Method for treating tetra-negative breast cancer by inhibiting CYP1B1 through quercetin and combining with Palbociclib

Abstract

The invention provides a method for treating tetra-negative breast cancer through combination of quercetin inhibiting CYP1B1 and Palbociclib, which comprises the following steps: verifying the influence of AR expression quantity on the curative effect of a CDK4 / 6 inhibitor in TNBC, screening related Chinese herbal medicine sets for treating TNBC by applying an HERB herbal medicine identification database, obtaining effective components and target spots thereof, analyzing the influence of quercetin inhibiting CYP1B1 expression in a QNBC cell line, and determining the curative effect of the CYP1B1 in the QNBC cell line. By knocking down an influence mechanism of CYP1B1 on AR expression in a QNBC cell line, analyzing a mechanism that the QNBC cell line is insensitive to Palbociclib and improving sensitivity, starting from the key target gene CYP1B1, how to improve the sensitivity of the QNBC cell line by regulating and controlling AR, Rb and other genes and related signal channels is deeply analyzed, so that the QNBC cell line is obtained. The multi-dimensional and multi-level research thought is beneficial to more comprehensive and deep understanding of the pathogenesis and the treatment target of QNBC; according to the present invention, the QNBC inhibition effect of quercetin is verified, and through the synergistic effect of the combination of the traditional Chinese medicine effective components and the clinical first-line western medicines, the new thought and the new drug selection are provided for the QNBC treatment, and the innovativeness and the practicability are provided;

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] Breast cancer, as the most common cancer in women, has become the main cause of cancer death in women. In 2022, the number of newly diagnosed cases of breast cancer in women worldwide exceeded 2.3 million, accounting for about 11.6% of all newly diagnosed cancer patients. In recent years, the incidence and mortality of breast cancer in China have shown an increasing trend year by year. In 2022, the number of newly diagnosed cases of breast cancer in Chinese women was about 357,200, and the number of death cases was about 75,000. Triple-negative breast cancer refers to a special type of breast cancer in which estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 are all negative. There are 170,000 cases of TNBC worldwide every year. Patients have a poor prognosis and overall survival, and the tumor cells are highly invasive, with a high metastasis and recurrence rate. At present, some scholars have proposed to divide TNBC into AR-positive TNBC and AR-negative TNBC according to the expression of androgen receptor. Among them, AR-TNBC is also named quadruple-negative breast cancer. As a subtype of TNBC, it accounts 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, the treatment effect is limited, and the prognosis is poor. This makes the treatment of QNBC particularly difficult and becomes a difficult point in the current treatment of breast cancer.

[0004] At present, the treatment methods of 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 metastases. 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 current treatment of QNBC. Summary of the Invention

[0005] The present invention provides 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: S1. Use the HERB Materia Medica Group Identification Database to screen the relevant Chinese herbal medicine sets for treating TNBC, and obtain the active ingredients and their targets; S2. Investigate the effect of quercetin on inhibiting the expression of CYP1B1 in the QNBC cell line; S3. Explore the mechanism of the effect of knocking down CYP1B1 on the expression of AR in the QNBC cell line; S4. Analyze the mechanism of the QNBC cell line being insensitive to Palbociclib; S5. Improve the sensitivity of QNBC to Palbociclib by inhibiting CYP1B1; S6. Knocking down CYP1B1 can inhibit the growth of triple-negative breast cancer xenografts in nude mice and improve the efficacy of Palbociclib; S7. Quercetin combined with Palbociclib has a synergistic effect on the treatment of QNBC.

[0007] As a preferred embodiment of the method, the process of screening using the HERB Materia Medica Group Identification Database in S1 includes: 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; 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; S13. Screen the corresponding targets for each drug active ingredient using the online prediction database platform for small molecule drug targets, Swiss TargetPrediction; S14. Substitute the drug active ingredients and their action targets into the Cytoscape software to construct a traditional Chinese medicine-active ingredient-target network diagram.

[0008] As a preferred embodiment of the method, the process of quercetin inhibiting CYP1B1 in the QNBC cell line in S2 includes: S21. Preparation of the quercetin treatment group: IC50: 25.06 μmol / L (231) for 24 h; S22. Detection of cell growth inhibition: After 24 h of adding the drug, the growth of cells in each group was detected by the 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 assay: Scratches were made on the cell monolayer, and the migration of cells in each group after scratching was observed and recorded to evaluate the effect of quercetin on the migration ability of QNBC cells after inhibiting CYP1B1.

[0009] As a preferred embodiment of the method, the process of knocking down CYP1B1 on AR expression in the QNBC cell line described in S3 includes: S31. Construction of a stable cell line: The expression of CYP1B1 was knocked down in the human breast cancer cell lines MDA-MB-231 and MDA-MB-453 to construct the CYP1B1-KD cell line, which was cultured in DMEM high-glucose medium containing 10% FBS. The knockdown efficiency and the effect of knocking down CYP1B1 on AR expression were detected by Western blot and qPCR.

[0010] As a preferred embodiment of the method, the process of analyzing insensitivity described in S4 includes: S41. Grouping: 231-DMSO, 231-Pal. After 72 h of drug treatment, the absorbance values of the two groups were detected by CCK-8. The cell survival rates of the DMSO group and the Palbociclib group were calculated and compared to obtain the effect of Palbociclib alone on the proliferation of the 231 cell line. S42. After 72 h of drug treatment, the cells in the two groups were collected respectively, resuspended in PBS, centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in 70% ethanol and fixed for 2 h. Then, they were centrifuged at 300 g for 5 min, the supernatant was discarded, washed once with PBS, centrifuged and the supernatant was discarded. The cells were resuspended in the prepared PI staining solution (containing RNase), incubated in a water bath at 37 °C for 30 min, and the effect of Palbociclib alone on the cell cycle was detected by flow cytometry. S43. After 72 h of drug treatment, the two samples 231-DMSO and 231-Pal were collected, and transcriptome sequencing was performed to analyze the mechanism of insensitivity of Palbociclib in the treatment of QNBC alone.

[0011] As a preferred embodiment of the method, the process of improving sensitivity described in S5 includes: 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. S52. After drug treatment for 72 h, detect the effect of the four treatments on the survival rate of the 231 cell line by CCK-8. S53. Analyze the effects of the four treatments on the cell cycle and apoptosis by flow cytometry. S54. Detect the effect of the combination of quercetin and palbociclib on the proliferation of the MDA-MB-231 cell line by CK-8.

[0012] As a preferred embodiment of the method, 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 described in S6 includes: 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 per mouse subcutaneously under the armpit of the mice to form tumors. Observe and measure the body weight and tumor volume of the mice every 3 days. S62. Extract RNA and protein from the tumor tissues of the mice, and detect the expression of CYP1B1 and AR in the tumor tissues by qPCR and Western blot. S63. Perform HE staining on the tumor tissues, 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. S64. Perform IHC staining on the tumor tissues, 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.

[0013] As a preferred embodiment of the method, the specific content of the synergistic effect of quercetin combined with Palbociclib on the treatment of QNBC described in S7 includes: S71. Select nude mice aged 4 to 6 weeks, 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 armpit of the mice to form tumors. Observe and measure the body weight and tumor volume of the mice every 3 days. S72. Take RNA and protein from the tumor tissues of the mice, and detect the expression of CYP1B1 and AR in the tumor tissues by qPCR and Western blot. Detect the changes in the expression of related pathway proteins. S73. Histological examination was performed 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. 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.

[0014] 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.

[0015] Quercetin verifies its inhibitory effect on QNBC, and through the synergistic effect of the active ingredients of traditional Chinese medicine in combination with the current first-line western medicine in clinical practice, it provides new ideas and drug options for the treatment of QNBC, and has innovation and practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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 following drawings 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.

[0017] Figure 1 Schematic diagram of 11 traditional Chinese medicines for HERB screening in the treatment of TNBC of the present invention; Figure 2 Schematic diagram of the id, name and meridian tropism of traditional Chinese medicines for HERB screening in the treatment of TNBC of the present invention; Figure 3 Schematic diagram of the molecular docking results of the present invention (left: quercetin, right: kaempferol); Figure 4 Schematic diagram of the IC50 value of quercetin detected by CCK-8 in the MDA-MB-231 cell line of the present invention; Figure 5 Schematic diagram of the activation effect of AR expression after knocking down CYP1B1 of the present invention; Figure 6 Schematic diagram of the effect of adding drugs for 72 h on the cell cycle of MDA-MB-231 cells (left: DMSO, right: Pal) of the present invention; Figure 7Schematic diagram of the effect of the combination of quercetin and palbociclib on the apoptosis of MDA-MB-231 cell line detected by flow cytometry according to the present invention; Figure 8 Schematic diagram of the effect of the combination of quercetin and palbociclib on the proliferation of MDA-MB-231 cell line detected by cell colony formation assay according to the present invention. Detailed implementation manners

[0018] 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 creative efforts shall fall within the protection scope of the present invention.

[0019] A method for treating triple-negative breast cancer by quercetin inhibiting CYP1B1 combined with Palbociclib, the method comprising: S1. Use the HERB herbal group identification database to screen relevant Chinese herbal medicine sets for treating TNBC, and obtain the active ingredients and their targets; S2. Influence of quercetin on inhibiting the expression of CYP1B1 in QNBC cell line; S3. Influence mechanism of knocking down CYP1B1 on the expression of AR in QNBC cell line; S4. Analyze the mechanism of insensitivity of QNBC cell line to Palbociclib; S5. Improve the sensitivity of QNBC to Palbociclib by inhibiting CYP1B1; S6. Knocking down CYP1B1 can inhibit the growth of triple-negative breast cancer xenograft tumors in nude mice and improve the efficacy of Palbociclib; S7. Quercetin combined with Palbociclib has a synergistic effect on the treatment of QNBC.

[0020] As Figure 1-3 shown, the process of screening using the HERB herbal group identification database in S1 includes: S11. Respectively screen out relevant Chinese herbal medicine sets that can treat ER-, PR-, and HER2-breast cancers through the disease partition, and take the intersection of the three data sets to screen out 11 Chinese herbal medicines that can simultaneously treat ER-, PR-, and HER2-breast cancers; S12. The active ingredients of Chinese herbal medicines are retrieved based on the Traditional Chinese Medicine Pharmacology Database and Analysis Platform TCMSP, 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 corresponding targets (possible > 0.3) of the active ingredients of each drug are screened using the online prediction database platform for small molecule drug targets, Swiss TargetPrediction; S14. The active ingredients of the drug and their action targets are substituted into the Cytoscape software to construct a traditional Chinese medicine-active ingredient-target network diagram.

[0021] As Figure 4 shown, the process of quercetin inhibiting CYP1B1 in the QNBC cell line in S2 includes: S21. Prepare the quercetin treatment group: IC50: 25.06 μmol / L (231) for 24 h; S22. Detection of cell growth inhibition: After adding the drug for 24 h, the growth of cells in each group is detected by the CCK-8 kit, and the cell growth inhibition rate is recorded; S23. Western blot detection: The inhibitory effect of quercetin on the expression level of CYP1B1 and the activating effect on the expression of AR; S24. Scratch assay: Make scratches on the cell monolayer, observe and record the migration of cells in each group after scratching to evaluate the effect of quercetin inhibiting CYP1B1 on the migration ability of QNBC cells.

[0022] As Figure 5 shown, the process of knocking down CYP1B1 on the expression of AR in the QNBC cell line in S3 includes: S31. Construct 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 construct 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.

[0023] As Figure 6 shown, the process of analyzing insensitivity in S4 includes: S41. Grouping: 231-DMSO, 231-Pal, add the drug and treat 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; S42. After 72 h of drug addition treatment, cells in the two groups were collected respectively, resuspended with PBS, centrifuged at 300 g for 5 min, the supernatant was discarded, resuspended with 70% ethanol, fixed for 2 h, centrifuged at 300 g for 5 min, the supernatant was discarded, washed once with PBS again, centrifuged and the supernatant was discarded, resuspended with the prepared PI staining solution (containing RNase), incubated in a water bath at 37 °C for 30 min, and the effect of Palbociclib monotherapy on the cell cycle was detected by flow cytometry; S43. After 72 h of drug addition treatment, two samples, 231-DMSO and 231-Pal, were collected, and transcriptome sequencing was performed to analyze the mechanism of insensitivity of Palbociclib in the treatment of QNBC alone.

[0024] As Figure 7-8 shown, the process of improving sensitivity in S5 includes: S51. Four cell lines were detected, namely 231-DMSO, 231-Pal, 231-QUE, and 231-Pal+QUE. Among them, for Pal, IC50: 1 μM, for 3 days; for QUE, IC50: 20 μmol / L, for 3 days; S52. After 72 h of drug addition treatment, the effect of the four treatments on the viability of 231 cell line was detected by CCK-8; S53. Flow cytometry was used to analyze the effects of the four treatments on the cell cycle and apoptosis; S54. CCK-8 was used to detect the effect of the combination of quercetin and palbociclib on the proliferation of MDA-MB-231 cell line.

[0025] Furthermore, the specific content of knocking down CYP1B1 in S6 to inhibit the growth of xenograft tumors in nude mice with triple-negative breast cancer and improve the efficacy of Palbociclib includes: 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. Tumors were formed by injecting 5×106 cells subcutaneously into the axilla of each mouse. The body weight and tumor volume of the mice were observed and measured every 3 days; S62. RNA and protein were extracted from the tumor tissues of the mice, and the expression levels of CYP1B1 and AR in the tumor tissues were detected by qPCR and Western blot; S63. HE staining was performed on the tumor tissues to observe the morphology, mitotic figures of tumor cells and the structural characteristics of the tumor tissues, and to evaluate the effect 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.

[0026] Furthermore, the specific content of the synergistic effect of quercetin combined with Palbociclib on QNBC treatment in S7 includes: S71. Select nude mice aged 4 to 6 weeks 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 every 3 days. S72. Take the RNA and protein of mouse tumor tissues, and detect the expression of CYP1B1 and AR in tumor tissues by qPCR and Western blot. Detect the changes in the expression of related pathway proteins. S73. Observe the morphological changes of tumor tissues by histological examination, including cell size, morphology, nuclear-cytoplasmic ratio, mitotic figures, etc., and evaluate the anti-tumor effect of the drug. 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 tumor tissues after drug treatment by immunohistochemical staining, and evaluate the anti-tumor effect of the drug.

[0027] The present invention comprehensively applies various technical means such as network pharmacology, molecular docking technology, bioinformatics analysis, molecular biology experiments, cell biology experiments, and animal experiments to comprehensively analyze the action mechanisms of key molecules such as CYP1B1 and AR in QNBC from multiple levels including molecules, cells, tissues, and animals. This comprehensive multi-level research method improves the accuracy and reliability of the research.

[0028] The present invention provides a method for treating triple-negative breast cancer by inhibiting CYP1B1 with quercetin combined with Palbociclib. The above is only the preferred embodiment of the present invention and is 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 within 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 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 treatment groups 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. Take RNA and protein from mouse tumor tissues, and use qPCR and Western blot to detect the expression of CYP1B1 and AR in tumor tissues. Detect changes in the expression of proteins in related pathways; 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.

Images