Method for treating tetra-negative breast cancer by inhibiting CYP1B1 through quercetin and combining with Palbociclib
By inhibiting CYP1B1 and using Palbociclib in combination, the problem of poor response to traditional treatment of four-negative breast cancer was solved, and significant tumor growth inhibition and treatment effect were achieved, providing new treatment ideas.
Patent Information
- Application Number
- CN202510217051.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-26
- Publication Date
- 2025-05-30
AI Technical Summary
Patients with quad-negative breast cancer (QNBC) have poor responses to traditional endocrine therapy and targeted therapy, limited treatment effects, and poor prognosis, which makes their treatment particularly difficult.
Quaternary negativity breast cancer is treated by inhibiting CYP1B1 with quercetin and combined with Palbociclib. The method includes screening the Chinese herbal medicine set, inhibiting the expression of CYP1B1, analyzing the sensitivity mechanism of Palbociclib, knocking down CYP1B1 to inhibit tumor growth, and evaluating the synergistic effect of quercetin and Palbociclib.
Quercetin increases the sensitivity of QNBC to Palbociclib by inhibiting CYP1B1, significantly inhibits the growth of quad-negative breast cancer, improves the treatment effect, and provides new treatment ideas and drug choices.
Smart Images
Figure CN120053455A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of breast cancer, and particularly relates to a method for treating triple-negative breast cancer by quercetin inhibiting CYP1B1 in combination with Palbociclib. Background Art
[0002] Currently, some scholars have proposed to classify TNBC into AR-positive TNBC and AR-negative TNBC according to the expression of androgen receptor. Among them, AR-TNBC, also known as triple-negative breast cancer, is a subtype of TNBC, accounting for 63% - 87% of TNBC. Due to its high invasiveness and poor prognosis, and compared with other breast cancer subtypes, including the AR+TNBC subtype, the overall survival, progression-free survival, and recurrence-free survival of QNBC patients are shorter.
[0003] Due to the lack of expression of ER, PR, HER-2, and androgen receptor (AR), QNBC patients do not respond well to traditional endocrine therapy and targeted therapy, with limited therapeutic effects and poor prognosis, which makes the treatment of QNBC particularly difficult and becomes a difficult point in current breast cancer treatment.
[0004] Currently, the treatment methods for QNBC mainly include surgical treatment, chemotherapy, radiotherapy, etc. However, these treatment methods have many limitations. Although surgical treatment can remove tumors, it has poor effects on QNBC patients with metastasis. Although chemotherapy and radiotherapy can kill some tumor cells, they will also damage normal human cells, cause a series of side effects, and there is a problem of drug resistance. Therefore, QNBC may be classified as an independent breast cancer subtype. Finding highly effective targeted drugs, screening drug action molecular targets, clarifying the molecular mechanism of drug-targeted anti-tumor, and improving the curative effect of current clinical drugs are undoubtedly scientific problems that need to be solved urgently in the process of treating QNBC. Summary of the Invention
[0005] The present invention proposes a method for treating triple-negative breast cancer by quercetin inhibiting CYP1B1 in combination with Palbociclib, which solves the problems in the prior art.
[0006] The technical solution of the present invention is realized as follows: A method for treating triple-negative breast cancer by quercetin inhibiting CYP1B1 in combination with Palbociclib, the method comprising:
[0007] S1. Using the HERB herbal group identification database to screen the relevant Chinese herbal medicine sets for treating TNBC, and obtaining the active ingredients and their targets;
[0008] S2. The influence of quercetin on inhibiting the expression of CYP1B1 in QNBC cell lines;
[0009] S3. The mechanism of the effect of knocking down CYP1B1 on AR expression in the QNBC cell line;
[0010] S4. Analyze the mechanism of insensitivity of the QNBC cell line to Palbociclib;
[0011] S5. Improve the sensitivity of QNBC to Palbociclib by inhibiting CYP1B1;
[0012] S6. Knocking down CYP1B1 can inhibit the growth of xenograft tumors in nude mice with triple-negative breast cancer and improve the efficacy of Palbociclib;
[0013] S7. Quercetin combined with Palbociclib has a synergistic effect on the treatment of QNBC.
[0014] As a preferred implementation of the method, the process of screening using the HERB Materia Medica Group Identification Database described in S1 includes:
[0015] S11. Respectively screen out the relevant Chinese herbal medicine sets that can treat ER-, PR-, and HER2-breast cancer through the disease partition, and take the intersection of the three data sets to screen out the Chinese herbal medicines that can treat ER-, PR-, and HER2-breast cancer simultaneously;
[0016] S12. Retrieve the active ingredients of Chinese herbal medicines based on the Traditional Chinese Medicine Pharmacology Database and Analysis Platform TCMSP, and predict the bioavailability and oral bioavailability of potential drug molecules according to Lipinski's Rule of Five, and conduct a preliminary evaluation of the molecules in the early screening stage to screen out potential candidate drug molecules;
[0017] S13. Screen the corresponding targets for each active ingredient of the drug using the online prediction database platform for small molecule drug targets, Swiss TargetPrediction;
[0018] S14. Substitute the active ingredients and action targets of the drug into the Cytoscape software to construct a traditional Chinese medicine-active ingredient-target network diagram.
[0019] As a preferred implementation of the method, the process of quercetin inhibiting CYP1B1 in the QNBC cell line described in S2 includes:
[0020] S21. Prepare the quercetin treatment group: IC50: 25.06 μmol / L (231) 24 h;
[0021] S22. Detection of cell growth inhibition: After adding the drug for 24 h, detect the growth of cells in each group by the CCK-8 kit, and record the cell growth inhibition rate;
[0022] S23. Western blot assay: the inhibitory effect of quercetin on the expression level of CYP1B1 and the activating effect on the expression of AR;
[0023] S24. Scratch assay: create scratches on the cell monolayer, observe and record the migration of cells in each group after scratching, so as to evaluate the effect of quercetin on the migration ability of QNBC cells after inhibiting CYP1B1.
[0024] As a preferred implementation manner of the method, the process of knocking down CYP1B1 on the expression of AR in the QNBC cell line described in S3 includes:
[0025] S31. Establish a stable cell line: Knock down the expression of CYP1B1 in the human breast cancer cell lines MDA-MB-231 and MDA-MB-453 to establish the CYP1B1-KD cell line, culture it with DMEM high-glucose medium containing 10% FBS, and detect the knockdown efficiency and the effect on the expression of AR after knocking down CYP1B1 by Western blot and qPCR.
[0026] As a preferred implementation manner of the method, the process of analyzing insensitivity described in S4 includes:
[0027] S41. Grouping: 231-DMSO, 231-Pal, drug treatment for 72 h, detect the absorbance values of the two groups by CCK-8, calculate the cell survival rates of the DMSO group and the Palbociclib group and compare them to obtain the effect of Palbociclib alone on the proliferation of the 231 cell line;
[0028] S42. After drug treatment for 72 h, collect the cells of the two groups respectively, resuspend them with PBS, centrifuge at 300 g for 5 min, discard the supernatant, add 70% ethanol to resuspend, fix the samples for 2 h, centrifuge at 300 g for 5 min, discard the supernatant, wash with PBS again once, centrifuge and discard the supernatant, add the prepared PI staining solution (containing RNase) to resuspend the samples, incubate in a water bath at 37 °C for 30 min, and detect the effect of Palbociclib alone on the cell cycle by flow cytometry;
[0029] S43. After drug treatment for 72 h, collect the two samples 231-DMSO and 231-Pal, and perform transcriptome sequencing to analyze the mechanism of insensitivity when Palbociclib is used alone to treat QNBC.
[0030] As a preferred implementation manner of the method, the process of improving sensitivity described in S5 includes:
[0031] S51. Detect four groups of cell lines, namely 231-DMSO, 231-Pal, 231-QUE, and 231-Pal+QUE. Among them, Pal: IC50: 1 μM, for 3 days; QUE: IC50: 20 μmol / L, for 3 days.
[0032] S52. After 72 hours of drug treatment, use CCK-8 to detect the effect of the four treatments on the survival rate of the 231 cell line.
[0033] S53. Use flow cytometry to analyze the effects of the four treatments on the cell cycle and apoptosis.
[0034] S54. Use CK-8 to detect the effect of the combination of quercetin and palbociclib on the proliferation of the MDA-MB-231 cell line.
[0035] As a preferred embodiment of the method, the specific content of knocking down CYP1B1 in S6 to inhibit the growth of transplanted tumors in nude mice with triple-negative breast cancer and improve the efficacy of Palbociclib includes:
[0036] S61. Select nude mice aged 4 to 6 weeks, divide them into 4 groups (NC-ctrl, NC-Pal, sh-ctrl, sh-Pal), with 6 mice in each group. Inject 5×106 cells into the subcutaneous area under the axilla of each mouse to form tumors. Observe and measure the body weight and tumor volume of the mice every 3 days.
[0037] S62. Extract RNA and protein from the tumor tissues of the mice, and use qPCR and Western blot to detect the expression of CYP1B1 and AR in the tumor tissues.
[0038] S63. Perform HE staining on the tumor tissues to observe the morphology, mitotic figures of the tumor cells, and the structural characteristics of the tumor tissues, and evaluate the effects of knocking down CYP1B1 on the proliferation of tumor cells and the structure of the tumor tissues.
[0039] S64. Perform IHC staining on the tumor tissues to detect the expression and localization of key proteins in the tumor tissues, further verify the results of qPCR and Western blot, and observe the distribution and expression levels of these proteins in the tumor tissues.
[0040] As a preferred embodiment of the method, the specific content of the synergistic effect of quercetin combined with Palbociclib on the treatment of QNBC in S7 includes:
[0041] S71. Select nude mice at 4 to 6 weeks of age and divide them into 4 groups (231-ctrl, 231-QUE, 231-Pal, 231-Pal+QUE), with 6 mice in each group. Inject 5×106 cells per mouse subcutaneously under the axilla of the mice to form tumors, and observe and measure the body weight and tumor volume of the mice once every 3 days.
[0042] S72. Take the RNA and protein of the mouse tumor tissue, and detect the expression of CYP1B1 and AR in the tumor tissue by qPCR and Western blot. Detect the changes in the expression of related pathway proteins.
[0043] S73. Observe the morphological changes of the tumor tissue by histological examination, including cell size, morphology, nuclear-cytoplasmic ratio, mitotic figures, etc., and evaluate the anti-tumor effect of the drug.
[0044] S74. Select specific antibodies related to tumor proliferation, apoptosis, angiogenesis, etc. by immunohistochemistry, such as Ki-67, TUNEL, CD31 or CD34; observe the changes in the expression of these markers in the tumor tissue after drug treatment by immunohistochemical staining, and evaluate the anti-tumor effect of the drug.
[0045] After adopting the above technical solutions, the beneficial effects of the present invention are as follows: The present invention starts from the key target gene CYP1B1 and deeply analyzes how it affects the malignant progression of QNBC by regulating genes such as AR and Rb and related signaling pathways. This multi-dimensional and multi-level research idea helps to more comprehensively and deeply understand the pathogenesis and treatment targets of QNBC.
[0046] Quercetin verifies its inhibitory effect on QNBC, and provides new ideas and drug choices for the treatment of QNBC through the synergistic effect of the effective components of traditional Chinese medicine in combination with the current first-line western medicine in clinical practice, which is innovative and practical. BRIEF DESCRIPTION OF THE DRAWINGS
[0047] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.
[0048] Figure 1 It is a schematic diagram of 11 traditional Chinese medicines for HERB screening and treatment of TNBC of the present invention;
[0049] Figure 2 It is a schematic diagram of the id, name and meridian tropism of the traditional Chinese medicines for HERB screening and treatment of TNBC of the present invention;
[0050] Figure 3 Schematic diagram of the molecular docking results of the present invention (left: quercetin, right: kaempferol);
[0051] Figure 4 Schematic diagram of the IC50 value of quercetin detected by CCK-8 in the MDA-MB-231 cell line in the present invention;
[0052] Figure 5 Schematic diagram of the activation effect on AR expression after knocking down CYP1B1 in the present invention;
[0053] Figure 6 Schematic diagram of the effect on the cell cycle of MDA-MB-231 after 72 hours of drug addition in the present invention (left: DMSO, right: Pal);
[0054] Figure 7 Schematic diagram of the effect of the combination of quercetin and palbociclib on apoptosis of the MDA-MB-231 cell line detected by flow cytometry in the present invention;
[0055] Figure 8 Schematic diagram of the effect of the combination of quercetin and palbociclib on the proliferation of the MDA-MB-231 cell line detected by cell colony formation assay in the present invention. Detailed implementation manners
[0056] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.
[0057] A method for quercetin to inhibit CYP1B1 combined with Palbociclib in the treatment of triple-negative breast cancer, the method comprising:
[0058] S1. Use the HERB herbal group identification database to screen the relevant Chinese herbal medicine sets for the treatment of TNBC, and obtain the active ingredients and their targets;
[0059] S2. The influence of quercetin on the expression of CYP1B1 in the QNBC cell line;
[0060] S3. The influence mechanism of knocking down CYP1B1 on the expression of AR in the QNBC cell line;
[0061] S4. Analyze the mechanism of insensitivity of the QNBC cell line to Palbociclib;
[0062] S5. Improve the sensitivity of QNBC to Palbociclib by inhibiting CYP1B1;
[0063] S6. Knockdown of CYP1B1 can inhibit the growth of xenografted tumors in nude mice with triple-negative breast cancer and improve the efficacy of Palbociclib;
[0064] S7. Quercetin combined with Palbociclib has a synergistic effect on the treatment of QNBC.
[0065] As Figure 1-3 shown, the process of screening using the HERB Chinese herbal medicine group identification database in S1 includes:
[0066] S11. Respectively screen the relevant Chinese herbal medicine sets that can treat ER-, PR-, and HER2-breast cancer through the disease partition, and take the intersection of the three data sets to screen out 11 Chinese herbal medicines that can treat ER-, PR-, and HER2-breast cancer simultaneously;
[0067] S12. Retrieve the active ingredients of Chinese herbal medicines based on the Traditional Chinese Medicine Pharmacology Database and Analysis Platform TCMSP, and predict the bioavailability and oral bioavailability of potential drug molecules according to Lipinski's Rule of Five. Conduct a preliminary evaluation of the molecules in the early screening stage to screen out potential candidate drug molecules;
[0068] S13. Screen the corresponding targets (possible>0.3) for each active ingredient of the drug using the online prediction database platform for small molecule drug targets, Swiss TargetPrediction;
[0069] S14. Substitute the active ingredients of the drug and their action targets into the Cytoscape software to construct a Chinese medicine-active ingredient-target network diagram.
[0070] As Figure 4 shown, the process of quercetin inhibiting CYP1B1 in the QNBC cell line in S2 includes:
[0071] S21. Prepare the quercetin treatment group: IC50: 25.06 μmol / L (231) 24 h;
[0072] S22. Cell growth inhibition detection: After adding the drug for 24 h, detect the growth of cells in each group through the CCK-8 kit, and record the cell growth inhibition rate;
[0073] S23. Western blot detection: The inhibitory effect of quercetin on the expression level of CYP1B1 and the activation effect on the expression of AR;
[0074] S24, Scratch assay: Make a scratch on the cell monolayer, observe and record the migration of cells in each group after the scratch, so as to evaluate the effect of quercetin on the migration ability of QNBC cells after inhibiting CYP1B1.
[0075] As Figure 5 shown, the process of knocking down CYP1B1 on AR expression in the QNBC cell line in S3 includes:
[0076] S31, Establish a stable cell line: Knock down the expression of CYP1B1 in human breast cancer cell lines MDA-MB-231 and MDA-MB-453 to establish a CYP1B1-KD cell line, culture it with DMEM high-glucose medium containing 10% FBS, and detect the knockdown efficiency and the effect on AR expression after knocking down CYP1B1 by Western blot and qPCR.
[0077] As Figure 6 shown, the process of analyzing insensitivity in S4 includes:
[0078] S41, Grouping: 231-DMSO, 231-Pal, drug treatment for 72 h, detect the absorbance values of the two groups by CCK-8, calculate the cell survival rates of the DMSO group and the Palbociclib group and compare them to obtain the effect of Palbociclib monotherapy on the proliferation of the 231 cell line;
[0079] S42, After drug treatment for 72 h, collect the cells of the two groups respectively, resuspend them with PBS, centrifuge at 300 g for 5 min, discard the supernatant, add 70% ethanol to resuspend, fix the samples for 2 h, centrifuge at 300 g for 5 min, discard the supernatant, wash with PBS once again, centrifuge and discard the supernatant, add the prepared PI staining solution (containing RNase) to resuspend the samples, incubate in a water bath at 37 °C for 30 min, and detect the effect of Palbociclib monotherapy on the cell cycle by flow cytometry;
[0080] S43, After drug treatment for 72 h, collect the two samples 231-DMSO and 231-Pal, and perform transcriptome sequencing to analyze the mechanism of insensitivity when Palbociclib is used alone to treat QNBC.
[0081] As Figure 7-8 shown, the process of increasing sensitivity in S5 includes:
[0082] S51, Detect the four cell lines, and the four cell lines are 231-DMSO, 231-Pal, 231-QUE, 231-Pal+QUE respectively, where, Pal: IC50: 1 μM, 3 days; QUE: IC50: 20 μmol / L, 3 days;
[0083] S52. After 72 hours of drug addition treatment, the CCK-8 assay was used to detect the effect of the four treatments on the survival rate of the 231 cell line;
[0084] S53. Flow cytometry was used to analyze the effect of the four treatments on the cell cycle and apoptosis;
[0085] S54. The CCK-8 assay was used to detect the effect of the combination of quercetin and palbociclib on the proliferation of the MDA-MB-231 cell line.
[0086] Furthermore, the specific content of suppressing the growth of xenograft tumors in nude mice with quadruple-negative breast cancer and improving the efficacy of Palbociclib by knocking down CYP1B1 in S6 includes:
[0087] S61. Nude mice aged 4 to 6 weeks were selected and divided into 4 groups (NC-ctrl, NC-Pal, sh-ctrl, sh-Pal), with 6 mice in each group. The mice were inoculated subcutaneously under the axilla with 5×106 cells per mouse, and the body weight and tumor volume of the mice were observed and measured every 3 days;
[0088] S62. RNA and protein were extracted from the tumor tissues of the mice, and qPCR and Western blot were used to detect the expression of CYP1B1 and AR in the tumor tissues;
[0089] S63. HE staining was performed on the tumor tissues to observe the morphology, mitotic figures of the tumor cells, and the structural characteristics of the tumor tissues, and to evaluate the effect of knocking down CYP1B1 on the proliferation of tumor cells and the tumor tissue structure;
[0090] S64. IHC staining was performed on the tumor tissues to detect the expression and localization of key proteins in the tumor tissues, further verify the results of qPCR and Western blot, and observe the distribution and expression levels of these proteins in the tumor tissues.
[0091] Furthermore, the specific content of the synergistic effect of quercetin combined with Palbociclib on the treatment of QNBC in S7 includes:
[0092] S71. Nude mice aged 4 to 6 weeks were selected and divided into 4 groups (231-ctrl, 231-QUE, 231-Pal, 231-Pal+QUE), with 6 mice in each group. The mice were inoculated subcutaneously under the axilla with 5×106 cells per mouse, and the body weight and tumor volume of the mice were observed and measured every 3 days;
[0093] S72. RNA and protein were taken from the tumor tissues of the mice, and qPCR and Western blot were used to detect the expression of CYP1B1 and AR in the tumor tissues. The expression changes of related pathway proteins were detected;
[0094] S73. Histological examination was used to observe the morphological changes of tumor tissues, including cell size, morphology, nuclear-cytoplasmic ratio, mitotic figures, etc., to evaluate the anti-tumor effect of the drug.
[0095] S74. Immunohistochemistry was used to select specific antibodies related to tumor proliferation, apoptosis, angiogenesis, etc., such as Ki-67, TUNEL, CD31 or CD34; through immunohistochemical staining, the expression changes of these markers in tumor tissues after drug treatment were observed to evaluate the anti-tumor effect of the drug.
[0096] The present invention comprehensively applies various technical means such as network pharmacology, molecular docking technology, bioinformatics analysis, molecular biology experiments, cell biology experiments, animal experiments, etc., to comprehensively analyze the action mechanisms of key molecules such as CYP1B1 and AR in QNBC from multiple levels including molecules, cells, tissues, animals, etc. This comprehensive multi-level research method improves the accuracy and reliability of the research.
[0097] The present invention provides a method for treating triple-negative breast cancer by quercetin inhibiting CYP1B1 combined with Palbociclib. The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for treating quadruple-negative breast cancer by inhibiting CYP1B1 with quercetin combined with Palbociclib, characterized in that: The method includes: S1. Use the HERB Materia Medica Database to screen relevant Chinese herbal medicines for the treatment of TNBC and obtain the active ingredients and their targets; S2. Effect of quercetin on inhibiting the expression of CYP1B1 in QNBC cell line; S3. The mechanism of the effect of CYP1B1 knockdown on AR expression in QNBC cell line; S4. Analysis of the mechanism of QNBC cell line insensitivity to Palbociclib; S5, improves the sensitivity of QNBC to Palbociclib by inhibiting CYP1B1; S6. Knockdown of CYP1B1 can inhibit the growth of quadruple-negative breast cancer xenografts in nude mice and improve the efficacy of Palbociclib. S7. Quercetin combined with Palbociclib has a synergistic effect in the treatment of QNBC.
2. The method of claim 1 for treating quadruple-negative breast cancer by inhibiting CYP1B1 with quercetin combined with Palbociclib, characterized in that: The process of screening using the HERB Materia Medica database described in S1 includes: S11. Use disease partitioning to screen out relevant Chinese herbal medicine sets that can treat ER-, PR-, and HER2-breast cancer, and take the intersection of the three data sets to screen out Chinese herbal medicines that can treat ER-, PR-, and HER2-breast cancer at the same time; S12. The active ingredients of Chinese herbal medicines are retrieved based on the TCMSP database and analysis platform for Chinese herbal medicine pharmacology, and the bioavailability and oral bioavailability of potential drug molecules are predicted according to Lipinski's Rule of Five. The molecules are preliminarily evaluated in the early screening stage to screen out potential candidate drug molecules; S13, the active ingredients of each drug were screened for corresponding targets using the small molecule drug target online prediction database platform Swiss TargetPrediction; S14. Substitute the active ingredients and targets of the drugs into the Cytoscape software to construct a network diagram of traditional Chinese medicine-active ingredients-targets.
3. The method of claim 1 for treating quadruple-negative breast cancer by inhibiting CYP1B1 with quercetin combined with Palbociclib, characterized in that: The process described in S2 for inhibiting CYP1B1 in QNBC cell line with quercetin includes: S21, preparation of quercetin treatment group: IC50: 25.06 μmol / L (231) 24h; S22. Cell growth inhibition detection: 24 hours after drug addition, the growth of cells in each group was detected by CCK-8 kit, and the cell growth inhibition rate was recorded; S23, Western blot detection: the inhibitory effect of quercetin on the expression level of CYP1B1 and the activating effect on AR expression; S24. Scratch experiment: Scratch was made on the cell monolayer, and the migration of each group of cells after scratch was observed and recorded to evaluate the effect of quercetin on the migration ability of QNBC cells after inhibiting CYP1B1.
4. The method of claim 1 for treating quadruple-negative breast cancer by inhibiting CYP1B1 with quercetin combined with Palbociclib, characterized in that: The process of knocking down CYP1B1 on AR expression in QNBC cell line described in S3 includes: S31. Construction of stable cell lines: Knockdown the expression of CYP1B1 in human breast cancer cell lines MDA-MB-231 and MDA-MB-453, construct CYP1B1-KD cell lines, and culture them in DMEM high-glucose medium containing 10% FBS. The knockdown efficiency and the effect of CYP1B1 knockdown on AR expression were detected by Western blot and qPCR.
5. The method of claim 1 for treating quadruple-negative breast cancer by inhibiting CYP1B1 with quercetin combined with Palbociclib, characterized in that: The analytically insensitive processes described in S4 include: S41. Grouping: 231-DMSO and 231-Pal, treatment for 72 hours, CCK-8 detection of absorbance of the two groups, calculation and comparison of cell survival rates of DMSO group and Palbociclib group, to obtain the effect of Palbociclib monotherapy on proliferation of 231 cell line; S42. After 72 hours of drug treatment, the two groups of cells were collected, resuspended in PBS, centrifuged at 300g for 5 minutes, the supernatant was discarded, and 70% ethanol was added to resuspend the samples, fixed the samples for 2 hours, centrifuged at 300g for 5 minutes, the supernatant was discarded, washed again with PBS, centrifuged and the supernatant was discarded, the prepared PI dye solution (containing RNase) was added to resuspend the samples, and the samples were incubated in a 37°C water bath for 30 minutes. Flow cytometry was used to detect the effect of Palbociclib monotherapy on the cell cycle; S43. After 72 hours of drug treatment, two groups of samples, 231-DMSO and 231-Pal, were collected, and transcriptome sequencing was used to analyze the mechanism of Palbociclib's insensitivity to QNBC alone.
6. The method of claim 1 for treating quadruple-negative breast cancer by inhibiting CYP1B1 with quercetin combined with Palbociclib, characterized in that: The process of increasing sensitivity described in S5 includes: S51. Four groups of cell lines were tested, namely 231-DMSO, 231-Pal, 231-QUE, and 231-Pal+QUE, among which Pal: IC50: 1 μM, 3 days; QUE: IC50: 20 μmol / L, 3 days; S52, 72h after drug treatment, CCK-8 was used to detect the effects of the four groups of treatment on the survival rate of 231 cell lines; S53, flow cytometry analysis of the effects of the four groups of treatments on cell cycle and apoptosis; S54 and CK-8 were used to detect the effect of quercetin combined with palbociclib on the proliferation of MDA-MB-231 cell line.
7. The method of claim 1 for treating quadruple-negative breast cancer by inhibiting CYP1B1 with quercetin combined with Palbociclib, characterized in that: As described in S6, knocking down CYP1B1 can inhibit the growth of quadruple-negative breast cancer xenografts in nude mice, and the specific contents of improving the efficacy of Palbociclib include: S61. Nude mice aged 4 to 6 weeks were selected and divided into 4 groups (NC-ctrl, NC-Pal, sh-ctrl, sh-Pal), with 6 mice in each group. 5×106 cells were injected into the subcutaneous tissue of the axilla of each mouse to form tumors. The weight of the mice and the tumor volume were observed and measured every 3 days. S62, extract RNA and protein from mouse tumor tissues, and detect the expression of CYP1B1 and AR in tumor tissues by qPCR and Western blot; S63. HE staining was performed on tumor tissues to observe the morphology, nuclear division figures and structural characteristics of tumor cells, and to evaluate the effects of knocking down CYP1B1 on tumor cell proliferation and tumor tissue structure; S64. Perform IHC staining on tumor tissues to detect the expression and localization of key proteins in tumor tissues, further verify the results of qPCR and Western blot, and observe the distribution and expression levels of these proteins in tumor tissues.
8. The method of claim 1 for treating quadruple-negative breast cancer by inhibiting CYP1B1 with quercetin combined with Palbociclib, characterized in that: The specific contents of the synergistic effect of quercetin combined with Palbociclib on the treatment of QNBC described in S7 include: S71. Nude mice aged 4 to 6 weeks were selected and divided into 4 groups (231-ctrl, 231-QUE, 231-Pal, 231-Pal+QUE), with 6 mice in each group. 5×106 cells were injected into the subcutaneous tissue of the axilla of each mouse to form tumors. The weight and tumor volume of the mice were observed and measured every 3 days. S72. RNA and protein were collected from mouse tumor tissues, and qPCR and Western blot were used to detect the expression of CYP1B1 and AR in tumor tissues. Changes in the expression of proteins in related pathways were detected; S73. Histological examination to observe the morphological changes of tumor tissue, including cell size, morphology, nuclear-cytoplasmic ratio, nuclear division figures, etc., to evaluate the anti-tumor effect of the drug; S74. Immunohistochemistry: select specific antibodies related to tumor proliferation, apoptosis, angiogenesis, etc., such as Ki-67, TUNEL, CD31 or CD34; observe the expression changes of these markers in tumor tissues after drug treatment through immunohistochemical staining to evaluate the anti-tumor effect of the drug.