A method for constructing endocrine therapy resistance in breast cancer and screening drugs

Abstract

The present invention discloses a method for constructing endocrine therapy resistance of breast cancer and screening drugs, which relates to the field of medical technology, including: 1) obtaining CDK4 / 6 inhibitor-resistant cells and fulvestrant-resistant cells by constructing resistant breast cancer cells; 2) taking CDK4 / 6 inhibitor-resistant cells and fulvestrant-resistant cells and MCF-7 parent strains for mixed inoculation, adding drugs to be screened for treatment and cultivation, and using a living cell analysis system with a 10X objective lens to analyze the cell proliferation state, and performing drug screening by heat map. The method can obtain a variety of new drugs that can be used to overcome endocrine therapy resistance, and in particular discloses the use of I-BRD9 or SGC-CBP30 or UNC1999 alone and in combination, which can significantly inhibit the growth of resistant cells and effectively treat CDK4 / 6 inhibitor-acquired or fulvestrant-acquired resistant breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a method for constructing endocrine therapy resistance for breast cancer and screening drugs. Background Art

[0002] CDK4 / 6 inhibitors and fulvestrant are the preferred first-line treatments for ER-positive advanced breast cancer. After first-line treatment progresses, the American Society of Clinical Oncology (ASCO) guidelines recommend PIK3CA inhibitors or mTOR inhibitors. However, these two drugs are not available or approved for breast cancer indications in my country, leaving patients with limited options for chemotherapy, which carries greater side effects. New drugs are urgently needed to overcome resistance to CDK4 / 6 inhibitors and fulvestrant. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for constructing endocrine therapy resistance in breast cancer and screening drugs. The method can obtain a variety of drugs that can be used to overcome endocrine therapy resistance. Disclosed is I-BRD9 or SGC-CBP30 or UNC1999, used alone or in combination, which can significantly inhibit the growth of resistant cells and effectively treat CDK4 / 6 inhibitor-acquired or fulvestrant-acquired breast cancer.

[0004] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0005] A method for establishing endocrine therapy resistance in breast cancer and screening drugs, comprising:

[0006] 1) Generate CDK4 / 6 inhibitor-resistant cells and fulvestrant-resistant cells by constructing drug-resistant breast cancer cells;

[0007] 2) Inoculate CDK4 / 6 inhibitor-resistant cells, fulvestrant-resistant cells, and the parental MCF-7 strain, add the drug to be screened, and analyze cell proliferation using a live cell analysis system with a 10X objective lens to screen for drugs that are sensitive to the resistant cells.

[0008] The drugs include BRD9 inhibitors or CBP / p300 inhibitors or EZH1 / 2 inhibitors. The present invention successfully constructed a breast cancer cell line resistant to CDK4 / 6 inhibitors and Fulvestrant by inducing ER-positive MCF-7 breast cancer cells, and screened with a small molecule drug library to obtain a variety of new drugs that can be used to overcome endocrine therapy resistance. The method provided by the present invention is convenient and fast, and is suitable for rapid screening of drugs; the screened drugs have a sensitizing effect and enhanced cancer treatment effect on drug-resistant breast cancer cells, such as CDK4 / 6 inhibitor-resistant cells and Fulvestrant-resistant cells. At the same time, according to the screening method provided by the present invention, matching sensitizing drugs can be accurately, quickly and efficiently obtained for specific drug-resistant drugs, which is more conducive to precision medical treatment for patients with specific drug-resistant breast cancer, and has broad application prospects.

[0009] Furthermore, the above method is specifically as follows:

[0010] Step 1: Induction of drug-resistant cell lines

[0011] MCF-7 breast cancer cells in the logarithmic growth phase were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. After the MCF-7 breast cancer cells adhered and grew normally, a CDK4 / 6 inhibitor was added to the culture medium to bring the concentration to 0.5 μM. The culture medium was replaced every 2-3 days to maintain the concentration of the CDK4 / 6 inhibitor. Initially, a large number of adherent cells were killed and floated into the culture medium, and only a few cells remained adherent. After several changes of culture medium, only a few adherent cells remained in the culture dish. The cell growth status was observed regularly until cells began to proliferate. When MCF-7 cells showed a sustained proliferation state at a CDK4 / 6 inhibitor concentration of 0.5 μM, they were passaged at a ratio of 1:2 to 1:3 and the inhibitor concentration was maintained to obtain a cell line that could be stably passaged. The concentration of CDK4 / 6 inhibitor was gradually increased from 0.5 μM to 2 μM. MCF-7 breast cancer cells could still proliferate and be stably passaged at high concentrations. The sensitivity of the cell line at this time to the parental line of CDK4 / 6 inhibitor was tested by the MTT method. When the difference in IC50 reached 3-5 times or more, the drug-resistant cell line was considered to be successfully constructed.

[0012] Following the above method, fulvestrant-resistant MCF-7 breast cancer cells were also successfully constructed, with the final drug maintenance concentration being 1 μM;

[0013] Step 2: Drug Screening

[0014] 1000 breast cancer cells were seeded per well on a multi-well plate. After the cells adhered to the plate, a small molecule inhibitor dissolved in DMSO was added to a final concentration of 5 μM; DMSO served as a control. The cells were then placed in an incubator for 5 days, and the cell proliferation status was analyzed using a live cell analysis system with a 10X objective lens to screen for drugs that were sensitive in drug-resistant cells.

[0015] The present invention further discloses the use of a BRD9 inhibitor or a CBP / p300 inhibitor or an EZH1 / 2 inhibitor in the preparation of a drug for treating CDK4 / 6 inhibitor acquired-resistant cells and / or fulvestrant acquired-resistant cells. The present invention discloses the use of a BRD9 inhibitor or a CBP / p300 inhibitor or an EZH1 / 2 inhibitor alone, as well as the combined use of a CBP / p300 inhibitor and an EZH1 / 2 inhibitor, as a CDK4 / 6 inhibitor acquired-resistant cell inhibitor and / or fulvestrant acquired-resistant cell inhibitor, which can significantly inhibit the growth of resistant cells, effectively treat CDK4 / 6 inhibitor acquired and / or fulvestrant acquired-resistant breast cancer, and the combined use has a better therapeutic effect; the drug raw materials screened by the present invention are easy to obtain and the method of use is simple. The medication regimen provided by the present invention can effectively reverse CDK4 / 6 inhibitor resistance and / or fulvestrant resistance, provide a new medication regimen for the treatment of resistant breast cancer, and provide a new idea for clinical treatment.

[0016] Furthermore, the CDK4 / 6 inhibitor acquired-resistant cells or fulvestrant acquired-resistant cells are obtained by drug screening and culture of MCF-7 cells.

[0017] Furthermore, the dosage of I-BRD9, SGC-CBP30, or UNC1999 is the standard clinical dosage.

[0018] It should be noted that the above-mentioned drug is administered to patients at a dose of 200-500 mg / day.

[0019] Furthermore, the above-mentioned drug is administered to the patient at a dose of 200 mg / day, 300 mg / day, 400 mg / day or 500 mg / day.

[0020] Drugs screened based on the methods of the present invention can be administered to a patient once or multiple times. In those embodiments in which the compounds are administered multiple times, they can be administered at set intervals, such as daily, every other day, weekly, or monthly. Alternatively, they can be administered at irregular intervals, for example, as needed based on symptoms, patient health, and the like. A therapeutically effective amount of the combination can be administered once daily (qd) for 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 15 days.

[0021] It should be noted that the administration routes include, but are not limited to, topical administration, oral administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intravesical infusion, subcutaneous administration, transdermal administration, and transmucosal administration.

[0022] Furthermore, CDK4 / 6 inhibitors include Palbociclib.

[0023] Furthermore, BRD9 inhibitors include I-BRD9.

[0024] Furthermore, CBP / p300 inhibitors include SGC-CBP30.

[0025] Furthermore, EZH1 / 2 inhibitors include UNC1999.

[0026] The present invention also discloses the use of a combination of SGC-CBP30 and UNC1999 in the preparation of a drug for treating CDK4 / 6 inhibitor acquired-resistant cells and / or fulvestrant acquired-resistant cells.

[0027] The present invention further discloses the use of a ferulic acid derivative and an I-BRD9 inhibitor in combination for preparing a drug for treating CDK4 / 6 inhibitor acquired drug-resistant cells and / or fulvestrant acquired drug-resistant cells.

[0028] Furthermore, the ferulic acid derivative is a ferulic acid ester derivative, which is obtained by modifying ferulic acid with geranylgeraniol through an esterification reaction. The present invention uses geranylgeraniol to esterify ferulic acid to prepare a ferulic acid derivative, which has good biological activity and a certain inhibitory effect on the growth of drug-resistant cells. When used in combination with an I-BRD9 inhibitor, it can effectively treat CDK4 / 6 inhibitor-acquired drug-resistant breast cancer, and the combined use has a better therapeutic effect.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention discloses a method for constructing endocrine therapy resistance in breast cancer and screening drugs. CDK4 / 6 inhibitors and fulvestrant are the first-line drugs for the treatment of hormone receptor (ER)-positive advanced breast cancer. When treatment resistance occurs, the available drugs are still very limited. The present invention first constructs a drug-resistant cell line of the two drugs. By using large doses of the drugs, the majority of the parental cells are killed, while the surviving cells are retained. By gradually increasing the drug concentration, cells that can be stably propagated are induced, and finally a drug-resistant cell line is obtained. By screening a library of multiple small molecule inhibitors, several new drugs that can be used after endocrine resistance are obtained, such as I-BRD9, SGC-CBP30, UNC1999 and other small molecule inhibitors with different mechanisms of action. The present invention constructs an endocrine-resistant breast cancer cell line and screens through a library of small molecule inhibitors to obtain a new drug that can be used to treat endocrine therapy resistance.

[0031] Therefore, the present invention provides a method for constructing endocrine therapy resistance in breast cancer and screening drugs. The method can obtain a variety of drugs that can be used to overcome endocrine therapy resistance. Disclosed is I-BRD9 or SGC-CBP30 or UNC1999 used alone or in combination, which can significantly inhibit the growth of resistant cells and effectively treat CDK4 / 6 inhibitor-acquired or fulvestrant-acquired breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a picture of cell proliferation during the process of inducing cell endocrine drug resistance;

[0033] Figure 2 These are the IC50 test results of the CDK4 / 6 inhibitor-resistant strain (CDK-MCF7) and the parental strain (MCF-7);

[0034] Figure 3 These are the IC50 test results of the fulvestrant-resistant strain (FUL-MCF7) and the parental strain (MCF-7);

[0035] Figure 4 It is a heat map for screening drugs that can be used after endocrine therapy resistance;

[0036] Figure 5 This is the cell proliferation curve test result of the I-BRD9 inhibitor validation experiment in fulvestrant-resistant cells;

[0037] Figure 6 The inhibitory effects of SGC-CBP30 and UNC1999 alone or in combination on CDK4 / 6 inhibitor-resistant cell lines;

[0038] Figure 7The inhibitory effect of SGC-CBP30 and UNC1999 alone or in combination on fulvestrant-resistant cell lines;

[0039] Figure 8 The inhibitory effect of ferulic acid derivatives and I-BRD9 inhibitors alone or in combination on CDK4 / 6 inhibitor-resistant cell lines;

[0040] Figure 9 This is the infrared spectrum test result of ferulic acid derivatives. DETAILED DESCRIPTION

[0041] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, each embodiment of the present invention will be described in detail below with reference to the embodiments. However, it will be understood by those skilled in the art that in each embodiment of the present invention, many technical details are provided to help readers better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical solutions claimed in the present application can still be implemented.

[0042] The MCF-7 breast cancer cell line used in the examples was obtained from ATCC. Preparation of the breast cancer cell line: MCF-7, ER-positive breast cancer cells, were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Fresh medium was replaced 2-3 times per week, and cells were trypsinized at room temperature for 1-2 minutes. The cells were passaged at a 1:2 to 1:3 ratio.

[0043] Endocrine drug preparation: CDK4 / 6 inhibitors were purchased from Selleck. Palbociclib (PD0332991) was manufactured under license from Pfizer, catalog number: S1579. Fulvestrant, an estrogen receptor (ER) antagonist, Fμlvesrant (ICI-182780), catalog number: S1191. All drugs were dissolved in DMSO.

[0044] The small molecule inhibitor library used for drug screening in the embodiments of the present invention contains a variety of different types of drugs, most of which are commercially available small molecule inhibitors.

[0045] Example 1:

[0046] A method for constructing endocrine therapy resistance in breast cancer and screening drugs:

[0047] Step 1: Induction of drug-resistant cell lines

[0048] Induction of CDK4 / 6 inhibitor-resistant cells: MCF-7 breast cancer cells in the logarithmic growth phase were taken and cultured in a 37°C, 5% CO2 incubator using DMEM medium containing 10% fetal bovine serum. When the MCF-7 breast cancer cells adhered to the culture medium and grew normally, CDK4 / 6 inhibitors were added to make the concentration in the culture medium reach 0.5μM. The culture medium was replaced every 2 days and the concentration of CDK4 / 6 inhibitors was maintained. Initially, a large number of adherent cells were killed and floated into the culture medium, and only a few cells remained adherent. After several changes of the culture medium, only a few adherent cells remained in the culture dish. The cell growth status was observed regularly until the cells began to proliferate, such as Figure 1 As shown in the figure (A represents the situation of adding a large dose of drug to retain adherent cells, B represents the situation of maintaining the drug concentration to observe the cells to show tolerance and begin to proliferate, and C represents the situation of rapid cell proliferation and increase in number for passaging), when MCF-7 cells show a sustained proliferation state at a CDK4 / 6 inhibitor concentration of 0.5μM, they are passaged at a ratio of 1:2 to 1:3 and the inhibitor concentration is maintained to obtain a cell line that can be stably passaged; gradually increase the CDK4 / 6 inhibitor concentration from 0.5μM to 2μM, and MCF-7 breast cancer cells can still maintain proliferation and stable passage at high concentrations. The sensitivity of the cell line and the parental line to the CDK4 / 6 inhibitor at this time is detected by the MTT method. When the difference in IC50 reaches 3-5 times or more, it can be determined that the drug-resistant cell line is successfully constructed, as shown in the figure. Figure 2 As shown;

[0049] Induction of Fulvestrant-resistant cells: Following the induction process of CDK4 / 6 inhibitors, the same method was used to construct Fulvestrant-resistant cells. First, a higher drug concentration (0.5 μM) was used to kill MCF-7 cells, retaining the adherent cells. After the proliferation was observed, the Fulvestrant concentration was maintained and gradually increased, and the final drug maintenance concentration was 1 μM. The IC50 of the resistant strain and the parent strain was detected by MTT assay, and the Fulvestrant-resistant cells were successfully constructed. Figure 3 As shown;

[0050] Step 2: Drug Screening

[0051] 1000 breast cancer cells were seeded per well of a 384-well plate: the parental MCF-7 cell line, a CDK4 / 6 inhibitor-resistant cell line, and a fulvestrant-resistant cell line. After cells adhered to the plate, small-molecule inhibitors dissolved in DMSO were added to a final concentration of 5 μM. Each inhibitor concentration was replicated four times, with DMSO serving as a control. The cells were then placed in an incubator for 5 days, and cell proliferation was analyzed using a live cell analysis system (Sartoriμs, Incμcyte S3) with a 10X objective. Figure 4A heat map was created to screen drugs that can be used after endocrine therapy resistance occurs (in the figure, X represents CDK4 / 6-resistant strains, Y represents fulvestrant-resistant strains, and Z represents the parental strain). From this, it was found that multiple drugs (such as I-BRD9, SGC-CBP30, and UNC1999) can be used as new options for the treatment of breast cancer after resistance to CDK4 / 6 inhibitors and fulvestrant.

[0052] Example 2:

[0053] Validation experiment of drug I-BRD9 inhibitor in fulvestrant-resistant cells

[0054] I-BRD9 inhibitors were selected for validation in fulvestrant-resistant cells. 1000 breast cancer cells were seeded per well on a 384-well plate: the parental MCF-7 cell line and the fulvestrant-resistant cell line. After cells adhered to the plate, small-molecule inhibitors (I-BRD9, JQ1) dissolved in DMSO were added to a final concentration of 5 μM. Each inhibitor concentration was replicated four times, with DMSO serving as a control. The cells were then placed in an incubator for 5 days, and cell proliferation was analyzed using a live cell analysis system (Sartorius, Incucyte S3) with a 10X objective.

[0055] The test results are as follows Figure 5 As shown in the cell proliferation curve, I-BRD9 has a more significant inhibitory effect on drug-resistant strains, verifying that I-BRD9 is more sensitive in the fulvestrant-resistant cell line (FUL-MCF) and can be used to prepare drugs to improve breast cancer resistance.

[0056] Example 3:

[0057] Study on the effect of SGC-CBP30 and UNC1999 alone or in combination on the growth inhibition of drug-resistant cells

[0058] CDK4 / 6 inhibitor-resistant cell lines or fulvestrant-resistant cell lines in the logarithmic growth phase were washed twice with PBS, added with DMEM medium containing 10% fetal bovine serum, and seeded into 96-well plates. The cell concentration per well was adjusted to 4 × 10 3 cells / well in a total volume of 100 μL; after culturing for 24 h, the cells were treated with SGC-CBP30 or UNC1999 alone or in combination for 6, 12, 24, and 48 h, respectively, with a final drug concentration of 5 μM; 20 μL of 5 mg / mL MTT working solution was added to each well, mixed well, and cultured at 37°C and 5% CO2 saturated humidity for 4 h, then 100 μL of triple solution (10% sodium dodecyl sulfate + 5% isobutanol + 0.12 M hydrochloric acid) was added, completely dissolved, and placed in a microplate reader to detect the absorbance value at 570 nm. All experiments were repeated four times.

[0059] The drug groups included: A1: SGC-CBP30; A2: UNC1999; A3: SGC-CBP30+UNC1999 (mass ratio 1:1).

[0060] The test results are as follows Figure 6-Figure 7 shown. Figure 6 The inhibitory effect of drug treatment on CDK4 / 6 inhibitor-resistant cell lines can be seen from the analysis in the figure. SGC-CBP30 and UNC1999 alone have excellent inhibitory effects on CDK4 / 6 inhibitor-resistant cell lines, and the combined use of SGC-CBP30 and UNC1999 has a better inhibitory effect on CDK4 / 6 inhibitor-resistant cell lines, which has a synergistic enhancement effect. Figure 7 As for the inhibitory effect of drug treatment on fulvestrant-resistant cell lines, the analysis in the figure shows that SGC-CBP30 and UNC1999 alone have excellent inhibitory effects on fulvestrant-resistant cell lines, and the combined use of SGC-CBP30 and UNC1999 has a better inhibitory effect on fulvestrant-resistant cell lines, which has a synergistic enhancement effect.

[0061] Compatibility calculation method

[0062] Calculate the q value according to Jin's formula, q=E A +B / (E A +E B -E A ×E B ) is used to determine whether the effect of two drugs combined is superior to that of either drug alone. If q < 0.55, the two drugs are significantly antagonistic; if 0.55 ≤ q < 0.85, the two drugs are antagonistic; if 0.85 ≤ q < 1.15, the two drugs have a simple additive effect; if 1.15 ≤ q < 20, the two drugs have a synergistic effect; and if q ≥ 20, the two drugs have a significantly enhancing effect.

[0063] from Figure 6 The data analysis showed that 48 hours after treatment, the viability reduction rates of drug-resistant cells in groups A1, A2, and A3 were 34.8%, 37.4%, and 69.3%, respectively, compared with those before treatment. According to the King's formula, q=69.3% / (34.8%+37.4%-34.8%×37.4%)=1.153, which complies with 1.15≤q<20, indicating that the combined use of SGC-CBP30 and UNC1999 has a synergistic enhancement effect on the inhibitory effect of CDK4 / 6 inhibitor-resistant cell lines.

[0064] from Figure 7The data analysis showed that 48 hours after treatment, the viability reduction rates of the drug-resistant cells in groups A1, A2, and A3 were 36.2%, 39.1%, and 74.3%, respectively, compared with those before treatment. According to the King's formula, q=74.3% / (36.2%+39.1%-36.2%×39.1%)=1.215, which complies with 1.15≤q<20, indicating that the combined use of SGC-CBP30 and UNC1999 has a synergistic enhancement effect on the inhibitory effect of fulvestrant-resistant cell lines.

[0065] Example 4:

[0066] Study on the effect of ferulic acid derivatives and I-BRD9 inhibitors alone or in combination on the growth inhibition of drug-resistant cells

[0067] The CDK4 / 6 inhibitor-resistant cell lines in the logarithmic growth phase were taken, washed twice with PBS, added with DMEM medium containing 10% fetal bovine serum, and seeded into 96-well plates. The cell concentration per well was adjusted to 4 × 10 3 cells / well in a total volume of 100 μL; after culturing for 24 h, the cells were treated with ferulic acid derivatives, I-BRD9 inhibitors, or a combination of drugs for 6, 12, 24, and 48 h, respectively, with a final drug concentration of 5 μM; 20 μL of 5 mg / mL MTT working solution was added to each well, mixed evenly, and cultured at 37°C and 5% CO2 saturated humidity for 4 h, then 100 μL of triple solution (10% sodium dodecyl sulfate + 5% isobutanol + 0.12 M hydrochloric acid) was added, completely dissolved, and placed in a microplate reader to detect the absorbance at 570 nm. All experiments were repeated four times.

[0068] The medication groups included: M1: ferulic acid derivative; M2: I-BRD9 inhibitor; M3: ferulic acid derivative + I-BRD9 inhibitor (mass ratio 1:1).

[0069] The test results are as follows Figure 8 As shown in the figure, the analysis shows that the I-BRD9 inhibitor alone has an excellent inhibitory effect on CDK4 / 6 inhibitor-resistant cell lines, while the ferulic acid derivative alone has a poor inhibitory effect on CDK4 / 6 inhibitor-resistant cell lines. However, the combined use of ferulic acid derivatives and I-BRD9 inhibitors has a better inhibitory effect on fulvestrant-resistant cell lines, achieving a synergistic enhancement effect.

[0070] Compatibility calculation method

[0071] Calculate the q value according to Jin's formula, q=E A +B / (E A +E B -E A ×E B) is used to determine whether the effect of two drugs combined is superior to that of either drug alone. If q < 0.55, the two drugs are significantly antagonistic; if 0.55 ≤ q < 0.85, the two drugs are antagonistic; if 0.85 ≤ q < 1.15, the two drugs have a simple additive effect; if 1.15 ≤ q < 20, the two drugs have a synergistic effect; and if q ≥ 20, the two drugs have a significantly enhancing effect.

[0072] from Figure 8 The data analysis showed that 48 hours after treatment, the viability reduction rates of drug-resistant cells in groups A1, A2, and A3 were 9.7%, 29.5%, and 64.2%, respectively, compared with those before treatment. According to King's formula, q=64.2% / (9.7%+29.5%-9.7%×29.5%)=1.767, which complies with 1.15≤q<20, indicating that the combined use of ferulic acid derivatives and I-BRD9 inhibitors has a synergistic enhancing effect on the inhibitory effect of CDK4 / 6 inhibitor-resistant cell lines.

[0073] Preparation of ferulic acid derivatives:

[0074] Ferulic acid is dissolved in acetone, and then geranylgeraniol and p-toluenesulfonic acid are added. The mixture is stirred in a water bath at 75-85°C for 10-12 hours. After the reaction is completed, the mixture is washed with distilled water, dried with anhydrous magnesium sulfate, and distilled under reduced pressure. The mixture is then separated and purified using a C18 column, and distilled under reduced pressure and dried to obtain a ferulic acid derivative.

[0075] Furthermore, the molar ratio of ferulic acid to geranylgeraniol is 1:1-1.2; and the amount of p-toluenesulfonic acid used accounts for 6-9wt% of the total amount of reactants.

[0076] In this embodiment, the preparation of ferulic acid derivatives is specifically as follows:

[0077] Ferulic acid is dissolved in acetone (at a concentration of 0.25 g / mL), followed by the addition of geranylgeraniol (the molar ratio of ferulic acid to geranylgeraniol is 1:1.2) and p-toluenesulfonic acid (the amount used is 7.8 wt% of the total reactants), and the mixture is stirred in an 80°C water bath for 12 hours. After the reaction is completed, the mixture is washed with distilled water, dried over anhydrous magnesium sulfate, and distilled under reduced pressure. The mixture is then separated and purified using a C18 column (gradient elution with a methanol solution of 10-60% concentration as the eluent, at a flow rate of 1 mL / min), and distilled under reduced pressure and dried to obtain a ferulic acid derivative.

[0078] The above ferulic acid derivatives were tested by infrared spectroscopy, and the results were as follows: Figure 9 As shown in the figure, we can see that 3000cm -1 ~2800cm -1 The characteristic absorption peaks of methyl and methylene are in the range of 1686 cm -1Nearby is the characteristic absorption peak of the carbonyl group in the ester group. Compared with the carbonyl peak position of the carboxyl group, there is an obvious red shift, indicating that the esterification reaction has occurred; 1595cm -1 The characteristic absorption peak of the double bond is near it. The above results indicate that the ferulic acid derivative was successfully prepared.

[0079] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0080] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Use of a CBP / p300 inhibitor and an EZH1 / 2 inhibitor in the preparation of a drug for treating CDK4 / 6 inhibitor acquired-resistant cells and / or fulvestrant acquired-resistant cells, wherein the CBP / p300 inhibitor is SGC-CBP30, the EZH1 / 2 inhibitor is UNC1999, the resistant cells are resistant breast cancer cells, and the CDK4 / 6 inhibitor is Palbociclib.

2. The use according to claim 1, characterized in that The CDK4 / 6 inhibitor acquired resistance cells or fulvestrant acquired resistance cells are obtained by MCF-7 cell drug screening culture.

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