Application of a compound T-MAS in the preparation of drugs for treating breast cancer

Abstract

The present invention discloses for the first time that the compound T-MAS has the effect of treating breast cancer and can be used in the preparation of drugs for treating breast cancer. The drug is effective when used alone and can also be used in combination with other anti-tumor drugs, such as paclitaxel and carboplatin, in the preparation of drugs for treating breast cancer. It can inhibit tumor cell proliferation and promote tumor cell apoptosis, and has broad clinical significance.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to application of a compound T-MAS in preparing a drug for treating breast cancer. Background Art

[0002] The process of mammalian oocyte formation, growth and maturation is inseparable from the meiotic process. In vivo, oogonia stop mitosis, start and enter meiosis to transform into oocytes, and then are surrounded by somatic cells to form follicles. Oocyte meiosis then stagnates at the prophase of the first meiotic division. After a long growth and development process in the follicle, it resumes meiosis and matures under certain stimulation conditions. Therefore, the regulation of meiotic resumption is crucial to understanding the formation, growth, development and maturation of oocytes. Meiosis-activating sterols (MAS) can induce oocytes cultured in vitro to resume meiotic maturation. Therefore, MAS has attracted the attention of researchers since its discovery, but its specific role and mechanism are still controversial.

[0003] There are two types of MAS, namely FF-MAS (follicle fluid-meiosisactivatingsterol) and -TMAS (testis-meiosisacitviatingsetorl), both of which are intermediates in the cholesterol biosynthesis pathway. FF-MAS is synthesized by lanosterol 14α-demethylase (CY5P1), and then further converted into T-MAS by △14-reductase. FF-MAS accumulates in the ovary. Inhibitors of CYP51 enzymes, such as azalanstat (RS-21607) and RS-21745, can inhibit the synthesis of MAS and reduce the accumulation of MAS; while inhibitors of △14-reductase, such as AY9944-A-7, can inhibit the conversion of FF-MAS to -TMAS and cause the accumulation of FF-MAS; and some substances, such as nystatin, can prevent the downstream metabolism of MAS and cause its accumulation.

[0004] Breast cancer is the most common malignant tumor in women and the leading cause of cancer death in women. The latest global cancer burden data in 2020 show that breast cancer has become the cancer with the highest incidence rate in the world. Breast cancer can be molecularly classified according to the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 proliferation index. HER2-positive breast cancer is highly malignant, easy to metastasize, easy to relapse, and has a poor prognosis. It is the focus and difficulty of breast cancer treatment.

[0005] It can be seen that in the prior art, no successful examples of using steroid hormone drugs to treat breast cancer have been found. Summary of the invention

[0006] In order to overcome the defects of the prior art, the present invention provides the following technical solutions:

[0007] The present invention provides the use of the compound T-MAS in preparing a drug for treating breast cancer;

[0008] Furthermore, the compound T-MAS is as shown in Formula I:

[0009]

[0010] Furthermore, the compound can be used in combination with anti-tumor drugs to prepare drugs for treating breast cancer;

[0011] Furthermore, the anti-tumor drug is paclitaxel;

[0012] Furthermore, the anti-tumor drug is carboplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Western Blot results of UPR pathway in MDA-MB-231 cell line treated with different concentrations of T-MAS

[0014] Figure 2 .Thioflavin T staining results of MDA-MB-231 cell line treated with different concentrations of T-MAS

[0015] Figure 3 .Flow cytometry apoptosis results of MDA-MB-231 cell line treated with T-MAS and 4-PBA

[0016] Figure 4.Flow cytometry results of apoptosis in MDA-MB-231 cells treated with paclitaxel, carboplatin and T-MAS

[0017] Figure 5 .T-MAS sensitization experiment on the chemotherapy drug carboplatin.

[0018] Beneficial Effects

[0019] The present invention discloses for the first time that the compound T-MAS has the effect of treating breast cancer and can be used in the preparation of drugs for treating breast cancer. The drug is effective when used alone and can also be used in combination with other anti-tumor drugs, such as paclitaxel and carboplatin, in the preparation of drugs for treating breast cancer. It can inhibit tumor cell proliferation and promote tumor cell apoptosis, and has broad clinical significance. DETAILED DESCRIPTION

[0020] Example 1 Western Blot Experiment

[0021] like Figure 1 As shown, according to the experimental needs, cells were pre-treated. For T-MAS, 0, 1 μg / ml, 2ug / ml, and 4 μg / ml were selected to treat cells for 2 days and then proteins were collected; SDS-PAGE gel was prepared; the loading comb was pulled out, and the protein amount was adjusted according to the protein concentration and then loaded using a pipette; 1X electrophoresis buffer was poured into the electrophoresis tank, and the power was turned on. Electrophoresis was performed at a constant voltage of 75V. After the protein Marker was layered, the voltage was adjusted to 120V, and the electrophoresis was stopped after bromophenol blue ran to the appropriate position; the PVDF membrane was activated, and the membrane was transferred at 250mA horizontal flow, and the appropriate transfer time was set according to the molecular weight of the protein; after the transfer, it was blocked with 5% skimmed milk powder and blocked at room temperature for 1 hour; after the blocking, the bands were placed in the corresponding primary antibody and incubated overnight at 4°C; the membrane was washed with TBST the next day, and then the corresponding secondary antibody was added and incubated at room temperature for 1 hour; the membrane was washed with TBST, and the exposure solution was prepared and developed using an imager. Figure 1 The cells were treated with culture medium without T-MAS, culture medium containing 1μg / ml, 2μg / ml and 4μg / ml T-MAS, respectively. Western Blot results showed that T-MAS could induce endoplasmic reticulum stress and activate the PERK / p-eIF2α / ATF-4 / CHOP pathway.

[0022] Example 2 Thioflavin T staining experiment

[0023] Plant an appropriate number of cells on the cell slide, observe under a microscope after 24 hours, and treat the cells according to experimental needs after the cells adhere to the wall. For T-MAS, choose 2μg / ml and 4μg / ml to treat the cells for 2d; after the cell pretreatment is completed, use paraformaldehyde to fix the cells at room temperature for 1h; wash the slide with PBS slowly; prepare 10μM concentration of Thioflavin T, protect from light, and incubate at room temperature for 10min; wash the slide with PBS slowly; counterstain with DAPI and seal the slide; observe under a fluorescence microscope and take pictures. Thiflavin T is a cell membrane permeable benzothiazole dye that binds to stacked protein β-folds to cause maximum excitation wavelength shift and emission enhancement. Its maximum excitation wavelength is: (free); (combined); the maximum emission wavelength is: (free); (Combination). After Thiflavin T staining, the proportion of fluorescence-positive cells and the fluorescence intensity detected by confocal microscopy can reflect the level of unfolded proteins in cells. Figure 2 The cells were treated with medium without T-MAS, medium containing 2 μg / ml and 4 μg / ml T-MAS, respectively. Figure 2 In the figure, the small circles represent fluorescent spots stained with Thioflavin T. Thioflavin T staining experiments show that the content of unfolded proteins in cells increases after T-MAS treatment. Figure 1 The results suggest that T-MAS can promote the accumulation of unfolded proteins and thus induce the unfolded protein response.

[0024] Example 3: Flow cytometry apoptosis assay

[0025] like Figure 3 As shown, cells were treated with different concentrations of drugs in advance. For T-MAS, 4 μg / ml was used to treat cells for 2 days; for 4-PBA, 1 mM 4-PBA was used to pre-treat cells for 3 hours and then normal culture medium was replaced.

[0026] Annexin V-FITC / PI Apoptosis Detection Kit (Vazyme) was used to detect cell apoptosis: cells were digested with EDTA-free trypsin, centrifuged at 1000 rpm, 4°C for 5 min, and the supernatant was discarded; cells were washed twice with pre-cooled PBS, centrifuged at 1000 rpm, 4°C for 5 min each time; 100 μl 1X Binding Buffer was added, and the mixture was gently blown to a single cell suspension; 5 μl Annexin V-FITC and 5 ul PI Staining Solution were added, and the mixture was gently blown to a single cell suspension; 5 μl Annexin V-FITC and 5 ul PI Staining Solution were added, and the mixture was gently blown to a single cell suspension; incubated at room temperature for 10 min in a dark environment, and 400 μl 1X Binding Buffer was added, and the mixture was gently mixed. After staining, the samples were detected by flow cytometry within 1 hour. Flow cytometric analysis: The excitation wavelength of the flow cytometer was 488 nm; the green fluorescence of FITC was detected in the FL1 channel; the red fluorescence of PI was detected in the FL2 or FL3 channel, and FL3 was recommended. 10,000 events were collected for each sample. Data analysis was performed using FlowJo and other software, with FL1 as the horizontal axis and FL3 as the vertical axis. The positive and negative boundaries of the two fluorescence parameters were determined based on the fluorescence values ​​of FITC and PI, and the cross gate was drawn. In a typical experiment, cells can be divided into three subpopulations: live cells are double negative (Annexin V-FITC- / PI-); early apoptotic cells are single positive for Annexin V-FITC (Annexin V-FITC+ / PI-); late apoptotic cells are double positive for Annexin V-FITC and PI (Annexin V-FITC+ / PI+).

[0027] Flow cytometry technology revealed that T-MAS could induce cell apoptosis, and the endoplasmic reticulum stress inhibitor 4-PBA could inhibit T-MAS-induced cell apoptosis, suggesting that T-MAS induces cell apoptosis through the endoplasmic reticulum stress pathway.

[0028] Experimental Example 4: Flow Cytometry Apoptosis Experiment of Chemosensitizing Drugs

[0029] like Figure 4 As shown, cells were treated with different concentrations of drugs in advance. For T-MAS, 4 μg / ml was selected to treat cells for 2 days; paclitaxel (PAX) was selected to treat cells for 2 days at 150 nM; carboplatin (CBP) was selected to treat cells for 2 days at 100 μM; T-MAS was combined with chemotherapy drugs paclitaxel or carboplatin, and cell apoptosis was detected by the same method as in Experimental Example 3. It was found that T-MAS can significantly increase the proportion of cell apoptosis induced by paclitaxel and carboplatin, indicating that T-MAS has a chemotherapy sensitization effect.

[0030] Example 5: Organoid Death / Survival Analysis

[0031] like Figure 5 As shown, breast cancer organoids were cultured in advance for 1 week in medium without drugs, medium containing 4μg / ml T-MAS, medium containing 300μM carboplatin (CBP), and medium containing 4μg / ml T-MAS and 300μM CBP. Carefully remove the medium, and add 40μl (for 96-well plates) Nexcelom ViaStain AO / PI dye (Nexcelom, CS2-0106) to stain the organoids (room temperature, incubate in the dark for 20min). Acridine orange (AO) is a fluorescent pigment that produces green fluorescence after binding to intracellular DNA. Propidium iodide (PI) cannot pass through the membrane of living cells, but can pass through damaged cell membranes to make dead cells appear red fluorescence. Images were taken using a confocal microscope, and the Z-Stack function was used to capture multi-plane fluorescence, and multiple different fields of view were stitched to obtain a complete image. The quantification of living and dead cells in organoids was achieved by calculating the ratio of the area of ​​each fluorescent dye to the total cell area.

[0032] Figure 5 In a, organoid fluorescence was captured at a low magnification field of view, and the area occupied by each fluorescent dye was analyzed by ImageJ. The ratio of the fluorescent dye area was used to reflect the death / survival of the organoid. Figure 5 b is a representative organoid fluorescence staining result under high-power microscope. In summary, the results of the organoid death / survival experiment suggest that the use of 4 μg / ml T-MAS alone in breast cancer organoids is not enough to cause obvious death of breast cancer organoids, but the combined use of T-MAS and the chemotherapy drug carboplatin can significantly induce organoid death, proving that T-MAS has a chemosensitizing effect in breast cancer organoids.

Claims

1. Application of the compound T-MAS in the preparation of drugs for treating breast cancer.

2. The use according to claim 1, characterized in that: The compound T-MAS is shown in Formula I:

3. The use according to claim 1 or 2, characterized in that: The compound is combined with an anti-tumor drug to prepare a drug for treating breast cancer.

4. The use according to claim 3, characterized in that: The anti-tumor drug is paclitaxel.

5. The use according to claim 3, characterized in that: The anti-tumor drug is carboplatin.

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