Use of androst-4,6,8(9),13(14)-tetraen-3,11,16-trione in the treatment of lymphoma

Abstract

The application discloses a new use of androsta-4,6,8(9),13(14)-tetraen-3,11,16-trione, namely, application of the androsta-4,6,8(9),13(14)-tetraen-3,11,16-trione in preparation of a medicine for treating lymphoma. 50 The IC value of the androsta-4,6,8(9),13(14)-tetraen-3,11,16-trione in the SR cell line is detected by a CCK-8 method, and the cytotoxicity of the androsta-4,6,8(9),13(14)-tetraen-3,11,16-trione on human umbilical vein endothelial cells Huvce and human immortalized keratinocytes Hacat is detected, in the body, the androsta-4,6,8(9),13(14)-tetraen-3,11,16-trione significantly inhibits tumor growth and shows good biological safety and no systemic toxicity; TUNEL staining, gamma-H2AX staining and neutral comet experiment prove that the compound of the application inhibits the growth of SR by causing serious damage to DNA, the application not only adds the diversity of the biological activity of the androsta-4,6,8(9),13(14)-tetraen-3,11,16-trione, but also provides a new direction for developing a new medicine for treating lymphoma.

Description

Technical Field

[0001] This invention relates to the application of androstenedin-4,6,8(9),13(14)-tetraene-3,11,16-trione in the preparation of drugs for treating lymphoma, and belongs to the field of pharmaceutical technology. Background Technology

[0002] Lymphoma is a malignant proliferative disease originating from lymphoid cells, primarily affecting lymphoid organs such as lymph nodes, spleen, and tonsils, but it can also invade extranodal lymphoid tissues or organs. During lymphocyte development and differentiation, genetic abnormalities can lead to malignant transformation of lymphocytes at a certain developmental stage. These abnormal cells (usually mature or relatively mature lymphocytes) accumulate abnormally in the lymphoreticular tissue, forming tumors. The occurrence of lymphoma is associated with multiple factors, including the activation of proto-oncogenes, the inactivation of tumor suppressor genes, and viral infection. These alterations cause lymphocytes to evade normal apoptosis regulation, gaining unlimited proliferative capacity and ultimately forming space-occupying lesions in lymphoid tissues.

[0003] Lymphoma can occur at any age and is mainly divided into two categories: Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Hodgkin lymphoma is reported to have a high cure rate, with long-term survival rates exceeding 80%-90%. However, the prognosis of non-Hodgkin lymphoma varies greatly depending on the specific subtype. For more aggressive subtypes (such as diffuse large B-cell lymphoma), the cure rate after standard chemotherapy is approximately 60%-70%; however, for some highly aggressive or indolent lymphomas that transform into aggressive lymphomas, especially in older patients (>60 years old) or those with underlying diseases, treatment becomes significantly more difficult, and the 5-year overall survival rate may be less than 30%-40%.

[0004] Cisplatin (DDP) is a platinum-based compound and a key drug for treating lymphoma, particularly relapsed / refractory lymphoma or certain subtypes of lymphoma, as a salvage regimen (such as the GDP regimen: gemcitabine + cisplatin + dexamethasone, or the DHAP regimen: dexamethasone + cisplatin + cytarabine). Combined with other chemotherapy drugs, it can achieve clinical remission. However, when lymphoma patients are given cisplatin-containing regimens, the risk of side effects increases significantly. Side effects include severe nephrotoxicity (requiring hydration and diuresis for prophylaxis), ototoxicity (hearing loss or tinnitus), peripheral neuropathy (limb numbness, pain), severe nausea and vomiting, and bone marrow suppression. Therefore, developing alternative drugs or optimizing existing regimens to reduce the dose-limiting toxicities of cisplatin is crucial for improving the quality of life and treatment adherence of lymphoma patients.

[0005] Androst-4,6,8(9),13(14)-tetraene-3,11,16-trione is a natural small molecule compound derived from the plant *Cynanchum paniculatum*, with the molecular formula C2.19 H 18 O3, with a molecular weight of 294.13, belongs to the androsteroid class of compounds. *Symplocos simonii* is a plant belonging to the Apocynaceae family (…). Apocynaceae ) is a species in the genus *Symplocos spp.*, distributed in Myanmar and Malaysia. In my country, only *Symplocos spp.* is found. Epigynum auritum One of them, produced in southern Yunnan, is a plant with heat-clearing, detoxifying, and analgesic effects. It has been recorded as a medicinal plant in the folk medicine of the Dai people. However, there are very few studies on the pharmacological activity of androstenedin-4,6,8(9),13(14)-tetraene-3,11,16-trione, and so far no one has proposed its use in the treatment of lymphoma. Summary of the Invention

[0006] This invention provides a novel use of androstened-4,6,8(9),13(14)-tetraene-3,11,16-trione, namely its use in the preparation of drugs for treating lymphoma.

[0007] The chemical structural formula of the androstrol-4,6,8(9),13(14)-tetraene-3,11,16-trione (ATT) is as follows: .

[0008] The drug component (or active ingredient) for treating lymphoma described in this invention is androstenedin-4,6,8(9),13(14)-tetraene-3,11,16-trione. One or more drug-acceptable excipients may also be added to improve the absorption effect of the drug or facilitate its use, and to prepare a suitable dosage form, such as capsules or pills, powders, tablets, granules, oral liquids and injections.

[0009] This invention discloses a novel use of androst-4,6,8(9),13(14)-tetraen-3,11,16-trione, namely its application in the preparation of drugs for treating lymphoma. This invention uses the atypical large cell lymphoma cell line SR and detects the IC50 of androst-4,6,8(9),13(14)-tetraen-3,11,16-trione in the SR cell line using the CCK-8 assay. 50The value and cytotoxicity of androstened 4,6,8(9),13(14)-tetraene-3,11,16-trione to human umbilical vein endothelial cells (Huvce) and immortalized human keratinocytes (Hacat) were measured. In vivo, it significantly inhibited tumor growth and showed good biocompatibility with no systemic toxicity. Mechanistically, γ-H2AX staining and comet assays demonstrated that androstened 4,6,8(9),13(14)-tetraene-3,11,16-trione inhibits SR growth by inducing severe DNA damage. Multiple recombinant chemoanalysis identified granzyme A (GZMA) as a direct target. It was demonstrated that ATT binds to GZMA, leading to cleavage of the SET complex. This releases DNase NM23-H1,26, which then executes DNA fragments. This invention not only adds to the diversity of the biological activities of androstened 4,6,8(9),13(14)-tetraene-3,11,16-trione, but also provides a new direction for the development of new drugs for the treatment of lymphoma.

[0010] Compared with the prior art, the present invention has the following advantages: 1. This invention has discovered new medical applications for the monomer compound of *Syzygium sibiricum*, and opened up a new research field. 2. Through experiments, this invention has found that the monomeric compound of *Syzygium sibiricum*, androst-4,6,8(9),13(14)-tetraene-3,11,16-trione, can effectively inhibit the proliferation of SR cells at low doses; the raw materials are derived from natural products and have high safety. 3. The monomeric compound of *Symplocos simaoensis*, androst-4,6,8(9),13(14)-tetraene-3,11,16-trione, has a better or comparable inhibitory effect on the proliferation of SR cell tumors than DDP. Moreover, the separation and purification process of this compound is simple and inexpensive, making it suitable for industrial production and market application. Attached Figure Description

[0011] Figure 1 The survival rate of normal cells (Hacat and Huvce) after culturing in different concentrations of androstened 4,6,8(9),13(14)-tetraene-3,11,16-trione for 48 h was calculated. Figure 2 The survival rate of lymphoma cells SR after culturing in different concentrations of androstenedin-4,6,8(9),13(14)-tetraene-3,11,16-trione for 24 h and 48 h was determined. Figure 3 TUNEL staining results of lymphoma cells SR in different concentrations of androstenedin-4,6,8(9),13(14)-tetraene-3,11,16-trione. The upper figure is an image under a fluorescent inverted microscope, and the lower figure is the positive cell rate. Figure 4γ-H2AX staining results of lymphoma cells SR in different concentrations of androstenedin-4,6,8(9),13(14)-tetraene-3,11,16-trione; Figure 5 The results of comet assays on lymphoma cells SR at different concentrations of androstened 4,6,8(9),13(14)-tetraene-3,11,16-trione are shown in the left image under a fluorescence inverted microscope and the right image is a statistical graph. Figure 6 A method for constructing an SR xenograft mouse model; Figure 7 Changes in mouse body weight during the experiment; Figure 8 Images of tumors removed in each experimental group; Figure 9 This is the tumor volume result; Figure 10 Results for tumor weight; Figure 11 Organ indices for the brain, heart, and liver; Figure 12 The organ indices are for the spleen, lungs, and kidneys; Figure 13 Serum inflammatory factor levels in mice Figure 14 The level of inflammatory factors in mouse tumor tissue homogenate; Figure 15 Immunofluorescence staining of Ki-67 in tumor tissue; Figure 16 TUNEL staining of tumor sections from NSG mice modeled with SR lymphoma cells; the top image shows the staining, and the bottom image shows the statistical graph. Figure 17 Caspase-3 staining of lysed tumor sections of SR lymphoma cells induced in NSG mice; the top image is the staining image, and the bottom image is the statistical graph. Figure 18 The image shows Bcl2 staining of tumor sections from lymphoma cells (SR) induced in NSG mice. The top image is the staining diagram, and the bottom image is the statistical graph. Detailed Implementation

[0012] The androstrol-4,6,8(9),13(14)-tetraene-3,11,16-trione used in the embodiments of this invention was prepared using existing phytochemical separation and purification techniques. The invention will be further illustrated below with reference to experimental data and accompanying drawings. These experimental examples are for illustrative purposes only and are not intended to limit the scope of application of this invention. After reading this invention, any equivalent modifications, alterations, and modifications made by those skilled in the art are within the scope defined by the claims of this invention. Unless otherwise specified, the reagents used in the embodiments are conventional commercially available products or reagents prepared using conventional methods. Unless otherwise specified, the methods used in the embodiments are conventional experimental methods.

[0013] In this example, the cell counting (CCK-8) and assay kits were purchased from Shanghai Baisai Biotechnology Co., Ltd.; the cell cycle assay kit was purchased from Beijing Sizhengbai Biotechnology Co., Ltd. RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin, and streptomycin were all purchased from Gibco; DMSO was purchased from Beijing Solarbio Biotechnology Co., Ltd. The BCA protein assay kit, Hoechst staining kit, and TUNEL staining kit were all purchased from Shanghai Beiyotech Biotechnology Co., Ltd. The tumor necrosis factor-α, interferon-γ, IL-1β, and IL-6 84 assay kits were purchased from Jiangsu Enzyme Immunoassay Industry Co., Ltd.

[0014] Example 1: Detection of cell viability of androstenedione-4,6,8(9),13(14)-tetraene-3,11,16-trione (ATT) in SR cells 1. Preparation of experimental materials Solution preparation: Androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone were prepared into a 30mM stock solution using cell-grade DMSO under sterile conditions. The stock solution was sealed and stored in a -20℃ refrigerator in the dark for later use. In the experiment, the solution was diluted to different concentrations according to the final concentration requirements after drug addition.

[0015] Cell lines: Lymphoma SR, human immortalized keratinocytes Hacat, human umbilical vein endothelial cells Huvce; SR cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin, while normal cell lines were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin at 37°C in a 5% CO2 incubator for later use.

[0016] 2. Experimental Methods Hacat and Huvce cells in the logarithmic growth phase were used to prepare a solution with a concentration of 2.5 × 10⁻⁶ cells in complete culture medium. 4Cell suspension of cells per mL was seeded into 96-well plates at a density of 200 μL per well. The experiment was divided into a drug group and a negative control group, with 5 replicates per group (or per drug concentration). After 24 hours of culture, the drug group was replaced with 200 μL of medium containing different concentrations of the compound androstenedin-4,6,8(9),13(14)-tetraen-3,11,16-trione (concentrations of 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, and 60 μmol / L), while the negative control group was replaced with 200 μL of fresh complete medium. After 46 hours of culture, 20 μL of CCK-8 reagent was added to each well, and the cells were returned to the incubator for another 2 hours. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The cell viability for different drug concentrations was calculated using the formula: viability = (OD value of drug group / OD value of negative control group) × 100%. The results are shown in the figure. Figure 1 At drug concentrations of 10-60 μmol / L, androst-4,6,8(9),13(14)-tetraene-3,11,16-trione had no damaging effect on normal cells. SR cells in the exponential growth phase were used at a rate of 5 × 10⁻⁶. 4 The density of the seeding medium was determined in 96-well plates. The final concentrations of the drug solution in the drug treatment groups were set at 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, and 50 μmol / L. An equal volume of complete culture medium was added to the negative control group. The total volume per well was 200 μL, with 5 replicates per group (or per concentration). After 44 h of incubation, 20 μL of CCK-8 solution was added to each well, and incubation continued for another 4 h. The absorbance of each well at 450 nm was measured using a microplate reader, and the viability and IC50 were calculated. 50 Survival rate = (OD value of drug group / OD value of negative control group) × 100%, results are shown in [link to results]. Figure 2 Androst-4,6,8(9),13(14)-tetraene-3,11,16-trione significantly reduced the viability of SR cells in a dose- and time-dependent manner within 24 h and 48 h.

[0017] Example 2: Effects of different concentrations of androstenedione-4,6,8(9),13(14)-tetraene-3,11,16-trione on TUNEL staining, γ-H2AX staining, and neutral comet assay of lymphoma cells 1. TUNEL staining After counting SR cells in the logarithmic growth phase, the count was performed at a rate of 2.5 × 10⁻⁶. 5 Cells were cultured at a density of cells per well in 12-well plates, with... Interventions were performed with ATT (final concentrations of 20 μmol / L, 40 μmol / L, and 60 μmol / L), while the control group received the same volume of fresh RPMI 1640 complete culture medium, with a total volume of 1 mL for all groups. Cells were cultured at 37°C for 48 h, then culture was terminated. Cells were gently aerated and transferred to centrifuge tubes for collection. The cells were then washed once with pre-chilled PBS, fixed with 4% paraformaldehyde for 30 min, centrifuged to remove the fixative, and then incubated with TUNEL dye in the dark for 1 h, followed by DAPI staining for 12 min. The cell suspension was then prepared into slides and photographed under a fluorescence inverted microscope in multiple fields of view. Results are shown below. Figure 3 As can be seen from the figure, apoptosis of SR cells also increases significantly with increasing drug concentration.

[0018] γ-H2AX staining After counting SR cells in the logarithmic growth phase, the count was performed at a rate of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 cells / well in 12-well plates, and different concentrations (20, 40, 60 μmol / L) of ATT were added for intervention. The control group received an equal volume of fresh RPMI 1640 complete medium, with a final volume of 1 mL. Cells were incubated at 37°C for 48 h. After culture, cells were gently pipetted and transferred to centrifuge tubes for collection. Cells were washed once with pre-cooled PBS, fixed with 4% paraformaldehyde for 30 min, centrifuged to remove the fixative, and then permeabilized with 0.2% Triton X-100 for 10 min. After washing with PBS, cells were blocked with 5% BSA at room temperature for 30 min. After discarding the blocking solution, anti-γ-H2AX primary antibody (diluted according to the recommended ratio) was added and incubated overnight at 4°C. The next day, cells were washed with PBS, and fluorescently labeled secondary antibody (diluted according to the recommended ratio) was added and incubated in the dark for 1 h. After washing with PBS, DAPI staining was added for 12 min. After centrifugation, the cell suspension was collected and prepared into cell slides. Multiple fields of view were randomly selected and photographed under a fluorescence inverted microscope. Results are shown below. Figure 4 As can be seen from the figure, DNA damage increases significantly with increasing drug concentration.

[0019] Neutral Comet Experiment After counting SR cells in the logarithmic growth phase, the count was performed at a rate of 2.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 cells / well in 12-well plates, and different concentrations (20, 40, 60 μmol / L) of ATT were added for intervention. The control group received an equal volume of fresh RPMI 1640 complete medium, with a final volume of 1 mL. Cells were incubated at 37°C for 48 h. After culture, cells were collected and resuspended in pre-chilled PBS to adjust the cell density to 1 × 10⁻⁶ cells / well. 5The cell count is approximately [number] cells / mL. To prepare the three-layer gel: First, spread 100 μL of 0.5% normal melting point agarose (NMA) on a ground glass slide as the base gel and solidify at 4°C for 5-10 min. Next, mix 20 μL of cell suspension with 80 μL of 0.5% low melting point agarose (LMA) and spread this mixture on top of the first gel layer, solidifying at 4°C for 5-10 min as the second gel layer. Then, spread 80 μL of 0.5% LMA as the third gel layer and solidify at 4°C. Immerse the prepared gel layer in pre-chilled cell lysis buffer and lyse at 4°C for 2-3 h or overnight. After lysis, remove the gel layer, rinse with ultrapure water, and place it in a horizontal electrophoresis tank. Add pre-chilled alkaline electrophoresis buffer (pH>13) or neutral electrophoresis buffer (selected according to the detection purpose) and unwind at 4°C in the dark for 20-60 min. Electrophore at 25 V, 300 mA at 4°C for 20-30 min. After electrophoresis, the gel was neutralized three times in neutralization buffer (Tris-HCl, pH 7.5), 10 min each time. The neutralization buffer was discarded, and the gel was air-dried. Fluorescent dye was added (stained in the dark for 5-30 min), and the gel was rinsed with distilled water. Multiple fields of view were randomly selected for photographic analysis under a fluorescence inverted microscope. Results are shown below. Figure 5 As can be seen from the figure, the number of double-strand breaks in DNA increases significantly with the increase of drug concentration.

[0020] Example 3: Effects of different concentrations of androstened 4,6,8(9),13(14)-tetraene-3,11,16-trione on a xenograft lymphoma mouse model Reference Figure 6 The procedure shown involves transferring SR lymphoma cells (5 × 10⁻⁶) to the target cell line. 6 After successful tumor modeling, mice were injected subcutaneously on the right side of their bodies with 200 μL PBS. The tumor-bearing mice were then randomly divided into 5 groups (n=6): normal control group (NC), model group (TC), low-dose group (ATT-L, 2.5 mg / kg), medium-dose group (ATT-M, 5 mg / kg), high-dose group (ATT-H, 10 mg / kg), and cisplatin-positive control group (CDDP, 5 mg / kg). Mice in the ATT and CDDP groups received their respective drugs twice daily, while the NC and TC groups received the same volume of saline. Tumor size and body weight were measured every two days using calipers. Tumor volume (V) was calculated as V = (Long × Width) 2 () / 2, where Long is the tumor length and Width is the tumor width; after the experiment, serum was separated and stored at -80℃, and whole blood was stored at 4℃. Major organs and tumor tissues were collected for H&E staining, immunohistochemistry, and Western blot analysis. A portion of the tumor tissue was immediately and rapidly frozen in liquid nitrogen for subsequent analysis; results of mouse body weight, tumor size, tumor volume, tumor weight, and major organ weight during the experiment are shown in […]. Figure 7-12The figure shows that ATT treatment did not lead to a significant decrease in body weight, while cisplatin caused a severe decrease. We then analyzed the tumors; ATT treatment significantly inhibited tumor growth. We also examined the weight of six major organs. The liver and spleen of the model group mice were enlarged, but ATT treatment normalized these organs. The cisplatin group showed signs of kidney and spleen toxicity.

[0021] 2. Detection of inflammatory factor levels in mouse serum and tumor tissue homogenate The assay was performed using the Yaxin ELISA kit. Before the experiment, the kit was brought to room temperature, 1× wash buffer was prepared, and the standard was diluted according to the instructions. At the same time, biotin-labeled antibody working solution and enzyme complex working solution were prepared at a ratio of 1:100.

[0022] Samples were pretreated according to type. For testing, the required strips were removed, and 100 μL of sample or standard was added to each well. After sealing, the plate was incubated at 37°C for 90 minutes. After discarding the liquid and patting dry, 100 μL of biotin-labeled antibody working solution was added to each well. After sealing, the plate was incubated at 37°C for 60 minutes. The plate was washed 5 times and patted dry, and 100 μL of enzyme complex working solution was added to each well. After sealing, the plate was incubated at 37°C in the dark for 30 minutes. The plate was washed 5 times again and patted dry, and 100 μL of TMB chromogenic reagent was added to each well. The plate was incubated at 37°C in the dark for 10-25 minutes. When a clear blue gradient appeared in the first few wells of the standard, 100 μL of stop solution was added to each well. After mixing, the OD value was immediately read at 450 nm (corrected to 540 or 570 nm). Finally, a standard curve (four-parameter fitting) was plotted based on the standard concentration and OD value. The corresponding concentration was calculated from the sample OD value. If the OD value exceeded the upper limit of the standard curve, the plate needed to be diluted and retested by the dilution factor. The results are shown in [link to results]. Figure 13 , 14 The anti-inflammatory effect of ATT can be seen from the figure. We examined cytokines in serum and tumor tissue. Serum levels of TNF-α, IL-6, IL-1β, and INF-γ were abnormally elevated in the model group rats. For example, TNF-α reached as high as 1013.52 pg / mL. ATT treatment significantly reduced the levels of all these pro-inflammatory cytokines in serum and tumor homogenate. This indicates that ATT effectively alleviates the systemic and local inflammatory response in tumor-bearing mice.

[0023] 3. Immunofluorescence staining of Ki-67 in tumor tissue Tumor tissue was embedded in paraffin and sectioned to a thickness of 4 μm. After dewaxing with xylene and hydration with a gradient of ethanol (100%, 95%, 85%, 75%), antigen retrieval was performed: the sections were immersed in pH 9.0 EDTA-Tris buffer (or pH 6.0 citrate buffer) and autoclaved for 2.5 minutes or in a 95-100°C water bath for 25 minutes. After natural cooling, endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes, followed by washing with PBS. Normal non-immunized animal serum was added for blocking for 10-15 minutes, which was then discarded. Mouse anti-human Ki-67 monoclonal antibody (clone MIB-1, working concentration 1:100-1:200) was added and incubated overnight at 4°C or for 60 minutes at 37°C. After washing with PBS, biological... Incubate with horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (or ready-to-use EnVision / HRP secondary antibody) at 37°C for 30 minutes; wash with PBS, add horseradish peroxidase-labeled streptavidin (if using a biotin system) or directly inject into DAB for staining: prepare fresh DAB staining solution, incubate at room temperature for 3-10 minutes, control the staining intensity under a microscope, and stop the staining with distilled water; counterstain cell nuclei with hematoxylin for 30 seconds to 1 minute, dehydrate with graded ethanol, clear with xylene, and mount with neutral resin. A positive result is a brownish-yellow staining of cell nuclei, as shown in the figure. Figure 15 As can be seen from the figure, the proliferative activity of novel lymphoma tumors significantly decreased with increasing ATT dose.

[0024] 4. Tunel staining of tumor sections Paraffin-embedded tumor tissue sections were routinely dewaxed to water and then incubated with proteinase K working solution (20 μg / mL) at 37°C for 20 min for antigen retrieval. After washing three times with PBS, 50 μL of TUNEL reaction mixture (containing terminal deoxynucleotidyl transferase and fluorescein-labeled dUTP) was added, and the sections were incubated at 37°C for 60 min in the dark, followed by washing with PBS. DAPI staining solution (1 μg / mL) was then added, and the sections were stained at room temperature in the dark for 10 min, washed with PBS, and mounted with anti-fluorescence quenching mounting medium. Multiple fields of view were photographed under a fluorescence microscope. Apoptotic cells showed green fluorescence (TUNEL positive), and cell nuclei showed blue fluorescence. Results are shown in the table below. Figure 16 As can be seen from the figure, with the increase of ATT dose, the number of apoptotic cells in mouse lymphoma tumors increased, which corresponds to our cell experiments.

[0025] 5. Caspase-3 staining of tumor sections Paraffin-embedded tumor tissue sections were dewaxed to water using standard procedures, then subjected to high-pressure antigen retrieval in pH 6.0 citrate buffer for 2.5 min, allowed to cool naturally, and washed with PBS. Endogenous peroxidase was blocked with 3% hydrogen peroxide at room temperature for 10 min, followed by washing with PBS and then blocking with normal goat serum at room temperature for 15 min. The serum was discarded, and rabbit anti-human activated Caspase-3 monoclonal antibody (working concentration 1:200) was added and incubated overnight at 4°C. After washing with PBS, biotin-labeled goat anti-rabbit secondary antibody (1:200) was added and incubated at 37°C for 30 min. After washing with PBS, horseradish peroxidase-labeled streptavidin (1:200) was added and incubated at 37°C for 20 min. After washing with PBS, freshly prepared DAB chromogenic solution was prepared and incubated at room temperature for 3-5 min, controlling the chromogenic intensity under a microscope, and stopping with distilled water. Hematoxylin was counterstained for 30 s, followed by graded ethanol dehydration, xylene clearing, and mounting with neutral resin. Under an optical microscope, the cytoplasm of positive cells appeared brownish-yellow; results are shown below. Figure 17 As can be seen from the figure, the expression level of pro-apoptotic proteins in mouse lymphoma tumor tissue increases with the increase of ATT dose.

[0026] 6. Bcl-2 staining of tumor sections Paraffin-embedded tumor tissue sections were routinely dewaxed to water and then subjected to high-pressure antigen retrieval in pH 9.0 EDTA retrieval solution for 2.5 min. After natural cooling, the sections were washed with PBS. Endogenous peroxidase was blocked with 3% hydrogen peroxide at room temperature for 10 min. After washing with PBS, normal goat serum was added and blocked for 15 min at room temperature. The serum was discarded, and mouse anti-human Bcl-2 monoclonal antibody (working concentration 1:100) was added and incubated overnight at 4°C. After washing with PBS, biotin-labeled goat anti-mouse secondary antibody (1:200) was added and incubated at 37°C for 30 min. After washing with PBS, horseradish peroxidase-labeled streptavidin (1:200) was added and incubated at 37°C for 20 min. After washing with PBS, freshly prepared DAB chromogenic solution was prepared and chromogenic was performed at room temperature for 3-5 min. The chromogenic intensity was controlled under a microscope, and the staining was terminated with distilled water. Hematoxylin was counterstained for 30 s, followed by graded ethanol dehydration, xylene clearing, and mounting with neutral resin. Under an optical microscope, the cytoplasm of positive cells appeared brownish-yellow. Results are shown below. Figure 18 As can be seen from the figure, the expression level of anti-apoptotic proteins in mouse lymphoma tumor tissues decreases with increasing ATT dose.

[0027] In summary, androstenedione-4,6,8(9),13(14)-tetraen-3,11,16-trione exhibits inhibitory effects on acute lymphoblastic leukemia (SR) cell lines, with higher concentrations showing better inhibitory effects. Our study establishes ATT as an effective and safe drug for treating SR lymphoma. We demonstrated its effective inhibition of tumor growth both in vivo and in vitro, offering better safety compared to the clinical drug cisplatin, indicating its potential value in the development of novel drugs for treating leukemia.

Claims

1. Application of androstenedin-4,6,8(9),13(14)-tetraene-3,11,16-trione in the preparation of drugs for treating lymphoma.

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