Application of nobiletin in preparation of medicine for treating triple negative breast cancer

Abstract

The invention discloses application of nobiletin or medicinal salt thereof in preparation of a medicine for treating triple negative breast cancer. In animal experiments, low and high doses of nobiletin have an obvious anti-tumor growth effect on triple-negative breast in-situ tumor mice, have an obvious regulation effect on ferroptosis marker protein in tumor tissues of triple-negative breast cancer model mice, and do not cause obvious toxic and side effects while effectively inhibiting tumors.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to an application of nobiletin in the preparation of a drug for treating triple-negative breast cancer. Background Art

[0002] According to the latest data from the International Agency for Research on Cancer's "Cancer Today," breast cancer has become the second most common type of cancer worldwide, accounting for 11.6% of all cancer cases. Clinically, breast cancer treatment is typically selected based on stage and molecular classification. Among the various breast cancer subtypes, triple-negative breast cancer is more aggressive than other types. Triple-negative breast cancer does not express estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2), meaning its proliferation is independent of hormonal stimulation and typically exhibits a faster cell division rate. Currently, most breast cancer treatments primarily inhibit cancer cell growth by blocking the binding of hormones to receptors, such as with endocrine-based drugs. However, because triple-negative breast cancer lacks these receptors, endocrine-based drugs cannot be used, limiting their effectiveness. Furthermore, the limited treatment options available for triple-negative breast cancer complicate its clinical management. Therefore, the search for new and effective treatment strategies for triple-negative breast cancer is crucial.

[0003] Early clinical studies have shown that the "Tiaogan Bushen Xiaoji" formula can significantly improve the prognosis and prolong the survival of breast cancer patients. While citrus leaves have attracted considerable attention for their active ingredient, nobiletin, there are no reports on its use in triple-negative breast cancer. Summary of the Invention

[0004] The present invention aims to provide an application of nobiletin in the preparation of a medicament for treating triple-negative breast cancer.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a use of nobiletin or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating triple-negative breast cancer.

[0007] The CAS number of the nobiletin is 478-01-3, and its structure is shown below:

[0008]

[0009] The drug for treating triple-negative breast cancer uses nobiletin or a pharmaceutically acceptable salt thereof as the sole active ingredient.

[0010] The triple-negative breast cancer refers to MDA-MB-231 cells.

[0011] The pharmaceutically acceptable salt is an acid addition salt formed by nobiletin and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0012] The second aspect of the present invention provides a pharmaceutical preparation prepared from nobiletin or a pharmaceutically acceptable salt thereof and medically acceptable excipients.

[0013] The dosage form of the pharmaceutical preparation is selected from liquid medicine, tablet or capsule.

[0014] The pharmaceutical preparation can be administered orally, intravenously, or intraperitoneally.

[0015] The third aspect of the present invention provides a pharmaceutical composition prepared from nobiletin or a pharmaceutically acceptable salt thereof and other drugs for treating triple-negative breast cancer.

[0016] The main ingredient of the drug for treating triple-negative breast cancer is nobiletin, which comes from citrus leaves and has the effect of soothing the liver and regulating qi.

[0017] Nobiletin, a polymethoxylated flavonoid and a major component of citrus leaves, has been shown to induce cell cycle arrest and cell death in various cancer cell types. Notably, nobiletin has been reported to induce ferroptosis in human melanoma cells. However, whether nobiletin can induce ferroptosis in breast cancer cells remains unexplored.

[0018] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0019] The present invention provides an effective alternative for the treatment of triple-negative breast cancer. The present invention found that nobiletin has a significant inhibitory effect on the proliferation ability of triple-negative breast cancer cells from different sources, and has a significant promoting effect on the ferroptosis process. Nobiletin can significantly upregulate the expression of AKR1C1, and further proves that AKR1C1 is the direct binding target of nobiletin. The nobiletin-AKR1C1 complex can promote the ubiquitination degradation of GPX4, thereby promoting ferroptosis. In animal experiments, low and high doses of nobiletin have a significant anti-tumor growth effect on mice with triple-negative breast in situ tumors, and have a significant regulatory effect on ferroptosis marker proteins in the tumor tissue of triple-negative breast cancer model mice. It effectively inhibits tumors without causing obvious toxic side effects. The present invention uses natural products and is safe for use.

[0020] The present invention illustrates the safety of nobiletin from the perspectives of pharmacokinetics, in vivo hepato-renal toxicity, and blood biochemical testing. Cardiac, liver, and kidney function in animals treated with nobiletin were tested, and H&E staining was used for observation. Nobiletin had no significant effect on the heart, liver, and kidneys, and Swiss ADME prediction results further confirmed that nobiletin has good drugability. A comparison of the effects of luteolin, robiniaquinoxaline, sesamin, and nobiletin on triple-negative breast cancer cell viability revealed that nobiletin possessed the best anti-tumor proliferation activity, indicating that nobiletin has a strong anti-tumor proliferation ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the transcriptomic sequencing results of nobiletin in the treatment of triple-negative breast cancer.

[0022] Figure 2 This is a schematic diagram of the effect of nobiletin on the proliferation ability of triple-negative breast cancer cells.

[0023] Figure 3 This is a schematic diagram of the effects of nobiletin on the ferroptosis phenotype and molecular pathways of triple-negative breast cancer cells.

[0024] Figure 4 This is a schematic diagram of the anti-tumor effect of nobiletin on triple-negative breast cancer orthotopic model mice.

[0025] Figure 5 Schematic diagram of the effects of nobiletin on AKR1C1 in triple-negative breast cancer cells, the interaction between nobiletin and AKR1C1, and the ubiquitination and degradation of GPX4 mediated by the complex formed by nobiletin and AKR1C1.

[0026] Figure 6 This is a safety evaluation of nobiletin in the treatment of triple-negative breast cancer, and a schematic diagram comparing the inhibitory effects of luteolin, robinia pigment, sesamin, and nobiletin on triple-negative breast cancer cell viability. DETAILED DESCRIPTION

[0027] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0028] Example 1

[0029] Transcriptomic sequencing of nobiletin in the treatment of triple-negative breast cancer

[0030] MDA-MB-231 cells, a triple-negative breast cancer cell line (from the Chinese Academy of Sciences Cell Bank), were cultured with nobiletin at concentrations of 0 and 40 μM for 48 hours. Total RNA was then extracted and quality assessed. The RNA was fragmented and reverse transcribed to generate complementary DNA. The complementary DNA was blunt-end repaired, tailed, and ligated with adapters. Finally, the PCR amplification products were sequenced by Illumina.

[0031] The results are as follows Figure 1 As shown, Figure 1 This is a schematic diagram of transcriptomic sequencing results of Nobiletin in the treatment of triple-negative breast cancer. A is a schematic diagram of the top 20 enriched pathways, B is a schematic diagram of the chord diagram of the enriched significantly changed genes, and C is a schematic diagram of the volcano diagram of the enriched significantly changed genes. As can be seen from the figure, after Nobiletin intervened in triple-negative breast cancer cells, the pathway was enriched in ferroptosis ( Figure 1 As shown in A in the middle, it shows that nobiletin can affect the ferroptosis of triple-negative breast cancer cells. After intervention with nobiletin, the expression of ferroptosis-related gene AKR1C1 (Aldo-Keto Reductase Family 1 Member C1) was significantly increased ( Figure 1 (B and C in the middle), indicating that nobiletin can promote ferroptosis by affecting the expression of AKR1C1.

[0032] Example 2

[0033] Pharmacodynamics of nobiletin on triple-negative breast cancer

[0034] 1. Effect of Nobiletin on the Proliferation of Triple-Negative Breast Cancer Cells

[0035] CCK8 assay: Triple-negative breast cancer cell line MDA-MB-231 cells and mouse breast cancer cell line 4T1 were co-cultured with 0-1000 μM concentration gradient of nobiletin for 24, 48, and 72 hours, respectively. After the incubation period, cell proliferation was detected using CCK8 reagent, and the absorbance was measured at 450 nm using a microplate reader. The IC at 48 hours was measured using Graph Pad. 50 value.

[0036] Plate cloning assay: Approximately 1,000 cells (triple-negative breast cancer cell line MDA-MB-231 and mouse breast cancer cell line 4T1) were co-cultured with nobiletin for 10 days. The cells were then fixed with 4% paraformaldehyde and stained with crystal violet solution. After the culture plate dried, the cells were imaged.

[0037] EDU experiment: 5×10 4Cells (triple-negative breast cancer cell line MDA-MB-231 and mouse breast cancer cell line 4T1) were cocultured with nobiletin for 48 hours. The cells were incubated at 37°C for 2 hours for EdU labeling. The acetylene group on EdU reacts covalently with a fluorescently labeled small molecule azide probe (Azide Alexa Fluor 555) catalyzed by monovalent copper ions, forming a stable triazole ring with an excitation maximum at 555 nm and an emission maximum at approximately 565 nm. After incubation, the cells were fixed with 4% paraformaldehyde and washed with 3% BSA. Next, the cells were treated with 0.3% Triton X-100 and washed again with PBS containing 3% BSA. After incubation at room temperature for 30 minutes in the dark, the click reaction solution was added. To assess cell proliferation, cell nuclei were stained with Hoechst 33342 and the stained cells were observed under a fluorescence microscope.

[0038] The results are as follows Figure 2 As shown, Figure 2Figure 1 is a schematic diagram of the effects of nobiletin on the proliferation of triple-negative breast cancer cells. Figure A is a CCK8 assay showing cell viability in MDA-MB-231 cells treated with 0-1000 μM nobiletin. As can be seen, MDA-MB-231 cell viability decreased with increasing nobiletin concentration and duration. Figure B is a CCK8 assay showing cell viability in 4T1 cells treated with 0-1000 μM nobiletin. As can be seen, 4T1 cell viability decreased with increasing nobiletin concentration and duration. Figure C is a schematic diagram showing plate cloning assay showing colony formation in MDA-MB-231 and 4T1 cells treated with 0, 10, 20, and 40 μM nobiletin. As can be seen, nobiletin inhibits colony formation in both MDA-MB-231 and 4T1 breast cancer cell lines. D is a schematic diagram of the statistical analysis of colony formation in 4T1 cells treated with nobiletin at concentrations of 0, 10, 20, and 40 μM. E is a schematic diagram of the statistical analysis of colony formation in MDA-MB-231 cells treated with nobiletin at concentrations of 0, 10, 20, and 40 μM. As shown in the figure, nobiletin inhibits colony formation in triple-negative breast cancer cell lines with statistically significant differences. F is a schematic diagram of fluorescence staining in 4T1 cells treated with nobiletin at concentrations of 0, 10, 20, and 40 μM. As shown in the figure, EDU expression in 4T1 cells decreases with increasing nobiletin concentration, and cell proliferation is weakened. G is a schematic diagram of fluorescence staining in MDA-MB-231 cells treated with nobiletin at concentrations of 0, 10, 20, and 40 μM. As shown in the figure, EDU expression in MDA-MB-231 cells decreases with increasing nobiletin concentration, and cell proliferation is weakened. H is a schematic diagram of the statistical analysis of EDU fluorescence staining of 4T1 cells after intervention with nobiletin at concentrations of 0, 10, 20, and 40 μM. I is a schematic diagram of the statistical analysis of EDU fluorescence staining of MDA-MB-231 cells after intervention with nobiletin at concentrations of 0, 10, 20, and 40 μM. It can be seen from the figure that nobiletin at concentrations of 0, 10, 20, and 40 μM can inhibit the fluorescence intensity of EDU, and there are statistical differences.

[0039] In summary, with the increase of nobiletin concentration and the extension of treatment time, cell viability decreased significantly ( Figure 2 IC of triple-negative breast cancer cell line MDA-MB-231 50 The value was 483.12 μM, while the IC 50 The value was 235.49 μM. Based on the above results, 0, 10, 20 and 40 μM of nobiletin were selected as non-toxic concentrations in subsequent experiments. Further experiments showed that nobiletin significantly inhibited breast cancer cell colony formation and cell proliferation ( Figure 2 In addition, EDU experiments showed that nobiletin treatment significantly inhibited DNA replication in breast cancer cells, and the effect was more pronounced at high concentrations ( Figure 2 The above shows that nobiletin effectively inhibits the proliferation of triple-negative breast cancer cell lines in triple-negative breast cancer cells. Nobiletin effectively inhibits the proliferation of breast cancer cells by inhibiting DNA replication and inducing ferroptosis.

[0040] 2. Effect of Nobiletin on Ferroptosis in Triple-Negative Breast Cancer Cells

[0041] Electron microscopy of mitochondria: After incubating the triple-negative breast cancer cell line MDA-MB-231 with nobiletin for 48 hours, cells were fixed at room temperature using electron microscopy fixative. The fixative was removed and fresh electron microscopy fixative was added. The cell pellets were then dispersed, resuspended, and fixed for another 30 minutes at room temperature in the dark. The cells were fixed with osmium tetroxide and PBS for 2 hours and washed three times with buffer. The cells were then dehydrated using a gradient of ethanol (50%-70%-80%-90%-95%-100%-100%) for 15 minutes each, permeabilized overnight with embedding medium (acetone:812 embedding medium = 1:1), and polymerized (60°C for 48 hours) for embedding. Finally, the samples were double-stained with 2% uranyl acetate in water and lead citrate (15 minutes each), and mitochondrial morphology was observed using transmission electron microscopy.

[0042] Ferrous ion staining experiment: Approximately 3,000 triple-negative breast cancer cell lines MDA-MB-231 and 4T1 cells were seeded into each well and treated with nobiletin for 48 hours. 100 μl of divalent ferric staining solution (Meilun Bio, MA0647) was then added to ensure that the monolayer of cells was completely immersed. After mixing, the cells were incubated and observed. This probe can bind to ferrous ions (Fe 2+ ) and exhibit orange-red fluorescence (excitation / emission wavelength = 543 / 580 nm).

[0043] Western blot analysis: After co-culture of triple-negative breast cancer cell lines MDA-MB-231 and 4T1 with nobiletin for 48 hours, cells were lysed using a lysis buffer containing protease and phosphatase inhibitors (Protein Lysate, New Saimei, WB2040) and heated to 100°C. 10% and 12.5% ​​gels were prepared using a Color PAGE rapid gel preparation kit and subjected to electrophoresis. The membranes were transferred to polyvinylidene fluoride (PVDF) membranes and blocked for 2 hours. Specific antibodies (β-Actin, Nrf-2, SLC7A11, and GPX4) were then added and incubated overnight. Secondary antibodies, either anti-rabbit IgG or anti-mouse IgG, were then added for incubation. The PVDF membranes were soaked in ECL solution and visualized using an automated chemiluminescence imaging system.

[0044] The results are as follows Figure 3 As shown, Figure 3 Figure 1 is a schematic diagram showing the effects of nobiletin on the ferroptosis phenotype and molecular pathways in triple-negative breast cancer cells. A is a schematic diagram of electron microscopy observations of mitochondrial changes in MDA-MB-231 cells treated with nobiletin at a concentration of 40 μM. The figure shows that nobiletin treatment leads to a reduction in mitochondrial size, increased membrane density, loss of cristae, and even outer membrane disruption. B is a schematic diagram showing the changes in ferrous ion fluorescence staining in MDA-MB-231 and 4T1 cells treated with nobiletin at concentrations ranging from 0 to 40 μM. The figure shows that ferrous ion fluorescence intensity increases with increasing nobiletin concentration, indicating that nobiletin can increase ferrous ion concentrations in breast cancer cells. C is a schematic diagram showing the statistical analysis of ferrous ion fluorescence changes in MDA-MB-231 and 4T1 cells treated with nobiletin at concentrations ranging from 0 to 40 μM. The figure shows that nobiletin significantly increases ferrous ion concentrations. D is a schematic diagram of Western-blot analysis of the effects of nobiletin on ferroptosis-related proteins in MDA-MB-231 and 4T1 cells. The figure shows that after intervention with nobiletin, the expression of Nrf-2, SLC7A11, and GPX4 was significantly downregulated. E is a schematic diagram of statistical analysis of Western-blot analysis of the effects of nobiletin on ferroptosis-related proteins. The figure shows that nobiletin reduces the expression of Nrf-2, SLC7A11, and GPX4, promoting ferroptosis.

[0045] Example 3

[0046] Effects of nobiletin on proliferation and ferroptosis in mice bearing triple-negative breast orthotopic tumors

[0047] To establish an orthotopic mouse breast tumor model, 4T1 mouse breast cancer cells were pre-cultured overnight in serum-free medium after full growth. The digested cells were resuspended in PBS and kept on ice. Mice were anesthetized with Zolpidem 50 and xylazine (150 μl / 20 mg). The skin around the fourth nipple was shaved and disinfected with iodine. Forceps were used to lift the skin adjacent to the fourth nipple on the right side, and an equal number of 4T1 cells were injected into the subfat pad using an insulin needle. One week later, nobiletin was administered orally to the mice. The mice were divided into three groups: a control group (administered PBS); a low-dose nobiletin group (30 mg / kg); and a high-dose nobiletin group (60 mg / kg), with feeding every two days. Tumor size and volume were measured regularly throughout the gavage period, and the overall health of the mice, including weight change, dietary habits, and activity, was observed. The experiment strictly adhered to the 3R principle (replacement, reduction, and optimization). The effects of nobiletin on tumors were assessed by measuring tumor size, proliferation, and ferroptosis-related markers.

[0048] The results are as follows Figure 4 As shown, Figure 4The following is a schematic diagram of the anti-tumor effect of nobiletin in an orthotopic triple-negative breast cancer mouse model. Figure A shows the gross tumor growth of the control, low-dose nobiletin, and high-dose nobiletin groups after oral administration. The figure shows that nobiletin inhibits tumor growth, with higher concentrations showing greater inhibitory effects. Figure B shows the statistical analysis of tumor weights in the control, low-dose nobiletin, and high-dose groups. The figure shows that the high-dose nobiletin group significantly slowed tumor growth. Figure C shows the weight monitoring of mice in the control, low-dose nobiletin, and high-dose groups. The figure shows that nobiletin had no significant effect on mouse body weight. Figure D shows H&E staining and immunohistochemistry for Ki-67, Nrf-2, and SLC7A11 in the control, low-dose nobiletin, and high-dose nobiletin groups. The figure shows that after nobiletin treatment, tumor cell nuclear size increased, chromatin content increased, and cell arrangement became disorganized in the control group. However, after treatment with nobiletin, tumor cell nuclear size was significantly reduced, cell arrangement became more orderly, and Ki-67, a proliferation marker, decreased, as did Nrf-2 and SLC7A11. This suggests that nobiletin promotes ferroptosis in mice with triple-negative breast cancer. Figure E shows a statistical analysis of Ki-67 percentages in mice treated with the blank, low-dose, and high-dose nobiletin groups. This figure demonstrates that nobiletin treatment inhibits the proliferation of orthotopic breast tumors. Figure F shows a statistical analysis of Nrf-2 percentages in mice treated with the blank, low-dose, and high-dose nobiletin groups. This figure demonstrates that nobiletin treatment significantly inhibited Nrf-2 expression. Figure G shows a statistical analysis of SLC7A11 percentages in mice treated with the blank, low-dose, and high-dose nobiletin groups. This figure demonstrates that nobiletin treatment significantly inhibited SLC7A11 expression.

[0049] Nobiletin has a significant inhibitory effect on the size of tumors ( Figure 4 A and B in the figure), but had no significant effect on their body weight ( Figure 4 Immunohistochemical analysis showed that nobiletin treatment could reduce the tumor proliferation index (Ki-67), especially at higher concentrations ( Figure 4 In the control group, the tumor cell nuclear volume increased, the chromatin content increased, and the cell arrangement was disordered. However, after nobiletin treatment, the tumor cell nuclear volume decreased significantly and the cell arrangement became more orderly ( Figure 4 In addition, after nobiletin treatment, the expression levels of Nrf-2 and SLC7A11 in tumor cells decreased, suggesting that nobiletin may inhibit the proliferation of triple-negative breast cancer cells by inducing ferroptosis ( Figure 4 (as shown in F and G).

[0050] Example 4

[0051] Nobiletin promotes GPX4 ubiquitination and degradation by interacting with AKR1C1 (Aldo-Keto Reductase Family 1 Member C1).

[0052] The expression of AKR1C1 in triple-negative breast cancer cells after nobiletin intervention was detected by Western-blot (the steps were the same as those in Example 2).

[0053] Molecular docking: The AKR1C1 protein structure was obtained from UniProt (ID: Q04828), and the nobiletin structure was obtained from PubChem (ID: 72344). Molecular docking analysis between AKR1C1 and nobiletin was performed using UCSF Chimera and AutoDock Vina.

[0054] Surface Plasmon Resonance: The binding kinetics of recombinant human AKR1C1 and nobiletin were measured at 25°C using a Biacore 8K SPR instrument. The flow buffer consisted of PBS, 0.005% Tween-20, and 5% DMSO. The flow cell of a CM5 sensor chip was first activated with a mixture of N-hydroxysuccinimide and N-ethyl-N'-(3-diethylaminopropyl)carbodiimide. AKR1C1 was diluted to 20 μg / mL in 10 mM sodium acetate buffer and immobilized on the CM5 chip to obtain a response signal. Unbound active sites on the chip were blocked with 1 M ethanolamine. Nobiletin (at concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM) was sequentially injected into the flow cell and allowed to bind to the immobilized target protein. Binding signals were measured using a multi-cycle approach. Binding affinity was calculated using Biacore 8K evaluation software.

[0055] Immunoprecipitation technique: about 1×10 6 293T cells transfected with Flag-AKR1C1 and Myc-GPX4 were co-cultured with nobiletin. The cells were lysed using IP lysis buffer containing protease inhibitors. After centrifugation, 50 μL of the supernatant was collected for quantitative analysis of cellular protein content. The remaining supernatant was mixed with Flag magnetic beads that had been pre-washed overnight with IP lysis buffer. The beads were collected using a magnetic rack to remove unbound sample. The beads were thoroughly washed with IP lysis buffer, and the protein was eluted from the beads and denatured. The beads were separated again using a magnetic rack, and the supernatant containing the target antigen was retained. The expression levels of Flag, Myc, and vinculin were detected by Western blot analysis.

[0056] In vivo ubiquitination assay: 293T cells transfected with Myc-GPX4 were co-cultured with nobiletin for 48 hours. 10 μM MG132 was added and incubated for another 4 hours. IP lysis buffer containing protease and phosphatase inhibitors was added to the cells and lysed on ice for 20 minutes. After centrifugation, 50 μL of the supernatant was collected for quantification of cellular protein content. The remaining protein-containing supernatant was added to Myc antibody-antibody magnetic beads that had been pre-washed with IP lysis buffer. The next day, the beads were collected using a magnetic rack to remove unbound sample. The beads were washed with IP lysis buffer, and the protein was eluted from the beads and denatured. The beads were separated again using a magnetic rack, and the supernatant containing the ubiquitin antibody was retained. Ubiquitin expression levels were assessed by Western blot analysis.

[0057] The results are as follows Figure 5 As shown, Figure 5Schematic diagrams showing the effects of nobiletin on AKR1C1 in triple-negative breast cancer cells, the interaction between nobiletin and AKR1C1, and the effect of the nobiletin-AKR1C1 complex on GPX4 ubiquitination and degradation. Figure A shows AKR1C1 protein expression in triple-negative breast cancer cells after treatment with nobiletin at concentrations of 0, 10, 20, and 40 μM. As can be seen, AKR1C1 expression increased after treatment with nobiletin. Figure B shows a statistical analysis of the differences in AKR1C1 protein expression in MDA-MB-231 cells after treatment with different concentrations of nobiletin. As can be seen, AKR1C1 expression increased with statistical significance after treatment with tangerine peel. Figure C shows a statistical analysis of the differences in AKR1C1 protein expression in 4T1 cells after treatment with different concentrations of nobiletin. As can be seen, AKR1C1 expression increased with statistical significance after treatment with tangerine peel. D is a schematic diagram of the surface plasmon resonance (SPR) results for nobiletin and AKR1C1 protein. The figure shows a binding affinity constant (KD value) of 6.50e-06 M. A smaller KD value indicates a stronger interaction between nobiletin and AKR1C1, reflecting a higher binding affinity. E is a schematic diagram of the molecular docking of nobiletin and AKR1C1. The figure shows strong binding between nobiletin and AKR1C1, with a binding energy of -8.187 kcal / mol. F is a schematic diagram of the co-IP assay results after nobiletin treatment of 293T cells transfected with Flag-AKR1C1 and Myc-GPX4. In the blank control group, AKR1C1 did not bind to GPX4, while in the nobiletin group, AKR1C1 and GPX4 did bind, indicating that nobiletin promotes the interaction between AKR1C1 and GPX4. Furthermore, the input group shows increased AKR1C1 expression and decreased GPX4 expression after nobiletin treatment. G is a schematic diagram of the ubiquitination experiment of 293T cells transfected with Myc-GPX4 intervened by nobiletin. The ubiquitination and degradation of GPX4 increased in the nobiletin intervention group, indicating that nobiletin can promote the ubiquitination and degradation of GPX4 and ultimately promote ferroptosis.

[0058] As can be seen from the figure, nobiletin can increase the expression of AKR1C1, and the expression increases with increasing concentration ( Figure 5 Molecular docking revealed that nobiletin has a strong binding ability with AKR1C1, with a binding energy of -8.187 kcal / mol ( Figure 5 Surface plasmon resonance analysis further revealed that the binding affinity constant (KD value) was 6.50e-06 M. A smaller KD value indicated a stronger interaction between nobiletin and AKR1C1, reflecting a higher binding affinity ( Figure 5As shown in D). Both molecular docking and surface plasmon resonance analysis showed that nobiletin had a strong binding ability with AKR1C1. Through co-IP, it was found that after nobiletin intervention, AKR1C1 and GPX4 were significantly bound, and the binding of nobiletin and AKR1C1 promoted the ubiquitination degradation of GPX4 ( Figure 5 The above results indicate that nobiletin can significantly increase the expression of AKR1C1 and further verify that AKR1C1 is the binding target of nobiletin. The nobiletin-AKR1C1 complex can promote the ubiquitination and degradation of GPX4, thereby promoting ferroptosis.

[0059] Example 5

[0060] Safety evaluation of nobiletin in the treatment of triple-negative breast cancer

[0061] Cardiac, liver, and kidney function testing and analysis: After collecting whole blood samples, allow them to settle and centrifuge them for testing. Prepare the following reagents: ALT working solution (specific ingredients: L-alanine, α-ketoglutarate, NADH, pyridoxal phosphate, Tris buffer); AST working solution (L-aspartate, α-ketoglutarate, NADH, malate dehydrogenase (MDH), Tris buffer); BUN working solution (urease, glutamate dehydrogenase (GLDH), NADH, α-ketoglutarate, ADP); Cr working solution (creatinase, creatinase, sarcosine oxidase, peroxidase (POD), chromogen (TOOS); CK working solution (creatine phosphate, ADP, hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), NADP⁺, AMP buffer). Mix serum and working solution according to the kit ratio (5 μl serum + 200 μl reagents) and incubate at 37°C for 30 minutes. After setting the appropriate test parameters on the automated biochemical analyzer, load the sample. The instrument will automatically measure and export the results after the test is completed. The final test will reflect the relevant indicators of heart, liver and kidney function.

[0062] H&E staining: Tumor tissue was fixed in 10% neutral formalin for 24 hours, then directly dehydrated and embedded in 75%, 80%, 90%, and 100% ethanol for 1 hour, followed by 100% ethanol for 1 hour. Tissue clearing was performed using a 1:1 mixture of ethanol and xylene for 2 hours, followed by 40 minutes each of xylene (I) and xylene (II). The clearing agent was then replaced by wax immersion using a 1:1 mixture of xylene and paraffin for 2 hours, followed by 1 hour of paraffin (I), and 2 hours of paraffin (II). Embedding: Melted pure wax was poured into a wax box. Because the bottom of the box was in contact with the ice box, the wax at the bottom solidified first. The tissue was held upright at the bottom with forceps and the box was continuously filled to facilitate the microtome's grip on the wax block. Embedding was completed after the wax block was completely solidified. Embedded tissue blocks were sliced ​​on a microtome to create 3-5 μm wax strips. These strips were then unfolded in a slide bleaching machine. The sections were then placed on polylysine-treated slides, numbered, and baked at 65°C for 2 hours. Dewaxing was performed with 1 mL of xylene and 1 mL of ethanol for 2 hours. Staining was performed with 2 mL of hematoxylin for 10 minutes, followed by differentiation with 2 mL of 0.7% hydrochloric acid alcohol for several seconds. The sections were rinsed with tap water until a blue color developed. Staining was then performed with alcohol eosin for 30 seconds, followed by treatment with ethanol and then carbolic xylene, and finally, mounted with neutral gum. Scanning was performed using a scanning microscope.

[0063] Pharmacokinetic prediction: The early pharmacokinetic properties of nobiletin were evaluated and analyzed using the SwissADME online server (www.swiss-adme.ch) through computational-assisted prediction of pharmacokinetic properties in drug design. The CCK8 assay was used to investigate the effects of 0-1000 μM luteolin, robinia luteolin, sesamin, and nobiletin on triple-negative breast cancer cells and their effects on cell proliferation.

[0064] The results are as follows Figure 6 As shown in Table 1, Figure 6 This is a safety evaluation of nobiletin in the treatment of triple-negative breast cancer, as well as a schematic diagram comparing the inhibitory effects of luteolin, robinia pigment, sesamin, and nobiletin on triple-negative breast cancer cell viability.

[0065] Table 1 is a schematic diagram of the early pharmacokinetic evaluation and analysis of nobiletin by the Swiss ADME online server (www.swiss adme.ch), which shows that nobiletin complies with the Lipinski drug similarity rule, has a high gastrointestinal absorption rate, and does not inhibit cytochrome P450 enzymes, indicating that it has good pharmacokinetic properties.

[0066] Table 1

[0067]

[0068] Among them, A is a schematic diagram of H&E staining of the heart, liver, and kidney after Nobiletin intervention in mice with triple-negative breast cancer orthotopic tumors. As can be seen from the figure, there is no significant difference in staining between the blank group, the low-dose Nobiletin group, and the high-dose Nobiletin group, indicating that Nobiletin has no significant effect on the heart, liver, and kidney of mice. B is a schematic diagram of the statistical analysis of Cr after Nobiletin intervention in mice. As can be seen from the figure, Nobiletin has no significant effect on Cr. C is a schematic diagram of the statistical analysis of ALT after Nobiletin intervention in mice. As can be seen from the figure, Nobiletin has no significant effect on ALT. D is a schematic diagram of the statistical analysis of CK after Nobiletin intervention in mice. As can be seen from the figure, Nobiletin has no significant effect on CK. E is a schematic diagram of the statistical analysis of AST after Nobiletin intervention in mice. As can be seen from the figure, Nobiletin has no significant effect on AST. F is a schematic diagram of the statistical analysis of BUN after Nobiletin intervention in mice. As can be seen from the figure, Nobiletin has no significant effect on BUN. G is a schematic diagram of the results of the inhibitory effects of luteolin, robiniaxin, sesamin, and nobiletin on MDA-MB-231 cell viability. As can be seen from the figure, compared with luteolin, robiniaxin, and sesamin, nobiletin significantly inhibited the growth viability of MDA-MB-231 cells and had a stronger ability to inhibit proliferation. H is a schematic diagram of the results of the inhibitory effects of luteolin, robiniaxin, sesamin, and nobiletin on 4T1 cell viability. As can be seen from the figure, compared with luteolin, robiniaxin, and sesamin, nobiletin significantly inhibited the growth viability of 4T1 cells and had a stronger ability to inhibit proliferation.

[0069] The literature (Phytomedicine.2023;110:154610.) studied the anti-cancer effect of nobiletin on breast cancer and confirmed that it has a significant inhibitory effect on liver metastasis of breast cancer. This literature is about the inhibitory effect of nobiletin on liver metastasis of ER PR-positive breast cancer, but in the present invention, nobiletin promotes ferroptosis of triple-negative breast cancer. Different mechanisms: In the literature, nobiletin inhibits liver metastasis of ER PR-positive breast cancer by inhibiting the JNK / ERK pathway, but in the present invention, nobiletin promotes ferroptosis of triple-negative breast cancer cells through AKR1C1-mediated ubiquitination and degradation of GPX4. Therefore, the present invention also conducted cell proliferation assay experiments on other subtypes of breast cancer using nobiletin, and the results are as follows: Figure 6 As shown in Figure 1, nobiletin had no significant effect on the proliferation of human breast ductal carcinoma BT-474 cells, human breast cancer cells SK-BR-3 cells, and human breast cancer cells AU565 cells, indicating that nobiletin has a strong inhibitory effect on the proliferation of triple-negative breast cancer cell line MDA-MB-231 cells.

[0070] The results of H&E staining of the heart, liver and kidney showed that nobiletin had no obvious toxicity to the heart, liver and kidney after intervention ( Figure 6As shown in middle A). After nobiletin intervention in mice with breast cancer in situ, there was no significant difference in Cr, ALT, AST, CK, and BUN compared with the control group ( Figure 6 Comparison of the effects of different Chinese herbal monomers, including luteolin, robiniaxin, sesamin, and nobiletin, on triple-negative breast cancer cell viability revealed that nobiletin possessed the best anti-tumor proliferation activity compared to luteolin, robiniaxin, and sesamin, particularly at higher concentrations, significantly inhibiting cell proliferation ( Figure 6 In MDA-MB-231 cells, at a concentration of 100 μM, the viability of luteolin on triple-negative breast cancer cells was 79.58%, the viability of acaciatin on triple-negative breast cancer cells was 75.91%, the viability of sesamin on triple-negative breast cancer cells was 98.27%, and the viability of nobiletin on triple-negative breast cancer cells was 69.75%. The above results indicate that at a concentration of 100 μM, the viability of nobiletin on MDA-MB-231 cells was the lowest, and the cell proliferation ability was significantly inhibited. In 4T1 cells, at a concentration of 100 μM, the activity value of luteolin on triple-negative breast cancer cells was 86.77%, the activity value of acacia yellow on triple-negative breast cancer cells was 82.79%, the activity value of sesamin on triple-negative breast cancer cells was 99.01%, and the activity value of nobiletin on triple-negative breast cancer cells was 70.29%. The above shows that at a concentration of 100 μM, the activity value of nobiletin on 4T1 cells was the lowest, and the cell proliferation ability was significantly inhibited. The above shows that nobiletin can significantly inhibit the proliferation ability of triple-negative breast cancer cells and can be used as a drug for the treatment of triple-negative breast cancer.

[0071] The present invention illustrates the safety of nobiletin from aspects of pharmacokinetics, in vivo liver and kidney toxicity, blood biochemical detection and the like.

[0072] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present invention can make slight changes or modifications to equivalent embodiments using the above-mentioned technical contents without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. Use of nobiletin or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating triple-negative breast cancer.

2. Use of nobiletin or a pharmaceutically acceptable salt thereof according to claim 1 in preparing a drug for treating triple-negative breast cancer, characterized in that: The drug for treating triple-negative breast cancer uses nobiletin or a pharmaceutically acceptable salt thereof as the sole active ingredient.

3. Use of nobiletin or a pharmaceutically acceptable salt thereof according to claim 1 in preparing a drug for treating triple-negative breast cancer, characterized in that: The triple-negative breast cancer refers to MDA-MB-231 cells.

4. Use of nobiletin or a pharmaceutically acceptable salt thereof according to claim 1 in preparing a drug for treating triple-negative breast cancer, characterized in that: The pharmaceutically acceptable salt is an acid addition salt formed by nobiletin and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

5. A pharmaceutical preparation, characterized in that The invention is prepared from nobiletin or its pharmaceutical salt and medically acceptable auxiliary materials.

6. The pharmaceutical preparation according to claim 5, characterized in that The dosage form of the pharmaceutical preparation is selected from liquid medicine, tablet or capsule.

7. The pharmaceutical preparation according to claim 5, characterized in that The pharmaceutical preparation can be administered orally, intravenously, or intraperitoneally.

8. A pharmaceutical composition, characterized in that The invention is prepared from nobiletin or its pharmaceutical salt and other drugs for treating triple-negative breast cancer.

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