Use of Ispins combined with tamoxifen in the treatment of breast cancer

Abstract

The present invention discloses the use of Ispance combined with tamoxifen in treating breast cancer, and belongs to the field of biomedical technology. The present invention provides an application of a pharmaceutical composition in the preparation of a drug for preventing and / or treating tamoxifen-resistant ERα-positive breast cancer; the active ingredients of the pharmaceutical composition are Ispance and tamoxifen. Experimental results show that Ispance combined with tamoxifen significantly inhibits the cell proliferation activity of tamoxifen-resistant breast cancer cells; further, Ispance combined with tamoxifen significantly inhibits the proliferation activity of organoids derived from tamoxifen-resistant breast cancer patients. It can be seen that Ispance has a positive application prospect in improving tamoxifen resistance in breast cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of Ispins combined with tamoxifen in treating breast cancer. Background Art

[0002] Breast cancer ranks first in the incidence and mortality of female cancers worldwide. About 70% of breast cancer patients are estrogen receptor (ER) positive (+). Such patients are sensitive to endocrine therapy, and tamoxifen (Tam) is their first-line clinical drug. Tam competitively binds to ER and blocks estrogen signals, thereby inhibiting the development of breast cancer. Tam is low-toxic, economical, and has a long-lasting effect, which effectively improves the prognosis of ER+ patients. However, about 30-50% of patients still experience recurrence and distant metastasis, mainly due to primary or acquired Tam resistance.

[0003] The possible mechanisms of tamoxifen resistance include multiple aspects. First, genetic mutations or expression changes of estrogen receptor (ER) may result in the inability of drugs to effectively bind to and inhibit ER. Second, abnormal activation of intracellular signaling pathways such as PI3K / Akt and MAPK can bypass ER and promote cell proliferation and survival. In addition, polymorphisms of drug-metabolizing enzymes such as CYP2D6 can affect the metabolism of tamoxifen and the production of active products, reducing the efficacy. Overexpression or abnormal activation of auxiliary signaling molecules such as HER2 and EGFR can also enhance cell proliferation and anti-apoptosis ability, leading to drug failure. Epigenetic changes (such as DNA methylation, histone modification, and abnormal expression of non-coding RNA) and abnormal expression or activation of cell cycle regulatory proteins can also lead to drug resistance. These mechanisms work together to reduce or eliminate the efficacy of tamoxifen. Developing new treatment strategies for tamoxifen resistance is crucial to improving the survival prognosis of breast cancer patients. Summary of the invention

[0004] The purpose of the present invention is to overcome the tamoxifen resistance of breast cancer and provide the use of Ispins combined with tamoxifen to treat breast cancer.

[0005] The present invention first provides an application of a pharmaceutical composition in the preparation of a drug for preventing and / or treating ERα-positive breast cancer;

[0006] The active ingredients of the pharmaceutical composition are ispins and tamoxifen.

[0007] The present invention also provides an application of a pharmaceutical composition in the preparation of a drug for preventing and / or treating tamoxifen-resistant ERα-positive breast cancer;

[0008] The active ingredients of the pharmaceutical composition are ispins and tamoxifen.

[0009] In the above application, the molar ratio of Ispence to Tamoxifen is 1:5-1:10000; specifically, it can be 1:500-1:8000; more specifically, it can be 1:2000-1:8000 or 1:5000.

[0010] In the above application, the structural formula of the Ispins is as follows: ;

[0011] The structural formula of tamoxifen is as follows: .

[0012] The present invention also provides an application of a pharmaceutical composition in preparing a product for inhibiting proliferation of tamoxifen-resistant cells;

[0013] The active ingredients of the pharmaceutical composition are ispins and tamoxifen.

[0014] In the above application, the tamoxifen-resistant cells are MCF-7 and / or T47D tamoxifen-resistant cells.

[0015] In the above application, the molar ratio of Ispence to Tamoxifen is 1:5-1:10000; specifically, it can be 1:500-1:8000; more specifically, it can be 1:2000-1:8000 or 1:5000.

[0016] Furthermore, the present invention provides a tamoxifen treatment sensitizer for ERα-positive breast cancer, wherein the active ingredient is Ispins.

[0017] Finally, the present invention provides a drug for preventing and / or treating ERα-positive breast cancer, wherein the active ingredients of the drug are ispins and tamoxifen.

[0018] In the above-mentioned drug, the molar ratio of Ispence to Tamoxifen is 1:5-1:10000; specifically, it can be 1:500-1:8000; more specifically, it can be 1:2000-1:8000 or 1:5000.

[0019] The ERα-positive breast cancer is tamoxifen-resistant ERα-positive breast cancer.

[0020] In the present invention, the drug resistance of breast cancer is caused by the endocrine therapy drug tamoxifen.

[0021] The beneficial effects of the present invention are as follows:

[0022] The experimental results showed that Ispins combined with tamoxifen significantly inhibited the cell proliferation activity of tamoxifen-resistant breast cancer cells; further, Ispins combined with tamoxifen significantly inhibited the proliferation activity of organoids derived from tamoxifen-resistant breast cancer patients. Therefore, Ispins has a positive application prospect in improving tamoxifen resistance in breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result diagram of drug sensitivity test;

[0024] Figure 2 This is a graph showing the results of a cell proliferation activity experiment;

[0025] Figure 3 Construction of organoids from patients with tamoxifen-resistant breast cancer; case 1 was obtained from a metastatic lesion in the liver of a tamoxifen-resistant patient, and case 2 was obtained from a metastatic lesion in the chest wall of another tamoxifen-resistant patient;

[0026] Figure 4 This is the result of the organoid drug sensitivity experiment. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0028] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] The biological materials, reagents and kits used in the present invention can all be purchased from conventional commercial sources, and the experimental techniques used are all conventional operations in the art or are performed with reference to the instructions of the corresponding products.

[0031] The MCF-7 and T47D cell lines used in the following examples are ERα-positive breast cancer cells from the Key Laboratory of Breast Cancer Diagnosis and Treatment Research, Affiliated Tumor Hospital of Shantou University Medical College; tamoxifen-resistant MCF-7 TamR and T47D TamRThe cell construction method is as follows: culture in DMEM medium (Biological Industries) containing 10% (volume percentage) carbon-adsorbed fetal bovine serum (Biological Industries) and without phenol red / glutamine. When the cells adhere to the wall and grow to the exponential growth phase, add tamoxifen (Selleck) with a final concentration of 0.25 μM to the culture medium for 72 hours, discard the culture medium, wash once with PBS, and continue to culture with fresh medium without drugs. Sensitive cells gradually die, and when the surviving cells recover normal growth to 80%, the next concentration of induction is performed. The tamoxifen induction concentration is gradually increased to 5 μM with a concentration gradient of 0.25, 0.5, 1, and 2 μM. Repeated induction, medium change, and continued culture, and clones that can grow stably in 5 μM tamoxifen culture medium are screened, that is, tamoxifen-resistant cells. The whole process lasts for 12 months. To maintain its drug resistance, the tamoxifen concentration in the culture medium is maintained at 100 nM. The above cultures were all cultured in an incubator at 37°C and 5.0% CO2.

[0032] The English name of tamoxifen used in the following examples is Tamoxifen, the Chinese chemical name is (Z)-2-[4-(1,2-diphenyl-1-butene)phenoxy]-N,N-dimethylethylamine, and the molecular formula is C 26 H 29 NO, the structural formula is as follows:

[0033]

[0034] The English name of Ispinesib is Ispinesib, and its Chinese chemical name is N-(3-aminopropyl)-N-[(1R)-1-(3-benzyl-7-chloro-4-oxoquinazolin-2-yl)-2-methylpropyl]-4-methylbenzamide. Its molecular formula is C 30 H 33 ClN4O2, the structural formula is as follows:

[0035]

[0036] Example 1: Cell proliferation detection

[0037] 1. CCK-8 drug sensitivity test: As mentioned above, MCF-7 and T47D tamoxifen-resistant cells were inoculated into 96-well plates, with about 3,000 cells per well and about 100 μL suspension (the culture medium was DMEM). Each cell was divided into four groups: control (Vehicle), Ispinesib, and combination (Tam+Ispinesib). 6-8 replicates were made for each group. After the cells adhered, the corresponding drug solvent DMSO (0.5 μL / mL, dimethyl sulfoxide, MCE) was added to the Vehicle group as a control, 0.1 nM, 1 nM, 10 nM, 100 nM or 1000 nM ispinesib was added to the Ispinesib group, and 5 μM tamoxifen and 0.1 nM, 1 nM, 10 nM, 100 nM or 1000 nM ispinesib was added to the Tam+Ispinesib group, and the cells were cultured for 0, 24, 48, and 72 hours, respectively (cultured in an incubator at 37°C, 5.0% CO2). Aspirate the original culture medium in the 96-well plate, add 100 μL of culture medium containing 10% v / v CCK-8 working solution (Biyuntian) to each well (can be prepared in advance according to the required total amount, the volume ratio of CCK-8 to DMEM culture medium is 1:9), incubate in an incubator at 37°C and 5.0% CO2 for 2 h, and then measure the absorbance at 0, 24, 48, and 72 h respectively. Set the wavelength of the microplate reader to 450 nm, measure its absorbance, and obtain the average value of the readings based on the duplicate wells. Draw the growth curve with time (h) as the horizontal axis and cell activity (450 nm absorbance) as the vertical axis.

[0038] The results are as follows Figure 1 As shown, Ispins can significantly enhance the efficacy of tamoxifen-resistant cells (MCF-7 TamR and T47D TamR ) of tamoxifen sensitivity; among them, the best effect was achieved when 5 μM tamoxifen and 1 nM ispins were combined.

[0039] 2. EdU cell proliferation detection experiment

[0040] As described above, MCF-7 and T47D and their corresponding tamoxifen-resistant cells were seeded in 24-well plates. After the cell density reached about 70%, each cell was divided into four groups: control (Vehicle), tamoxifen (Tam), Ispinesib, and combined drug (Tam+Ispinesib). 0.5 μL / mL of the corresponding drug solvent DMSO control was added to the Vehicle group, 5 μM tamoxifen was added to the Tam group, 1 nM Ispinesib was added to the Ispinesib group, and 5 μM tamoxifen and 1 nM Ispinesib were added to the Tam+Ispinesib group, and cultured for 0 or 72 hours (37°C, 5.0% CO2 incubator). Prepare 2× EdU working solution (Biyuntian), preheat to 37°C. For 24-well plates, the volume of DMEM culture medium in each well is 500 μL, and the final concentration of EdU is 10 μM (1×). After adding the preheated EdU working solution, continue to incubate the cells in a 37°C incubator for 2 hours. Remove the culture medium, add 500 μL of fixative solution (Biyuntian) to each well, and fix at room temperature for 15 minutes. Remove the fixative solution, wash each well with 500 μL of washing solution (Biyuntian) 3 times, 3-5 minutes each time. Remove the washing solution, add 500 μL of permeabilization solution (Biyuntian) to each well, and incubate at room temperature for 10-15 minutes. Remove the permeabilization solution, wash each well with 500 μL of washing solution 2 times, 3-5 minutes each time. Prepare the reaction solution according to the instructions. For 24-well plates, the volume of the reaction solution is 100 μL, and the reaction solution must be used within 15 minutes after preparation. After removing the washing solution, 100 μL of the prepared reaction solution was added to each well, and the culture plate was gently shaken to ensure that the reaction mixture could evenly cover the sample. Incubate at room temperature in the dark for 30 minutes. Remove the reaction solution and wash 3 times with the washing solution, 3-5 minutes each time. Use Hoechst 33342 (Biyuntian) to stain the cell nucleus. Dilute Hoechst33342 (1000×) with PBS at a volume ratio of 1:1000. After removing the washing solution, add 500 μL of 1× Hoechst 33342 to each well and incubate at room temperature in the dark for 10 minutes. Remove the staining solution and wash 3 times with the washing solution, 3-5 minutes each time, and then perform fluorescence detection. Hoechst 33342 is blue fluorescence with a maximum excitation wavelength of 346 nm and a maximum emission wavelength of 460 nm. EdU is labeled with Alexa Fluor 488 and is green fluorescence with a maximum excitation wavelength of 495 nm and a maximum emission wavelength of 519 nm. Image J software was used for analysis.

[0041] The results are as follows Figure 2As shown, in parental cells (MCF-7 and T47D), the inhibitory effect of Ispins combined with tamoxifen on breast cancer cell proliferation was not significantly different from that of tamoxifen alone; in tamoxifen-resistant cells (MCF-7 TamR and T47D TamR ), Ispins combined with tamoxifen has a significant inhibitory effect on the proliferation of breast cancer cells compared with tamoxifen alone.

[0042] Example 2: Drug sensitivity experiment of organoids from patients with tamoxifen-resistant breast cancer

[0043] Construction of patient-derived organoids: Metastatic lesions of patients with tamoxifen-resistant ERα-positive breast cancer were obtained by puncture (the sampling was approved by the Ethics Committee of our hospital, and all patients signed informed consent forms). Each tissue sample (<3-4cm 3) were transferred to a 15 mL centrifuge tube containing D-BSA culture medium (Thermo Fisher Scientific) and stored at 4°C for tissue separation within 24 h. First, non-epithelial components, including muscle and adipose tissue, were carefully removed from breast cancer tissue samples using ophthalmic scissors and forceps, and obvious necrotic parts were also removed. Subsequently, the pretreated tissue samples were transferred to a 50 mL centrifuge tube and rinsed several times with 4°C pre-cooled DPBS solution (ThermoFisher Scientific) containing antibiotics until the rinse solution became clear. Next, the tissue samples were moved to a 10 cm cell culture dish, minced with surgical scissors or a scalpel, and at least 10 mL of tissue digestion solution (Thermo Fisher Scientific) was added according to the amount of tissue, and digested in a 37°C water bath. During the digestion process, the mixture was mixed every 10 to 15 minutes until the tissue blocks were obviously dissociated and a large number of cell clusters or single cells were observed under an optical microscope. After that, the samples were centrifuged at 200 g for 5 minutes at 4°C, the supernatant was discarded, and the cell pellet was placed on ice. Then, the cell pellet was resuspended in D-BSA culture medium and filtered through a 70 μm or 100 μm cell strainer, centrifuged again at 200g for 5 minutes at 4°C, and the supernatant was removed to retain the cell pellet. Finally, the cell pellet was resuspended in an appropriate amount of Cultrex RGF BME, Type 2 (R&DSystems, cat. no. 3533-005-02) and seeded in a 24-well plate. After the scaffold material solidified, 500 μL of the corresponding breast cancer organoid culture medium (PreceDo Pharmaceuticals, cat. no. PRS-BCM-3D) was added to ensure that the culture medium covered the cells, and then the culture plate was placed in a carbon dioxide cell culture incubator at 37°C for culture. During the organoid culture process, the culture medium was replaced every 2 to 3 days depending on the growth situation.

[0044] The results are as follows Figure 3 As shown, case 1 was obtained from a metastatic lesion in the liver of a tamoxifen-resistant patient, and case 2 was obtained from a metastatic lesion in the chest wall of another tamoxifen-resistant patient.

[0045] Organoid drug sensitivity test: Taking a 24-well plate as an example, aspirate 350 μL of culture medium from the well containing the organoids, hold the plate at a 45° angle, and place the pipette tip on the side wall of the well, away from the BME droplet. Add 450 μL of TrypLE (Thermo Fisher Scientific) to the well for a final volume of 600 μL, aim at the BME droplet, and use a P1000 pipette to dissociate the BME by pipetting up and down 6-10 times in each well. Hold the plate at a 45° angle and pipette up and down the BME-TrypLE mixture containing the organoids 10 times. Examine the organoids using an inverted bright field microscope (×5, ×10, or ×20 objective). Until the desired size (20-50 μm or 5-20 / cell clusters) is reached. Use a P1000 pipette to transfer single cells or organoid fragments to a 15 mL centrifuge tube containing 4 mL ice-cold adDMEM / F12+++ (Thermo Fisher Scientific). Rinse each well with 1 mL of adDMEM / F12+++ to ensure that no detached organoids remain in the well (a total of 5 mL of culture medium). Organoids from up to 6 wells can be pooled in a 15 mL centrifuge tube. Centrifuge at 4°C, 300 g for 5-10 min. Inoculate organoids into 96-well plates and set up control (Vehicle), tamoxifen (Tam), Ispinesib, and combination (Tam+Ispinesib) groups, with 200 μL of culture medium per well. After 24 h of culture (37°C, 5.0% CO2 incubator), the Vehicle group was replaced with a culture medium containing the corresponding amount of drug carrier DMSO (0.5 μL / mL), the Tam group was replaced with a culture medium containing 5 μM tamoxifen, the Ispinesib group was replaced with a culture medium containing 1 nM Ispinesib, and the Tam+Ispinesib group was replaced with a culture medium containing 5 μM tamoxifen and 1 nM Ispinesib; the culture medium used above was breast cancer organoid culture medium (PreceDo Pharmaceuticals, cat. no. PRS-BCM-3D). After 72 h of culture (37°C, 5.0% CO2 incubator), the cell activity of the organoids was measured. According to the instructions, 100 μL CellTiter-Glo® 3DReagent (Promega) was added to each well, and the cells were lysed by vigorous pipetting up and down for 5 minutes. The cells were incubated at room temperature for 25 minutes, and the fluorescence intensity of each well was recorded by a fluorescence spectrophotometer, and the corresponding cell activity curve was drawn.

[0046] The results are as follows Figure 4 As shown, compared with monotherapy, the combination of Ispins and tamoxifen significantly inhibited the proliferation activity of organoids from tamoxifen-resistant patients.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for treating tamoxifen-resistant ERα-positive breast cancer; The active ingredients of the pharmaceutical composition are Ispins and Tamoxifen; The molar ratio of Ispins to tamoxifen is 1:5 to 1:10000; The tamoxifen-resistant ERα-positive breast cancer cells are MCF-7 and / or T47D tamoxifen-resistant cells.

2. The use according to claim 1, characterized in that: The structural formula of the Ispins is as follows: ; The structural formula of tamoxifen is as follows: ; The molar ratio of Ispins to tamoxifen is 1:2000-1:8000.

3. Use of a pharmaceutical composition in the preparation of a drug for inhibiting the proliferation of tamoxifen-resistant ERα-positive breast cancer cells; The active ingredients of the pharmaceutical composition are Ispins and Tamoxifen; The tamoxifen-resistant ERα-positive breast cancer cells are MCF-7 and / or T47D tamoxifen-resistant cells; The molar ratio of Ispins to tamoxifen is 1:5-1:10000.

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