Use of piperlongumine in the preparation of anticancer drugs

Abstract

The application discloses application of piperlongumine in preparation of anticancer drugs. The application first proposes and verifies that piperlongumine can induce ferroptosis by regulating a Dcp1a related pathway, significantly inhibit cancer cell activity, and then inhibit drug-resistant breast cancer progression. Meanwhile, the combination of piperlongumine and tamoxifen can effectively improve the treatment effect of tamoxifen on drug-resistant breast cancer, and provides a new direction for the treatment of tamoxifen-resistant breast cancer.

Description

Technical Field

[0001] This invention relates to the field of natural medicinal chemistry, and more particularly to the application of sanshool in the preparation of anticancer drugs. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, and its disease burden continues to increase. According to an analysis released by the International Agency for Research on Cancer (IARC) of the World Health Organization in 2025, there were approximately 2.3 million new cases of breast cancer and 670,000 deaths globally in 2022. Model predictions show that if current trends continue, by 2050, the annual number of new cases and deaths will climb to 3.2 million and 1.1 million respectively, with a particularly severe impact on developing countries. Among the various subtypes of breast cancer, hormone receptor-positive / HER2-negative is the most common, accounting for approximately 70% of all cases.

[0003] Tamoxifen is a first-line endocrine therapy for this type of breast cancer, belonging to the class of selective estrogen receptor modulators. Its core mechanism involves competitively binding to the estrogen receptor (ERα) within tumor cells, altering the receptor's conformation and thus preventing the effective activation of genes that promote cancer cell growth, thereby inhibiting tumor progression. However, the emergence of tamoxifen resistance is a major clinical challenge. Studies show that approximately 30%-40% of estrogen receptor-positive patients develop primary or secondary resistance. Once resistance develops and leads to tumor recurrence and metastasis, the five-year survival rate significantly decreases to approximately 20%-30%.

[0004] Sanshodiol is a natural compound isolated from the jujube plant *Zanthoxylum bungeanum*, but research on it is still limited and its pharmacological effects are largely unclear. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide the application of sanshool in the preparation of breast cancer treatment drugs, especially tamoxifen-resistant breast cancer, either alone or in combination with tamoxifen.

[0006] Technical solution: The application of sanshool as described in this invention in the preparation of anticancer drugs.

[0007] Preferably, the CAS number of the sanshool is 54854-91-0.

[0008] Preferably, the application is in the preparation of anti-breast cancer drugs; more preferably, the breast cancer is tamoxifen-resistant breast cancer.

[0009] Preferably, the drug contains sanshool or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

[0010] Preferably, the drug further contains tamoxifen as an active ingredient; more preferably, the mass ratio of senna to tamoxifen in the drug is 1~2:1.

[0011] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0012] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention proposes and verifies for the first time that salinomyol can induce ferroptosis by regulating the Dcp1a-related pathway, significantly inhibit the activity of cancer cells, and thus inhibit the progression of drug-resistant breast cancer; the combined use of salinomyol and tamoxifen can effectively improve the therapeutic effect of tamoxifen on drug-resistant breast cancer, providing a new direction for the treatment of tamoxifen-resistant breast cancer. Attached Figure Description

[0014] Figure 1 Figure 1 shows the IC50 values ​​of MCF7 cells and MCF7 / TR cells after treatment with salinomycin. Figure 2 The image shows the results of MCF7 / TR cell viability assay after treatment. Figure 3 The image shows the results of the MCF7 / TR cell proliferation assay after treatment. Figure 4 This is a graph showing the quantitative analysis results of the MCF7 / TR cell proliferation assay after treatment. Figure 5 The image shows the results of colony formation ability assays for MCF7 / TR cells after treatment. Figure 6 This is a quantitative analysis of the colony-forming ability of MCF7 / TR cells after treatment. Figure 7 Figure 1 shows the flow cytometry results of apoptosis levels in MCF7 / TR cells after treatment. Figure 8 This is a quantitative analysis diagram of the flow cytometry results of apoptosis levels in MCF7 / TR cells after treatment. Figure 9 The image shows the results of MCF7 / TR cell cycle analysis after treatment. Figure 10 This is a graph showing the quantitative analysis results of MCF7 / TR cell cycle detection after treatment; Figure 11 Figure showing the results of the scratch assay on treated MCF7 / TR cells; Figure 12 This is a graph showing the quantitative analysis results of the scratch assay on treated MCF7 / TR cells; Figure 13 The graph shows the flow cytometry results of mitochondrial membrane potential measurement in treated MCF7 / TR cells. Figure 14 This is a quantitative analysis graph of the flow cytometry results of mitochondrial membrane potential measurement in treated MCF7 / TR cells. Figure 15 Figure showing the results of MCF7 / TR cell viability assay after treatment with combined cell death inhibitors; Figure 16 The image shows the results of ferroptosis assay in MCF7 / TR cells after treatment. Figure 17 Figure 1 shows the flow cytometry results of ROS level measurement in MCF7 / TR cells after treatment. Figure 18 Fluorescence images of ROS levels in treated MCF7 / TR cells; Figure 19 This is a quantitative analysis of fluorescence images used to measure ROS levels in treated MCF7 / TR cells. Figure 20 Figure 1 shows the flow cytometry results of lipid peroxidation levels in MCF7 / TR cells after treatment. Figure 21 This is a graph showing the transcriptome sequencing results of treated MCF7 / TR cells; Figure 22 Figure 1 shows the results of DCP1A protein expression level measurement in MCF7 / TR cells after treatment; Figure 23 Figure 1 shows the results of Dcp1a mRNA expression level measurement in MCF7 / TR cells after treatment. Figure 24 Figure showing the results of DCP1A protein expression level measurement in Dcp1a knockdown MCF7 / TR cells; Figure 25 The image shows the results of the Dcp1a knockdown MCF7 / TR cell viability assay after treatment. Figure 26 The image shows the results of ferroptosis assay in Dcp1a knockdown MCF7 / TR cells after treatment. Figure 27 Figure 1 shows the results of DCP1A protein expression level measurement in MCF7 / TR cells overexpressing Dcp1a; Figure 28 This is a graph showing the results of the activity assay of Dcp1a-overexpressing MCF7 / TR cells after treatment; Figure 29The image shows the results of ferroptosis assay in Dcp1a-overexpressing MCF7 / TR cells after treatment. Figure 30 This is a graph showing the statistical results of tumor tissue weight in mice after treatment. Figure 31 This is a graph showing the statistical results of tumor tissue volume monitoring in mice after treatment. Figure 32 The image shows the results of detecting the expression levels of Ki67 and DCP1A proteins in mouse tumor tissues after treatment. Figure 33 The image shows the results of ferrodeogenesis in mouse tumor tissue after treatment. Detailed Implementation

[0015] The technical solution of the present invention will be further described below.

[0016] Example 1: Construction and validation of tamoxifen (TAM) resistant cell lines 1. Establishment of a tamoxifen-resistant cell model MCF7 cells were 1×10⁶ per flask 6 Cells were seeded at a density of 1000 μM in T25 flasks and cultured in RPMI-1640 complete medium containing 0.05 μM TAM. When the cells reached 90% confluence, the medium was replaced with medium containing 1 μM TAM. When the cells reached 90% confluence again, the medium was replaced with medium containing 5 μM TAM. The cells were passaged three times at this TAM concentration to obtain TAM-resistant MCF7 cells (MCF7 / TR cells) for subsequent experiments.

[0017] 2. Verification of tamoxifen-resistant cells MCF7 cells were mixed with the selected MCF7 / TR cells at a ratio of 1×10⁻⁶. 4 Cells were seeded at a density of 1 cell / well in 96-well plates and cultured overnight. Then, the original medium was replaced with RPMI-1640 complete medium containing TAM at final concentrations of 0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, or 640 µM. After 24 h of treatment, 100 μL of RPMI-1640 basal medium containing 10% CCK8 reagent was added to each well and the cells were incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0018] The results are as follows Figure 1 As shown, the IC50 value of TAM in normal MCF7 cells was 5.99 µM, while the IC50 value of drug-resistant MCF7 / TR cells was 26.70 µM, indicating that the drug-resistant cell line was successfully constructed.

[0019] Example 2: In vitro efficacy verification of sanshodiol (Sans) in the treatment of breast cancer 1. Sans is used for tamoxifen-resistant breast cancer cells MCF7 / TR. 1.1 Cell viability assay MCF7 / TR cells were loaded at 1×10 4 Cells were seeded at a density of 1 cell / well in 96-well plates and cultured overnight. The original medium was replaced with RPMI-1640 complete medium containing 1‰ DMSO, or 5 µM TAM, or 10 µM Sans (purchased from MedChemExpress LLC., catalog number HY-N1325), or both 5 µM TAM and 10 µM Sans. After 24 h of treatment, 100 μL of RPMI-1640 basal medium containing 10% CCK8 reagent was added to each well and the cells were incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0020] The results are as follows Figure 2 As shown, compared with the DMSO treatment group, TAM treatment alone had no significant inhibitory effect on the activity of MCF7 / TR cells, while Sans treatment alone significantly reduced the activity of MCF7 / TR cells. Moreover, the combined treatment of Sans and TAM showed a synergistic inhibitory effect on cell activity, which significantly enhanced the killing effect of TAM on MCF7 / TR cells.

[0021] 1.2. Cell proliferation capacity assay MCF7 / TR cells were loaded at 1×10 4 Seeds were planted at a density of cells / well in 96-well plates. After overnight incubation, the original medium was replaced with RPMI-1640 complete medium containing 1‰ DMSO, or TAM with a final concentration of 5 µM, or Sans with a final concentration of 10 µM, or both TAM and Sans with a final concentration of 5 µM and 10 µM, for 24 h. After treatment, 20 μL of EdU reagent (purchased from Novizan, catalog number A413-01) was added to each well, and the plates were incubated at 37°C for 2 h. After fixation with 4% paraformaldehyde solution at room temperature for 10 min, the plates were permeated with Beyotime immunostaining permeabilization buffer (catalog number P0096) at room temperature for 15 min. Then, Click reaction solution (purchased from Novizan, catalog number A413-01) was added, and the plates were incubated in the dark for 30 min. The plates were then mounted with Beyotime anti-fluorescence quenching mounting buffer (catalog number P0131), and the images were observed and quantitatively analyzed using a fluorescence microscope.

[0022] The results are as follows Figure 3 , 4As shown, compared with the DMSO treatment group, TAM treatment alone had no significant effect on the proliferation of MCF7 / TR cells, while Sans treatment alone significantly inhibited the proliferation of MCF7 / TR cells, and the cell proliferation capacity was lowest after the combined treatment of Sans and TAM.

[0023] 1.3. Cell clone formation ability assay MCF7 / TR cells were seeded at a density of 500 cells / well in 6-well plates and cultured overnight. Then, the original medium was replaced with RPMI-1640 complete medium containing 1‰ DMSO, or TAM with a final concentration of 5 µM, or Sans with a final concentration of 10 µM, or both TAM and Sans with a final concentration of 5 µM and 10 µM, for 24 h. After that, the cells were replaced with fresh complete medium and cultured for another 24 h. The cells were then washed with PBS, fixed with methanol, stained with Beyotime crystal violet reagent (catalog number C0121-100ml), observed under a microscope, and images were acquired for quantitative analysis.

[0024] The results are as follows Figure 5 , 6 As shown, consistent with the cell proliferation assay results, Sans treatment alone significantly reduced the clonogenic capacity of MCF7 / TR cells compared to the DMSO treatment group, and the clonogenic capacity was further reduced after Sans combined with TAM treatment.

[0025] 1.4 Measurement of Apoptosis Level MCF7 / TR cells were used at a rate of 1×10 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight. After adhesion, the cells were treated for 24 h with RPMI-1640 complete medium containing 1‰ DMSO, or 5 µM TAM, or 10 µM Sans, or both 5 µM TAM and 10 µM Sans. Cells were then collected and stained using the Annexin V-PE / 7AAD apoptosis detection kit (catalog number A213-01). The stained cells were analyzed using a Beckman CytoFLEX flow cytometer to quantify apoptosis levels.

[0026] The results are as follows Figure 7 , 8 As shown, compared with the DMSO treatment group, TAM treatment alone had a weaker apoptosis-inducing effect on MCF7 / TR cells, while Sans treatment alone significantly increased the apoptosis rate of MCF7 / TR cells, and the apoptosis level was further increased after Sans and TAM combined treatment.

[0027] 1.5 Cell cycle detection MCF7 / TR cells were used at a rate of 1×10 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured overnight. After adherence, the cells were treated for 24 h with RPMI-1640 complete medium containing 1‰ DMSO, or 5 µM TAM, or 10 µM Sans, or both 5 µM TAM and 10 µM Sans. Cells were then collected and fixed with pre-chilled 95% ethanol (4°C) for 10 h. The fixed cells were centrifuged at 300×g for 5 min, and the cell pellet was recovered. The cells were resuspended in PBS buffer containing 50 μg / mL propidium iodide (MedChemExpress LLC., catalog number HY-D0815) and 100 μg / mL RNase A (Shanghai Beyotime Biotechnology Co., Ltd., catalog number ST579) and incubated at 37°C in the dark for 30 min. Cell cycle analysis was performed using a Beckman CytoFLEX flow cytometer and ModFit LT software.

[0028] The results are as follows Figure 9 , 10 As shown, compared with the DMSO treatment group, the MCF7 / TR cell cycle status of the TAM-only treatment group did not change significantly, while the Sans-only treatment caused the MCF7 / TR cell cycle to arrest in the G0 / G1 phase. Furthermore, the proportion of cells in the G2 / M phase was reduced to the lowest in the Sans-TAM combined treatment group, suggesting that Sans may inhibit the growth of MCF7 / TR cells by affecting the cell cycle process.

[0029] 1.6 Cell Scratch Test MCF7 / TR cells were used at a rate of 1×10 5 Cells were seeded at a density of 1 cell per well in 6-well plates. Once the cell density reached 70%, cells were scratched in each well using a 10 µL sterile pipette tip. After removing detached cells, the original medium was replaced with RPMI-1640 basal medium containing 1‰ DMSO, or TAM with a final concentration of 5 µM, or Sans with a final concentration of 10 µM, or both TAM and Sans with a final concentration of 5 µM and 10 µM, and the cells were treated for another 24 h. The condition of each group of cells 24 h after scratching was observed under an optical microscope, and images were acquired for quantitative analysis.

[0030] The results are as follows Figure 11 , 12 As shown, compared with the DMSO treatment group, both Sans treatment alone and combined with TAM treatment significantly reduced the migration ability of MCF7 / TR cells, and the inhibitory effect of the combined treatment group was more obvious, indicating that Sans can not only weaken cell migration ability, but also overcome TAM resistance.

[0031] 1.7 Mitochondrial membrane potential measurement MCF7 / TR cells at 2×10 4 Inoculate the cells at a density of cells / well in 24-well plates, incubate overnight, and then replace the original medium with RPMI-1640 basal medium containing 1‰ DMSO, or containing 5 µM TAM, or containing 10 µM Sans, or containing both 5 µM TAM and 10 µM Sans for 24 h.

[0032] Cells were collected after treatment and incubated in PBS buffer containing 10 µg / mL JC-1 (purchased from MedChemExpress LLC., catalog number: HY-15534) at 37°C in the dark for 2 h. Fluorescence signals were detected using a Beckman CytoFLEX flow cytometer. The excitation wavelength for JC-1 monomer (green fluorescence) was set to 488 nm, and the emission wavelengths were detected at 525 / 50 nm. The emission wavelengths for JC-1 multimer (red fluorescence) were detected at 590 / 40 nm.

[0033] The results are as follows Figure 13 , 14 As shown, the DMSO-treated group had a higher JC-1 polymer / JC-1 monomer ratio, indicating a higher mitochondrial membrane potential and stable mitochondrial function. The TAM-only treatment group showed no significant change in this ratio, while the Sans-only treatment group showed a significant decrease in the JC-1 polymer / JC-1 monomer ratio, and the decrease was more pronounced in the combined treatment group. This indicates that Sans can disrupt the mitochondrial membrane potential of MCF7 / TR cells, affect mitochondrial function, and thus inhibit cell activity.

[0034] 1.7 Identification and Assessment of Cell Death Pathways MCF7 / TR cells were loaded at 1×10 4Cells were seeded at a density of cells / well in 96-well plates and cultured overnight. All cells were then divided into four groups: a cell death inhibitor-free treatment group, a Fer-1 treatment group (final concentration 10 µM, purchased from MedChemExpress LLC., catalog number HY-100579), a zVAD treatment group (final concentration 10 µM, purchased from MedChemExpress LLC., catalog number HY-16658B), a 3-MA treatment group (final concentration 5 mM, purchased from MedChemExpress LLC., catalog number HY-19312), a BAY treatment group (final concentration 10 µM, purchased from MedChemExpress LLC., catalog number HY-13453), and an NSA treatment group (final concentration 5 µM, purchased from MedChemExpress LLC., catalog number HY-100573). Cells within each treatment group were treated with either 1‰ DMSO, or 10 µM Sans, or 5 µM TAM, or 10 µM TAM. After replacing the original medium with µM Sans RPMI-1640 complete medium for 24 h, each well was replaced with 100 μL of RPMI-1640 basal medium containing 10% CCK8 reagent and incubated at 37℃ for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0035] The results are as follows Figure 15 As shown, the cell viability of cells treated with Sans was significantly restored after the addition of the ferroptosis inhibitor Fer-1, while other inhibitors had no significant effect, preliminarily indicating that Sans mainly kills drug-resistant MCF7 / TR cells through the ferroptosis pathway.

[0036] Further detection of ferroptosis-related markers was performed using ELISA: MCF7 / TR cells were cultured at a concentration of 2×10⁻⁶ cells / mL. 4 Cells were seeded at a density of cells / well in 6-well plates and cultured overnight. After adhesion, the culture medium was replaced with RPMI-1640 basal medium containing 1‰ DMSO, or containing 5 µM TAM, or containing 10 µM Sans, or both 5 µM TAM and 10 µM Sans for 24 h. After treatment, the cell culture supernatant was collected, and the levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and glutathione (GSH) in the supernatant were detected using the Beyotime Lipid Oxidation (MDA) Detection Kit (Catalog No. S0131S), the Sangon Biotech 4-Hydroxynonenal (4-HNE) ELISA Kit (Catalog No. D751041), and the Beyotime GSH and GSSG Detection Kit (Catalog No. S0053).

[0037] The results are as follows Figure 16As shown, compared with the DMSO treatment group, the above ferroptosis indicators did not change significantly after TAM treatment alone. However, after Sans treatment, the levels of MDA and 4-HNE in MCF7 / TR cells increased significantly, while the content of GSH decreased significantly. Moreover, the changes in the above indicators were more significant in the Sans and TAM combined treatment group.

[0038] 1.8 Detection of reactive oxygen species (ROS) and lipid peroxidation levels MCF7 / TR cells were loaded at 1×10 5 Cells were seeded at a density of cells / well in 6-well plates and cultured overnight. After adhesion, the cells were treated for 24 h with RPMI-1640 basal medium containing 1‰ DMSO, or TAM (total concentration 5 µM), or Sans (total concentration 10 µM), or both TAM and Sans (total concentrations 5 µM and 10 µM). After treatment, cells were stained with the DCFH-DA probe (MedChemExpress LLC., catalog number HY-D0940) at 37°C for 30 min; or with the BODIPY 581 / 591 C11 probe (MedChemExpress LLC., catalog number HY-D1301) at 37°C for 30 min. Intracellular ROS levels were detected by flow cytometry, and images were observed and quantitatively analyzed using a fluorescence microscope. Lipid peroxidation levels were detected by flow cytometry only.

[0039] ROS level detection results are as follows: Figures 17-19 As shown, TAM treatment alone did not cause a significant increase in intracellular ROS levels in MCF7 / TR cells, while Sans treatment alone significantly increased intracellular ROS levels, and the ROS levels further increased after combined TAM treatment.

[0040] The results of lipid peroxidation level detection are as follows: Figure 20 As shown, consistent with the ROS detection results, both Sans treatment alone and combined with TAM treatment significantly increased the lipid peroxidation level of MCF7 / TR cells.

[0041] 1.9 Transcriptome Sequencing MCF7 / TR cells were loaded at 1×10 5Cells were seeded at a density of cells / well in 6-well plates and cultured overnight. After adhesion, the original medium was replaced with RPMI-1640 basal medium containing 1‰ DMSO or 10 µM Sans for 24 h. After treatment, adherent cells were washed twice with pre-cooled PBS at 4°C, and 1 mL of Novizan FreeZol Reagent (catalog number R711-01) was added. After complete cell lysis, the lysate was collected and sent to Shanghai Paisenuo Biotechnology Co., Ltd. for transcriptome sequencing.

[0042] The results are as follows Figure 21 As shown, the expression of the Dcp1a (decapping enzyme 1a) gene was significantly reduced in MCF7 / TR cells treated with Sans.

[0043] 1.10. Western blot detection of DCP1A protein expression level MCF7 / TR cells were loaded at 1×10 5 Inoculate at a density of cells / well in 6-well plates, incubate overnight, and then replace the original medium with RPMI-1640 basal medium containing 1‰ DMSO, or containing 5 µM TAM, or containing 10 µM Sans, or containing both 5 µM TAM and 10 µM Sans for 24 h. After treatment, the sample was lysed with 0.1 volume of cyprotease inhibitor mixture (P001) and 0.1 volume of cyprotease inhibitor mixture (P003) in cyprotease RIPA lysis buffer (WB3100) at 4°C for 30 min. After centrifugation at 12000 rpm for 30 min at 4°C, the supernatant was collected and cyprotease RIPA lysis buffer (WB2001) was added. The sample was then boiled in a metal bath for 10 min, followed by SDS-PAGE electrophoresis. After transfer and blocking, DCP1A primary antibody (Proteintech, 22373-1-AP) diluted 1:5000 was added and incubated overnight at 4°C. After rinsing, the sample was incubated with secondary antibody (Proteintech, 10494-1-AP) diluted 1:20000 at room temperature for 2 hours. h, and finally, the chemiluminescence was developed using the new SEMER ultrasensitive ECL chemiluminescence kit (catalog number P10100), and the images were acquired.

[0044] 1.11. qPCR detection of Dcp1a mRNA expression level MCF7 / TR cells were loaded at 1×10 5Inoculate at a density of cells / well in 6-well plates, incubate overnight, and then replace the original medium with RPMI-1640 basal medium containing 1‰ DMSO, or containing 5 µM TAM, or containing 10 µM Sans, or containing both 5 µM TAM and 10 µM Sans for 24 h. Cells were collected, and total RNA was extracted using the Novizan FreeZol Reagent Kit (catalog number R711-01). The RNA was then reverse transcribed into cDNA using the Novizan HiScript IV RTSuperMix for qPCR (+gDNA wiper) reagent (catalog number R423-01) with the following primers: Dcp1a upstream primer: 5'-gttcatacgcgcaattcgttg-3'; Dcp1a downstream primer: 5'-gaatgtgatgaacgcgtcgagt-3'; 18S upstream primer: 5'-aaacggctaccacatccaag-3'; 18S downstream primer: 5'-cctccaatggatcctcgtta-3'. qPCR detection was performed using the Novizan ChamQ Universal SYBR qPCR Master Mix reagent (catalog number Q711-02), with reaction conditions of 95℃ pre-denaturation for 30 s and 95℃ denaturation for 10 s. Annealing and extension at 60℃ for 30 s, 35 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. Using 18S as an internal control, Ct values ​​were calculated, and the relative expression level of Dcp1a was calculated using the ΔΔCt method.

[0045] The results of DCP1A protein expression level detection are as follows: Figure 22 As shown in the figure, the results of Dcp1a mRNA expression level detection are as follows: Figure 23 As shown, the protein and mRNA levels of Dcp1a in MCF7 / TR cells changed significantly after Sans treatment. The protein expression level and mRNA level were the lowest in the group treated with Sans and TAM.

[0046] 2. Sans is used to knock down tamoxifen-resistant breast cancer cells MCF7 / TR using Dcp1a. 2.1 Construction and Verification of Dcp1a Knockdown of MCF7 / TR Based on the publicly available Dcp1a gene sequence (NCBI Reference Sequence: NM_001290205.2), Suzhou Genewise Biotechnology Co., Ltd. was commissioned to design and synthesize siRNA that interferes with Dcp1a expression (Si-Dcp1a) and control siRNA (Si-NC).

[0047] MCF7 / TR cells were loaded at 1×105 Cells were seeded at a density of cells / well in 6-well plates. After overnight culture and adhesion, the synthesized Si-Dcp1a or Si-NC was transfected into MCF7 / TR cells using the Novizan Lipomaster 2000 Transfection Reagent Kit (catalog number TL201-01). Cells were collected 24 h after transfection, and the expression level of DCP1A protein was determined based on the method described in 1.10.

[0048] The results are as follows Figure 24 As shown, Si-Dcp1a can effectively knock down the expression level of DCP1A protein in MCF7 / TR cells.

[0049] 2.2 Cell viability assay Cell viability was determined using cells transfected with Si-Dcp1a or Si-NC as described in 2.1, based on the method described in 1.1. The original culture medium was replaced with RPMI-1640 complete medium containing 1‰ DMSO or 10 µM Sans, while all other experimental conditions remained unchanged.

[0050] The results are as follows Figure 25 As shown, the killing effect of Sans on MCF7 / TR cells was weakened after the expression level of DCP1A protein decreased.

[0051] 2.3 ELISA method for detecting ferroptosis-related indicators Using cells transfected with Si-Dcp1a or Si-NC as described in 2.1, ferroptosis-related indicators were determined based on the method described in 1.7. The original medium was replaced with RPMI-1640 complete medium containing 1‰ DMSO or 10 µM Sans, while all other experimental conditions remained unchanged.

[0052] The results are as follows Figure 26 As shown, the decrease in DCP1A protein expression level alleviated the increase in MDA and 4-HNE and the decrease in GSH caused by Sans treatment.

[0053] 3. Sans was used to treat Dcp1a-overexpressing tamoxifen-resistant breast cancer cells MCF7 / TR. 3.1 Construction and validation of Dcp1a overexpression of MCF7 / TR Based on the publicly available Dcp1a gene sequence, Suzhou Genewise Biotechnology Co., Ltd. was commissioned to construct a Dcp1a gene overexpression plasmid based on the pcDNA3.1 vector.

[0054] MCF7 / TR cells were loaded at 1×10 5Cells were seeded at a density of 1 cell / well in 6-well plates. After overnight culture and adhesion, the synthesized Dcp1a gene overexpression plasmid or pcDNA3.1 vector was transfected into MCF7 / TR cells using the Novizan Lipomaster 2000 Transfection Reagent Kit (catalog number TL201-01). Cells were collected 24 h after transfection, and the DCP1A protein expression level was determined based on the method described in 1.10.

[0055] The results are as follows Figure 27 As shown, the Dcp1a gene overexpression plasmid can effectively upregulate the DCP1A protein expression level in MCF7 / TR cells.

[0056] 3.2 Cell viability assay Cells transfected with the Dcp1a gene overexpression plasmid or vector as described in 3.1 were used to determine cell viability based on the method described in 1.1. The original culture medium was replaced with RPMI-1640 complete medium containing 1‰ DMSO or 10 µM Sans, while all other experimental conditions remained unchanged.

[0057] The results are as follows Figure 28 As shown, upregulation of DCP1A protein expression enhanced the killing effect of Sans on MCF7 / TR cells.

[0058] 3.3 ELISA method for detecting ferroptosis-related indicators Cells transfected with the Dcp1a gene overexpression plasmid or vector as described in 3.1 were used to determine ferroptosis-related indicators based on the method described in 1.7. The original culture medium was replaced with RPMI-1640 complete medium containing 1‰ DMSO or 10 µM Sans, while the other experimental conditions remained unchanged.

[0059] The results are as follows Figure 29 As shown, after the expression level of DCP1A protein was upregulated, the increase in MDA and 4-HNE and the decrease in GSH induced by Sans treatment were more significant.

[0060] Example 3: In vivo efficacy verification of sanshinol in a mouse model of breast cancer Female Balb / c nude mice aged 6-8 weeks were purchased from the Experimental Animal Center of Nantong University and housed in an SPF-grade animal room at a temperature of 22±2℃ and a humidity of 50±10%, with a 12-hour day-night cycle. They were acclimatized for one week before the experiment.

[0061] MCF7 / TR cells were administered at a rate of 5 × 10⁵ cells per mouse. 5 A seeding dose of 100 cells was administered subcutaneously in the left axilla to induce tumor formation. On day 10 post-seeding of MCF7 / TR cells, when the tumor volume reached approximately 100 mm...3 At that time, tumor-bearing mice were randomly divided into 4 groups, including: saline group, TAM treatment group (dose of 20 mg / kg), Sans treatment group (dose of 20 mg / kg), and Sans+TAM combined treatment group (dose of 20 mg / kg), and drug treatment was started at the same time.

[0062] The drug was administered intraperitoneally every 3 days, while the saline group received the same volume of saline. Treatment continued until day 36. Upon reaching the experimental endpoint, the mice were euthanized, and tumor tissue was collected from each group for subsequent analysis of relevant indicators.

[0063] 1. Tumor volume monitoring Starting from day 10 of drug administration, the tumor volume of mice in each experimental group was measured every 3 days. The length and width of the tumor were recorded, and the tumor volume was then calculated and statistically analyzed according to the following formula: Tumor volume (mm) 3 = length × width × width / 2.

[0064] The statistical results of tumor tissue weight collected at the end of the experiment are as follows: Figure 30 As shown, the tumor volume monitoring results are as follows: Figure 31 As shown, there was no significant difference in tumor volume between the TAM-treated group and the saline group, indicating that TAM is ineffective in treating TAM-resistant breast cancer. However, the tumor volume and weight of mice in the Sans-treated group were significantly reduced, showing a significant difference from the saline group. The combined treatment of Sans and TAM showed better anti-tumor effects, indicating that Sans can significantly promote the inhibitory effect of TAM on the growth of TAM-resistant tumors, that is, Sans can enhance the therapeutic sensitivity of TAM-resistant tumors.

[0065] 4. Immunohistochemical detection of Ki67 and DCP1A protein expression in tumor tissues Tumor tissue samples were fixed overnight with 4% paraformaldehyde, graded dehydration, paraffin embedding, and cut into 4 μm sections. Antigen retrieval was performed for 20 min at 95°C with citrate buffer (pH 6.0). Endogenous peroxidase activity was inhibited for 10 min with 3% hydrogen peroxide solution, followed by blocking with 5% bovine serum albumin solution for 1 h. The sections were incubated overnight at 4°C with either Ki67 primary antibody (1:10000 dilution, Proteintech, catalog number 27309-1-AP) or DCP1A primary antibody (1:10000 dilution). After washing, the sections were incubated with horseradish peroxidase (HRP) conjugated secondary antibody (1:500 dilution, Proteintech, catalog number SA00004-2) at room temperature for 1 h. Signal detection was performed using the Beyotime DAB chromogenic reagent kit (catalog number P0202), and cell nuclei were counterstained with hematoxylin. Images were acquired using an optical microscope.

[0066] The results are as follows Figure 32 As shown, the KI67 positivity rate in the TAM-treated group was not significantly different from that in the saline group, while Sans treatment significantly reduced Ki67 expression and inhibited Dcp1a expression. In addition, the inhibition of Ki67 and Dcp1a protein expression levels was more significant in the Sans and TAM combined treatment group, further indicating that Sans can enhance the therapeutic sensitivity of TAM-resistant tumors.

[0067] 5. ELISA method for detecting ferroptosis-related indicators 100 mg of tumor tissue was taken, ground in liquid nitrogen using a mortar and pestle, and then 1 mL of Beyotime Western lysis buffer (product number P0013) containing protease inhibitors and phosphatase inhibitors was added to completely lyse the tissue. After lysis at 4°C for 30 min, the tissue was centrifuged at 12000 rpm for 30 min to obtain the supernatant. The levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and glutathione (GSH) in the supernatant were detected using Beyotime Lipid Oxidation (MDA) Detection Kit (product number S0131S), Sangon Biotech 4-Hydroxynonenal (4-HNE) ELISA Kit (product number D751041), and Beyotime GSH and GSSG Detection Kit (product number S0053).

[0068] The results are as follows Figure 33 As shown, the ferroptosis index in the TAM-treated group did not change significantly compared with the saline group, indicating that TAM alone cannot achieve the goal of tumor treatment by inducing ferroptosis. However, after Sans pretreatment, the levels of MDA and 4-HNE in the tumor tissue increased significantly, while the level of GSH decreased significantly. Furthermore, the ferroptosis level was even higher after Sans and TAM combined treatment, ultimately achieving a better anti-tumor therapeutic effect.

Claims

1. The application of sanshinol in the preparation of anticancer drugs, characterized in that, The CAS number of the sanshool is 54854-91-0, and its application is in the preparation of anti-breast cancer drugs.

2. The application according to claim 1, characterized in that, The breast cancer mentioned is tamoxifen-resistant breast cancer.

3. The application according to claim 1, characterized in that, The drug contains sanshool or its pharmaceutically acceptable salt as an active ingredient.

4. The application according to claim 3, characterized in that, The drug also contains tamoxifen as an active ingredient.

5. The application according to claim 4, characterized in that, The mass ratio of salinomycin to tamoxifen in the drug is 1~2:

1.

6. The application according to any one of claims 3 to 5, characterized in that, The drug also contains pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, The pharmaceutically acceptable excipients are any one or more of the following: excipients, diluents, lubricants, glidants, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

8. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, capsules, granules, powders, microcapsules, injections, suspensions, patches, suppositories, microemulsions, liposomes, and nanoparticles.

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