Application of bicalutamide and GDC-0994 in combined treatment of triple negative breast cancer

Abstract

The invention provides application of bicalutamide and GDC-0994 in combined treatment of triple-negative breast cancer, and a combined treatment method of bicalutamide and GDC-0994 provided by the invention can significantly enhance the treatment effect of triple-negative breast cancer, especially in the aspect of inhibiting tumor growth, migration and metastasis; through combined treatment, no obvious toxic reaction occurs in an in-vivo mouse model, and body weight and serum biochemical indexes are not abnormal, so that the combined treatment has good safety; in-vitro and in-vivo experiments show that the combined treatment can significantly inhibit the growth and metastasis of TNBC tumors, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of bicalutamide combined with GDC-0994 in treating triple-negative breast cancer. Background Art

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks estrogen receptors, progesterone receptors, and HER2 receptors, making it difficult to treat. Traditional treatments are mainly chemotherapy, but due to its high drug resistance and recurrence rate, there is still a lack of effective treatment strategies.

[0003] Bicalutamide is a known androgen receptor (AR) antagonist that is widely used in the treatment of prostate cancer. However, its effect alone is limited for triple-negative breast cancer (TNBC). Therefore, finding other drugs to be used in combination with bicalutamide to enhance the anti-AR therapeutic effect has become a hot topic in current research.

[0004] GDC-0994, as a MEK inhibitor, has been shown to exhibit anti-tumor activity in other cancer models. The present invention proposes that the combined use of bicalutamide and GDC-0994 can significantly enhance its anti-tumor effect in TNBC, and the synergistic effect can significantly inhibit tumor growth, migration and metastasis. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and propose the use of bicalutamide combined with GDC-0994 in the treatment of triple-negative breast cancer.

[0006] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: application of bicalutamide combined with GDC-0994 in the treatment of triple-negative breast cancer.

[0007] Preferably, the ratio of bicalutamide to GDC-0994 is 2:1.

[0008] Preferably, the combined treatment significantly enhances cell growth inhibition, inhibits cell migration and invasion in vitro, or shows significant changes in cell cycle distribution.

[0009] Preferably, the combined treatment significantly reduces the colony-forming ability of TNBC cells and inhibits cell migration in wound healing experiments.

[0010] Preferably, the combination therapy of bicalutamide and GDC-0994 exerts an anti-tumor effect by reducing the activity of the MEK / ERK signaling pathway of cells.

[0011] Preferably, the combination therapy of bicalutamide and GDC-0994 has significant anti-tumor effects, including inhibiting tumor growth, reducing tumor volume, inhibiting tumor cell proliferation and reducing in vivo toxicity.

[0012] Preferably, the combination therapy can effectively inhibit tumor recurrence and metastasis in the TNBC mouse model, especially in the metastatic sites of the lung and liver.

[0013] Preferably, the combined treatment of bicalutamide and GDC-0994 can significantly reduce the expression of Ki-67 by immunohistochemical analysis, indicating its effect in inhibiting tumor cell proliferation.

[0014] Compared with the prior art, the present invention has the following beneficial effects: the combined treatment method of bicalutamide and GDC-0994 provided by the present invention can significantly enhance the therapeutic effect of triple-negative breast cancer, especially in inhibiting tumor growth, migration and metastasis; the present invention has no obvious toxic reaction in an in vivo mouse model through combined treatment, and there is no abnormality in body weight and serum biochemical indicators, indicating that the combined treatment has good safety; the present invention shows through in vitro and in vivo experiments that the combined treatment can significantly inhibit the growth and metastasis of TNBC tumors, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of combined drug screening to identify compounds that synergistically enhance anti-AR therapeutic effects with bicalutamide in TNBC; Figure 2 It is a schematic diagram of the synergistic properties of bicalutamide combined with GDC-0994 in anti-tnbc action; Figure 3 Schematic diagram showing that the synergistic effect of bicalutamide and GDC-0994 has a significant anti-tumor effect in vitro; Figure 4 Schematic diagram showing that the combined treatment of bicalutamide and GDC-0994 can enhance the anti-tumor therapeutic effect in vivo; Figure 5 Schematic diagram showing that combined treatment with bicalutamide and GDC-0994 can prevent TNBC recurrence and metastasis in vivo. DETAILED DESCRIPTION

[0016] In order to provide a further understanding of the purpose, structure, features, and functions of the present invention, the following detailed description is given in conjunction with the embodiments.

[0017] Please refer to Figure 1 The present invention provides an application of bicalutamide combined with GDC-0994 in the treatment of triple-negative breast cancer.

[0018] Example 1: Combined drug screening experiment: By identifying compounds that synergize with bicalutamide to enhance the effects of anti-androgen receptor (AR) therapy.

[0019] Bicalutamide is an androgen receptor antagonist commonly used to treat diseases associated with androgen-dependent tumors, such as prostate cancer. In triple-negative breast cancer (TNBC), it is important to identify drugs that can work synergistically with bicalutamide to enhance its anti-AR effect. Figure 1 Detailed description of each part: Figure 1 A scatter plot shows the sensitivity index of the MAPK small molecule inhibitor library (n=64) in three TNBC cell lines to find effective synergy with bicalutamide; if the sensitivity index is greater than 1, it indicates that the small molecule inhibitor and bicalutamide have a synergistic effect in the cell line, that is, the inhibitor can effectively enhance the anti-cancer effect of bicalutamide.

[0020] Figure 1 B Scatter plot shows the synergy index of each inhibitor (n=64) in three TNBC cell lines to find effective synergy with bicalutamide; a synergy index greater than 1 indicates that there is an effective synergistic effect between the inhibitor and bicalutamide, indicating that when used in combination, the anticancer effect of the drug is greater than the sum of the effects of bicalutamide and the inhibitor used alone.

[0021] Figure 1 C. Inhibitors with selectivity index > 1 and sensitivity index > 0.12 were intersected to form a Venn diagram; d4 candidate drugs were listed in three TNBC cell lines to detect their sensitivity index and synergistic effect in combination with bicalutamide.

[0022] The Venn diagram shows the candidate drugs screened, which all have significant synergistic effects in three TNBC cell lines. This screen was able to identify which drugs may work together with bicalutamide to enhance the effect of anti-AR therapy.

[0023] Figure 1 D records in detail the response of each candidate drug in different cell lines, helping to evaluate the potential of these drugs and provide a basis for subsequent experiments.

[0024] Figure 1 E Expression of MEK and ERK proteins in Hs578t cells after treatment with bicalutamide and DHT; Figure 1 E shows the expression of MEK and ERK proteins in Hs578t cells after treatment with bicalutamide and DHT. These proteins are key molecules in the MAPK signaling pathway and are related to processes such as cell proliferation, differentiation, and drug resistance.

[0025] By detecting the expression of MEK and ERK proteins, we analyzed whether bicalutamide and its combination with other candidate drugs affect the MAPK pathway, thereby further revealing their mechanism in enhancing the anti-AR therapeutic effect.

[0026] The activation of MEK and ERK may mean that some drug candidates may have synergistic effects when combined with bicalutamide by activating or inhibiting the MAPK pathway.

[0027] Example 2: Anticancer effect of bicalutamide combined with GDC-0994 in triple-negative breast cancer (TNBC) cells and their synergistic effect: Figure 2 A: Effects of GDC-0994 on TNBC cells: Experimental design: This part uses CCK-8 method (cell counting kit 8 method) to measure the survival rate of TNBC cells by GDC-0994; the effects of different concentrations of GDC-0994 on TNBC cells are measured at 24 hours and 48 hours; IC50 values: IC50 (half inhibitory concentration) was calculated at 24 hours and 48 hours, respectively, reflecting the effective concentration of GDC-0994 in inhibiting TNBC cell growth at these time points. These concentration values ​​are listed in the table.

[0028] Figure 2 B: Combined effects of bicalutamide and GDC-0994: (i) Combination drug cell viability assay: Bicalutamide and GDC-0994 were used in combination at a 2:1 ratio to treat Hs578t (a human triple-negative breast cancer cell line) and 4T1 (a mouse triple-negative breast cancer cell line); the viability of these cells was measured and compared with that of the single drug group.

[0029] (ii) Fa-CI plot: The Fa-CI plot (Fractional Activity-Combination Index) is used to evaluate the synergistic effect of drug combinations. By calculating the Fa (proportion of inhibited cells) and CI (combination index) values, the interaction between two drugs can be quantitatively analyzed. A CI value <1 indicates that there is a synergistic effect between the two drugs, that is, their combined effect is stronger than when used alone.

[0030] (iii) Interaction analysis: The drug interactions when bicalutamide was used in combination with GDC-0994 were analyzed. Generally, the CI values ​​range as follows: CI<1: synergistic effect, indicating that the combined use of drugs can produce a stronger anti-tumor effect than when used alone.

[0031] CI = 1: additive effect, meaning the drug combination has the same effect as when used alone.

[0032] CI>1: Antagonistic effect, which means that the combined effect of the drugs is less effective than when used alone.

[0033] Figure 2 C: Synergy: Predicted and actual growth inhibition analysis of synergy: This part further analyzed the synergistic effect of bicalutamide and GDC-0994 by HSA (Highest Single Agent) score and Bliss score.

[0034] HSA score: By comparing the effects of combination therapy with those of monotherapy, it is determined whether the combination therapy shows a synergistic effect; if the effect of combination therapy is significantly better than that of a single drug, the HSA score is higher.

[0035] Bliss score: The Bliss model is a mathematical model that predicts the effect of drug combination. If the actual effect of the combined drug is higher than the effect predicted by Bliss, it means that there is a synergistic effect.

[0036] If the HSA and Bliss values ​​are greater than 0, it means that the drug combination has a synergistic effect and enhances the therapeutic effect.

[0037] In summary, the combination of bicalutamide and GDC-0994 is more effective than either drug alone and can more significantly inhibit the growth of TNBC cells.

[0038] Example 3: In vitro experiment on the anti-tumor effect of combined treatment of bicalutamide and GDC-0994: DMSO (solvent) was used as the control group; Figure 3 A: Effect of combined treatment of bicalutamide and GDC-0994 on cell proliferation (colony formation assay: Hs578t and 4T1 cells were used for 14 days for colony formation assay, treated with bicalutamide (20 μM) and GDC-0994 (5 μM) alone or in combination; The combination group showed a significant anti-tumor effect, namely, the formation of cell colonies was significantly reduced; this indicates that bicalutamide and GDC-0994 have a synergistic inhibitory effect on tumor cell proliferation in vitro.

[0039] Figure 3B: Effect of combined treatment of bicalutamide and GDC-0994 on cell migration (wound healing experiment): Cell migration was detected by wound healing experiment; the wound healing experiment was performed by creating an artificial wound in a culture dish to observe whether cells would migrate to the wound area for repair; the treatment groups were bicalutamide, GDC-0994 alone or combined treatment, and Hs578t and 4T1 cells were used to measure cell migration between 0, 24, and 48 hours; The combined treatment group showed significant inhibition of migration, indicating that the combined use of bicalutamide and GDC-0994 can significantly reduce the migration ability of tumor cells.

[0040] Figure 3 C. Figure 3 D: Effect of combined treatment of bicalutamide and GDC-0994 on cell invasion (Transwell assay): Transwell assay was used to evaluate the migration and invasion ability of cells. In the Transwell assay, Matrigel was added to the upper chamber to simulate the basement membrane and the invasion ability of cells under this "barrier" was tested; The invasive ability of tumor cells in the combined treatment group in the Transwell experiment was significantly reduced, indicating that the combined treatment of bicalutamide and GDC-0994 effectively inhibited the invasive ability of tumor cells.

[0041] Figure 3 E: Effect of combined treatment with bicalutamide and GDC-0994 on cell cycle (flow cytometric analysis): The cell cycle distribution of Hs578t and 4T1 cells was evaluated by flow cytometry after staining with propidium iodide (PI); The proportion of cells arrested in the G1 or S phase in the combined treatment group increased, indicating that the combined treatment of bicalutamide and GDC-0994 may inhibit cell proliferation by preventing cells from entering the S phase or G2 / M phase.

[0042] Example 4: In vivo experiments on the anti-tumor effect of combined treatment with bicalutamide and GDC-0994: including a DMSO control group, bicalutamide alone, GDC-0994 alone, and bicalutamide combined with GDC-0994; Figure 4 A: Orthotopic tumor formation and drug treatment in BALB / c mice: Tumor formation in mice during the experiment and the implementation of different drug treatment regimens. In situ tumor formation refers to the development of tumors in the natural location of the mouse body (such as mammary gland, skin, etc.) and drug intervention treatment.

[0043] Figure 4 B: Tumor size after ex vivo extraction: Shows the measurement of the size of the tumor after it is extracted from the mouse; changes in tumor size reflect the inhibitory effect of the drug on tumor growth; ex vivo extraction refers to the direct measurement of the size of the tumor tissue after it is removed from the mouse, usually in units of diameter or volume; helps to determine the direct effect of the drug on tumor growth; Figure 4 C: Tumor weight after in vitro extraction: The weight of the tumor after ex vivo extraction is shown; the weight of the tumor is also an important indicator of its growth; the heavier the tumor, the faster it grows in the mouse, and the therapeutic effect of the drug may be manifested as a significant reduction in tumor weight; by comparing the tumor weights of different treatment groups, the effect of the drug can be judged; Figure 4 D: Tumor growth curve: Tumor growth curves usually use tumor volume or size as the vertical axis and time as the horizontal axis to show the growth trend of tumors at different time points. They help determine how tumors change during treatment; the speed of tumor growth can reflect the efficacy of drugs. If the treatment is effective, the tumor growth curve will usually show a slowdown in growth or even cessation of growth, indicating that the drug has an inhibitory effect on tumor growth; Figure 4 E: Weight curve: The weight curve shows the changes in the weight of the mice during the experiment; changes in weight often reflect the side effects of drug treatment or overall health.

[0044] For example, some drugs may have an effect on the weight of mice, causing weight gain or loss. In general, negative effects of drugs on mice (such as toxicity) may be manifested as weight loss, while effective anti-tumor drugs may not have obvious weight changes, or weight may remain stable.

[0045] Figure 4 F: Serum biochemical indicators (ALT, AST, BUN) The effects of drug treatment on serum biochemical indicators of mice are shown. Common biochemical indicators include: ALT (alanine aminotransferase) and AST (aspartate aminotransferase): These are indicators of liver function. Elevations generally indicate liver damage. Drug toxicity may affect these indicators, especially liver toxicity.

[0046] BUN (blood urea nitrogen): BUN levels often reflect kidney function, and urea concentrations may be elevated if the kidneys are damaged.

[0047] The above indicators can help evaluate the effects of drugs on the overall physiological condition of mice, especially the potential toxicity of drugs during tumor treatment.

[0048] Figure 4 G: Representative 4T1 tumor sections immunohistochemically stained for Ki-67 expression: Ki-67 is a protein closely related to cell proliferation and is widely used in tumor biology research to evaluate the proliferation activity of tumor cells.

[0049] In this experiment, tumor sections were processed by immunohistochemical staining to show the expression of Ki-67; high expression of Ki-67 usually indicates rapid proliferation of tumor cells, while low expression of Ki-67 may indicate slowed or inhibited tumor growth.

[0050] By observing the expression intensity of Ki-67, it can be determined whether the drug effectively inhibits the proliferation of tumor cells.

[0051] Example 5: Combination therapy of bicalutamide and GDC-0994 can prevent recurrence and metastasis of TNBC in vivo: Figure 5 A: Schematic diagram of transfer model establishment and drug administration scheme The schematic diagram shows the establishment process and dosing regimen of the metastasis model in the study, including: Metastasis model: Tumor metastasis models may be established through xenografts (such as mouse xenografts) or autologous transplants.

[0052] After experimental animals (such as mice) are injected with tumor cells, researchers observe the tumor's in situ recurrence and distant metastasis (such as the lungs, liver, etc.).

[0053] Dosage regimen: The diagram may show different drug treatment regimens, including route of administration (oral, injection, etc.), dosage, and frequency of administration.

[0054] Figure 5 B: Bioluminescent imaging of in situ recurrence and metastasis: Bioluminescence Imaging (BLI) of in situ recurrence and metastasis: Bioluminescence imaging is a non-invasive technology that monitors the location and size of tumors in real time by emitting light from labeled tumor cells in vivo. This imaging method is often used for early detection and dynamic monitoring of tumors.

[0055] In situ recurrence: refers to the recurrence of the tumor from the primary site; BLI imaging can show whether there are signs of tumor recurrence at the primary site.

[0056] Metastasis: refers to the transfer of tumor cells to other parts of the body, such as the lungs and liver, through the blood or lymph; BLI imaging can intuitively display the distribution of tumor metastasis and the formation of metastatic nodules.

[0057] BLI imaging provides a real-time, non-invasive means of tumor monitoring. Through BLI imaging, changes in tumor burden can be quantitatively analyzed to evaluate the anti-metastatic effect of drugs.

[0058] The present invention has been described by the above-mentioned relevant embodiments, however, the above-mentioned embodiments are only examples for implementing the present invention. It must be pointed out that the disclosed embodiments do not limit the scope of the present invention. On the contrary, changes and modifications made without departing from the spirit and scope of the present invention are all within the scope of patent protection of the present invention.

Claims

1. Application of bicalutamide combined with GDC-0994 in the treatment of triple-negative breast cancer.

2. The use according to claim 1, characterized in that: The ratio of bicalutamide to GDC-0994 was 2:

1.

3. The use according to claim 1, characterized in that: The combined treatment significantly enhances cell growth inhibition, inhibits cell migration and invasion ability in vitro, or shows obvious changes in cell cycle distribution.

4. The use according to claim 1, characterized in that: The combined treatment significantly reduced the colony-forming ability of TNBC cells and inhibited cell migration in a wound healing experiment.

5. The use according to claim 1, characterized in that: The combined treatment of bicalutamide and GDC-0994 exerts anti-tumor effects by reducing the activity of the MEK / ERK signaling pathway in cells.

6. The use according to claim 1, characterized in that: The combination therapy of bicalutamide and GDC-0994 had significant anti-tumor effects, including inhibition of tumor growth, reduction of tumor volume, inhibition of tumor cell proliferation and alleviation of in vivo toxicity.

7. The use according to claim 6, characterized in that: The combined therapy effectively inhibited tumor recurrence and metastasis in the TNBC mouse model, especially in the metastatic sites of the lung and liver.

8. The use according to claim 1, characterized in that: The combined treatment of bicalutamide and GDC-0994 can significantly reduce the expression of Ki-67 by immunohistochemical analysis, indicating its effect in inhibiting tumor cell proliferation.

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