar Red stock solution with serum-free medium to prepare a working staining solution with a concentration of 5 μM; then add the above staining working solution to the Control group and drug administration group cells, and mix gently; incubate each group of cells at 37°C for 1 h, centrifuge at 800 rpm for 5 min, and sieve the cells with a 400-mesh cell sieve for cell flow cytometry detection, set the excitation wavelength to 532 nm, and collect 1000 events for each sample.

[0095] As shown in Figure 10 (+)-Strebloside treatment increased the concentration of ferrous ions in Raji and Jurkat cells, indicating that STEAP3, which may be up-regulated, mediated the enrichment of ferrous ions in NHL cells.

[0096] Iron chelators or ferroptosis inhibitors can alleviate the proliferation inhibition mediated by (+)-Strebloside

[0097] The trivalent iron selective chelator Deferasirox can selectively bind to free iron and form insoluble chelates.

[0098] Cell treatment: Take the log phase of Raji / Jurkat cells cultured in 6-well plates, centrifuge at 1000 rpm for 5 min after 24 h, collect the precipitate, i.e. cells, wash with PBS once, set the control group (Control group), (+) Strebloside administration group, (+) Strebloside + Deferasirox group, dilute (+) Strebloside with serum-free medium to the administration concentration ((+) Strebloside administration group, (+) Strebloside + Deferasirox group (+) Strebloside administration concentration is 0.1 μM), Deferasirox according to the manufacturer's instructions, then incubate in an incubator for 12, 24, 36, 48, 60, and 72 h. After incubation, detect the iron ion content and use the CCK8 method to detect the cell survival rate.

[0099] In addition, the ferroptosis inhibitor Ferrostatin-1 (Fer-1) can reverse the trend of cell damage induced by ferroptosis inducers. Ferrostatin-1 (Fer-1) was administered in the same manner as the trivalent iron selective chelator Deferasirox, with only the (+) Strebloside concentration adjusted to 0.5, 1, and 5 μM.

[0100] Compared with control cells, the chelator Deferasirox, together with (+)-Strebloside, downregulated the intracellular ferrous ion content in Raji cells (after 48 h of incubation, p < 0.05). Figure 11 A, B), and increased cell viability under chelation (p<0.05, Figure 11 C); similar results were obtained in Jurkat cells. Figure 11 D). These results indicate that the iron chelator Deferasirox may act upstream of iron transport or reduction, mitigating the inhibitory effect of (+)-Strebloside on cell proliferation by reducing the concentration of free iron ions in the cell.

[0101] Ferrostatin-1, an inhibitor of ferroptosis, also alleviated intracellular ferrous overload induced by (+)-Strebloside. Figure 12 A and B) rescued Raji cells from (+)-Strebloside-mediated proliferation inhibition. Figure 12 C).

[0102] The results suggest that the iron-dependent programmed cell death pathway may partially contribute to (+)-Strebloside toxicity.

[0103] Based on the above results, the inventors hypothesize that (+)-Strebloside may mediate ferroptosis in NHL cells through a mechanism involving iron metabolism. 3+ STEAP3 is converted into Fe within cells. 2+ It is released into the cytoplasm. Excess ferrous iron accumulates in the cell and forms an unstable iron pool. Free ferrous iron participates in the Fenton reaction, producing large amounts of reactive oxygen species (ROS) and leading to lipid peroxidation, resulting in loss of cell function and cell death.

[0104] The inventors further tested other indicators of ferroptosis: intracellular mitochondrial ROS, lipid peroxidation, and GPX4 levels.

[0105] ROS, lipid peroxidation and GPX4 detection

[0106] ROS detection was performed using a reactive oxygen species assay kit. Before the experiment, the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA) was diluted to a concentration of 10 mM with serum-free culture medium. The Raji cells in the logarithmic growth phase were cultured in a 6-well plate, and after 24 h of culture, the Ctrl group and the drug administration group were set up. The cells in the Ctrl group were replaced with serum-free culture medium, and the cells in the drug administration group were replaced with culture medium containing (+)-Strebloside at a final concentration of 0.05 mM, 0.1 mM, and 0.5 mM, respectively. The cells in each group were treated for 48 h, and then the cells were collected in an EP tube and centrifuged at 1000 rpm for 5 min. The precipitate was collected and then placed in 10 mM DCFH-DA. The mixture was incubated at 37°C for 30 min, and the mixture was inverted and mixed every 3-5 min during the incubation. After the incubation, the cells were washed once with serum-free culture medium, and then centrifuged at 1000 rpm for 5 min. Finally, the cells were washed twice with PBS, passed through a 400-mesh sieve, and subjected to flow cytometry. Each sample was collected for 10,000 events.

[0107] The lipid peroxidation level was measured using BODIPY-C11 dye (purchased from MedChemExpress). 1 mg of C11BODIPY581 / 591 was dissolved in 198.2554 mM DMSO to prepare a stock solution with a concentration of 10 mM. The Raji cells in the logarithmic growth phase were cultured in a 6-well plate, and after 24 h of incubation, the cells were centrifuged at 1000 rpm for 5 min, and the precipitate was collected and then washed once with PBS. The Ctrl group and the drug administration group were set up. The cells in the Ctrl group were cultured with serum-free culture medium, and the cells in the drug administration group were treated with (+)-Strebloside at different concentrations (diluted with serum-free culture medium to a final concentration of 0.05 mM, 0.1 mM, and 0.5 mM, respectively). The Raji cells in each group were incubated for 48 h, and then centrifuged at 1000 rpm for 5 min. The precipitate was collected and then resuspended with PBS. C11BODIPY581 / 591 was added to the cells to a final concentration of 5 mM, and the mixture was incubated at room temperature for 1 h. The mixture was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were gently resuspended with culture medium containing 5% FBS. All the cell samples were passed through a 400-mesh sieve, and then subjected to flow cytometry. Each sample was collected for 10,000 events.

[0108] GPX4 was detected by Western blot. The specific process is described in “Example 4: Effect of (+)-Strebloside on the expression of STEAP3 protein”.

[0109] As Figure 13A shows that compared with the Ctrl group, obvious accumulation of ROS was observed in Raji cells in each concentration of (+)-Strebloside treatment group, and any concentration of (+)-Strebloside promoted the production of ROS; after labeling with lipid peroxide probe BODIPY-C11, accumulation of intracellular lipid peroxide was also observed in Raji cells Figure 13 B). Similarly, a dose-dependent decrease in GPX4 expression level was observed in the same cells Figure 13 C).

[0110] Subsequently, the inventors studied the effect of (+)-Strebloside on the mitochondrial function of Raji cells.

[0111] Mitochondrial membrane potential (ΔΨm), ATP detection, and cytochrome C detection

[0112] Mitochondrial membrane potential detection experiment

[0113] Determination of intracellular mitochondrial membrane potential (ΔΨm): First, prepare a 200 μM Mito-Tracker Red CMXRos (mitochondrial red fluorescent probe) stock solution with DMSO, and culture Raji cells in the logarithmic growth phase in a 6-well plate for 24 h. Then, set up the control group and the drug administration group. The control group is replaced with serum-free medium without drugs, and the drug administration group is replaced with different concentrations of drug-containing medium (diluted with serum-free medium to a final concentration of 0.05 μM, 0.1 μM, and 0.5 μM of (+)-Strebloside). Each group of cells is treated for 48 h, centrifuged at 1000 rpm for 5 min, and the precipitate is taken as the cells. The cells are gently resuspended with a 37°C preheated Mito-Tracker Red CMXRos stock solution, incubated at room temperature for 15-30 min, centrifuged at 1000 rpm for 5 min after incubation, washed with PBS three times, passed through a 400-mesh sieve, and subjected to flow cytometry detection at a detection wavelength of 579 nm. Each sample collects 10,000 events.

[0114] ATP detection experiment

[0115] ATP (adenosine 5'-triphosphate) levels in cells were measured using an ATP detection kit. Cell processing: Raji cells in the logarithmic growth phase were cultured in a 6-well plate, incubated for 24 h, and then set up control and drug groups. The control group was replaced with serum-free medium without drugs, and the drug group was replaced with different concentrations of drug-containing medium (diluted with serum-free medium to a final concentration of 0.05 μM, 0.1 μM, and 0.5 μM of (+)-Strebloside). Each cell was treated for 48 h, centrifuged at 1000 rpm for 5 min, and the precipitate was completely lysed with 200 μL Vortex lysis buffer for 30 min. After lysis, centrifugation was performed at 4°C and 12000 rpm for 5 min, and the supernatant was collected. Standard curve preparation: The reagents were thawed on ice, and the ATP standard solution was diluted with ATP detection lysis buffer to an appropriate concentration gradient, i.e., 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM. Before detection, 100 μL of ATP detection working solution was added and incubated at room temperature for 3-5 min to consume background ATP and reduce the background. Then 20 μL of standard and sample were added to each detection well, and three sets of parallel experiments were set up for each group. The ATP concentration of the sample to be tested was detected by L (luminometer), and the data were processed and calculated by GraphPad Prism 9.3.0 software L (luminometer) to detect the ATP concentration of the sample to be tested, and the data were processed and calculated by GraphPad Prism 9.3.0 software

[0116] Western blot was used to detect the expression level of cytochrome C protein in Raji cells: the experimental process was the same as that in Example 4-(+)-Strebloside on STEAP3 expression detection process.

[0117] The results showed that (+)-Strebloside reduced the transmembrane potential in mitochondria ( Figure 14 A), and concentration-dependently down-regulated the ATP production level ( Figure 14 B), and released cytochrome C ( Figure 14 C). It is shown that mitochondrial dysfunction is involved in the iron metabolism-dependent pathway of (+)-Strebloside-mediated NHL ferroptosis.

[0118] To further study the important function of STEAP3, the inventors transfected Raji cells with different sequences of Steap3-siRNA (NC, STEAP3-Si1, STEAP3-Si2, purchased from Shengong Bioengineering Co., Ltd.) to inhibit STEAP3, and selected the highest inhibition efficiency of STEAP3-Si2 for further study ( Figure 15 ).

[0119] The sequence of STEAP3-Si2 is UUGUAGGCAUAGAAGCAGACGTT.

[0120] Screening of siRNA

[0121] Primer selection and synthesis: The primers were designed by primer premier 5, and the primer sequences with no abnormal structure, no dimer, G and C content of 40-60%, and high specificity were selected. The primers required in this experiment were synthesized by Sangon Biotech, and the purity was PAGE grade. The primers were dissolved in sterile ultrapure water, and the volume was adjusted to a concentration of 10 μM.

[0122] Cell treatment: The Raji cells in logarithmic growth phase were cultured in a 6-well plate, incubated for 24 h, centrifuged to remove the supernatant, and the cells were collected. The NC group, Steap3-si1 group and Steap3-si2 group were set. The siRNA (NC, Steap3-si1, Steap3-si2) premix was prepared using buffer (i.e. enzyme-free water), and the final concentration of siRNA was 1.5 μM. 10 μL of plus transfection reagent was added to the above siRNA premix, and immediately mixed by blowing with a pipette for dozens of times to prepare siRNA / plus complex. After transfection incubation for 24 h, centrifugation at 1000 rpm for 5 min, the supernatant was discarded, and the precipitate was used for PCR experiment. The total RNA was isolated from the cells using the total RNA extraction kit according to the instruction manual.

[0123] Complementary DNA synthesis: Before reverse transcription, the total RNA concentration was determined by NANO quantitative instrument, and then the RNA reverse transcriptase, target gene and internal reference primer were added according to the reverse transcription kit instruction. The RNA was reverse transcribed to cDNA by reverse transcription instrument.

[0124] Real-time fluorescent quantitative PCR: This process uses 2x SYBR Green Fastq PCR premix, and is performed according to the manufacturer's instructions on BIO-RADS1000 equipment (BIO-RAD, USA). Different programs are used to analyze the total RNA and cDNA synthesis RT. The mRNA level is normalized to β-actin, and the standard 2 -ΔΔCt Method calculation. The highest knockdown rate of STEAP3-Si2 was selected.

[0125] Cell transfection

[0126] Raji cells in logarithmic growth phase were cultured in 6-well plates, incubated for 24 h, then transfected for 24 h, and set up Si-Steap3 group, NC group, Si-Steap3+ drug group (Si-Steap3+S group), NC+ drug group (NC+S group). The transfection process was the same as "si-RNA screening". First, dilute NC and Si-Steap3 to a concentration of 1.5 μM with buffer, mix with plus transfection reagent, immediately blow with a gun for dozens of times, mix evenly, add to the cells and mix, and put into the incubator for 24 h. After incubation, the supernatant in the well plate was aspirated, the Si-Steap3 group and the NC group were replaced with serum-free medium, and the Si-Steap3+ drug group (Si-Steap3+S group) and the NC+ drug group (NC+S group) were replaced with drug-containing medium (dilute (+)-Strebloside to a final concentration of 0.5 μM with serum-free medium). After 48 h of incubation, the cells were collected, and WB experiments were performed according to Example 4. The data were calculated and processed by GraphPad Prism 9.3.0 software.

[0127] Jurkat cells were processed as above.

[0128] Cell viability detection experiment

[0129] Cell viability was detected by CCK8 experiment: first, process the cells, take the logarithmic growth phase of Raji / Jurkat cells, and culture them in 96-well plates. After 24 h of incubation, set up Si-Steap3 group, NC group, Si-Steap3+ drug group (Si-Steap3+S group), and NC+ drug group (NC+S group). Transfect the cells according to "Cell transfection", and the process. After transfection, give drug for 48 h according to "Cell transfection" ((+)-Strebloside drug concentration is 0.5 μM). Refer to Example 1, and detect the viability by CCK8.

[0130] Jurkat cells were processed as above.

[0131] As expected, STEAP3 knockdown significantly rescued the decrease in GPX4 expression level ( Figure 16 A, B). The decrease in cell viability was also reversed after STEAP3 knockdown ( Figure 16 C). In addition, the same results were also obtained in Jurkat cells by cell transfection ( Figure 17 A, B, C).

[0132] In summary, the cardiac glycoside compound (+)-Strebloside can inhibit the proliferation of NHL cells through STEAP3-mediated ferroptosis, and play an anti-tumor role.

Claims

1. Use of (+)-strebloside in the preparation of a medicament for treating non-Hodgkin's lymphoma.

2. Use according to claim 1, characterized in that: The use is the use of (+)-strebloside in the preparation of a medicament for treating non-Hodgkin's lymphoma by exerting at least one of the following effects: inhibiting the proliferation of non-Hodgkin's lymphoma cells; inhibiting the metastasis of non-Hodgkin's lymphoma cells; inducing ferroptosis of non-Hodgkin's lymphoma cells.

3. Use according to claim 1, characterized in that: The use is the use of (+)-strebloside in the preparation of a medicament for inducing ferroptosis of non-Hodgkin's lymphoma cells by STEAP3.

4. Use according to any one of claims 1 to 3, characterized in that: The non-Hodgkin's lymphoma cells include diffuse large B-cell lymphoma, Burkitt's lymphoma cells, human T-lymphocyte leukemia cells.

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