Use of indole-3-carbinol in the preparation of a medicament for the treatment of triple negative breast cancer

Abstract

The application provides application of indole-3-formaldehyde in preparation of a drug for treating triple-negative breast cancer. Researches of the application show that indole-3-formaldehyde (I3A) has good anti-triple-negative breast cancer (TNBC) effect, in-vitro cell experiment results show that I3A significantly inhibits the activity and migration ability of TNBC cells, and has no effect on normal MCF-10A cells; in-vivo animal experiment results show that I3A can significantly reduce the tumor volume and tumor weight after drug treatment, effectively inhibit the growth of TNBC transplanted tumor, reduce the expression of Ki67 in tumor tissues, inhibit the proliferation activity of tumor cells in the body, and can significantly change the alpha diversity and beta diversity of intestinal flora, reshape the community structure of the flora, and play an anti-TNBC role by regulating the intestinal microecology. The application provides more efficient and safe therapeutic drugs for the treatment of triple-negative breast cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of indole-3-carboxaldehyde in the preparation of drugs for treating triple-negative breast cancer. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, seriously threatening their physical and mental health. Its onset is related to multiple factors, including genetics, hormone levels, and lifestyle. Clinically, it can be classified into different subtypes based on receptor expression. Among them, triple-negative breast cancer (TNBC) is a particularly challenging subtype, accounting for approximately 15%-20% of all breast cancer cases. This subtype is negatively expressed for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2), resulting in no significant efficacy from traditional endocrine therapy and HER-2 targeted therapy. Clinically, it mainly relies on traditional chemotherapy, but recurrence and metastasis are common, and drug resistance is a significant problem, leading to a very poor prognosis and a significantly lower five-year survival rate than other subtypes. Therefore, developing anti-TNBC drugs with novel mechanisms and targets remains a key scientific problem that urgently needs to be solved in this field.

[0003] Currently, clinical treatment for triple-negative breast cancer (TNBC) primarily relies on chemotherapy. However, existing chemotherapy drugs have poor specificity, significant toxic side effects, and are prone to drug resistance, leading to treatment failure and poor patient prognosis. Novel targeted therapies and immunotherapies for TNBC suffer from low patient response rates and limited applicability, failing to meet the treatment needs of a broad range of patients. Therefore, there remains a lack of drugs available for treating TNBC.

[0004] Indole compounds are an important class of heterocyclic molecules, playing a crucial role in the development of antitumor drugs. Indole-3-carboxaldehyde (I3A), as an important member of the indole family, is a key product of tryptophan metabolism by intestinal flora and has been shown in recent years to possess both immunomodulatory and antitumor activities. Currently, targeted research on I3A in the treatment of TNBC is scarce, and no I3A-based drugs for the treatment of TNBC have been applied clinically. Therefore, this invention application is filed. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the shortcomings of existing drugs for treating triple-negative breast cancer. This invention aims to provide a novel application of indole-3-carboxaldehyde in the preparation of drugs for treating triple-negative breast cancer. This application can effectively inhibit the proliferation and migration of TNBC cells, effectively suppress tumor growth in vivo without significant toxicity to normal cells, and simultaneously regulate the intestinal microecology of tumor-bearing mice. Therefore, it provides a novel, highly effective, and low-toxicity therapeutic drug candidate and treatment strategy for triple-negative breast cancer.

[0006] The first objective of this invention is to provide a novel application of indole-3-carboxaldehyde in the preparation of drugs for treating triple-negative breast cancer.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: This invention reveals that indole-3-carboxaldehyde (I3A) exhibits significant anti-triple-negative breast cancer (TNBC) activity. In vitro cell experiments showed that I3A significantly inhibited the viability and migration ability of TNBC cells without affecting normal MCF-10A cells. In vivo animal experiments showed that I3A treatment significantly reduced tumor volume and weight, effectively inhibiting the growth of TNBC xenografts. It also reduced Ki67 expression in tumor tissue, inhibiting tumor cell proliferation in vivo. Furthermore, it significantly altered gut microbiota α- and β-diversity, reshaping the gut microbiota community structure and exerting its anti-TNBC effect by regulating the gut microbiota. This invention provides more highly effective and low-toxicity new drugs for the treatment of triple-negative breast cancer.

[0008] Therefore, the present invention provides the use of indole-3-carboxaldehyde in the preparation of drugs for treating triple-negative breast cancer.

[0009] The indole-3-carboxaldehyde provided by this invention has CAS number 487-89-8. Density 1.3 ± 0.1 g / cm³ 3 The molecular formula is C9H7NO, the molecular weight is 145.158, and the structural formula is: .

[0010] This invention provides the use of indole-3-carboxaldehyde in the preparation of products that inhibit triple-negative breast cancer cells in vitro.

[0011] Preferably, the drug or product can inhibit the growth of triple-negative breast cancer cells.

[0012] Preferably, the drug or product can reduce tumor volume and tumor weight.

[0013] Preferably, the drug can inhibit the proliferation and migration of triple-negative breast cancer cells in vivo.

[0014] Preferably, the drug can reduce the expression of the tumor marker Ki67.

[0015] More preferably, the drug can regulate gut microbiota diversity, reshape the microbiota community structure, and regulate the abundance of key bacterial genera.

[0016] Furthermore, the drug can upregulate beneficial bacteria. Ligilactobacillus The abundance of.

[0017] Preferably, the dosage form of the drug is capsules, tablets, pills, granules, oral liquid preparations, or injections.

[0018] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0019] In particular, the drug provided by this invention can be administered via existing pharmaceutically acceptable routes, such as intravenous injection, intraperitoneal injection, subcutaneous injection, transdermal administration, oral solid dosage forms (tablets, capsules), or oral liquid dosage forms. The dosage and frequency can be adjusted within a certain range (e.g., 10-200 mg / kg, once or multiple times daily) based on the specific dosage form, patient condition, and other factors to achieve the best therapeutic effect.

[0020] This invention provides the anti-TNBC effect of indole-3-carboxaldehyde, and it is foreseeable that its pharmaceutically acceptable salts, esters, prodrugs, or combinations thereof with other synergistic ingredients, as previously reported, may also achieve similar therapeutic effects.

[0021] The present invention has the following beneficial effects: This invention provides a novel application of indole-3-carboxaldehyde in the preparation of drugs for treating triple-negative breast cancer. This invention is the first to demonstrate that I3A not only significantly inhibits the in vitro proliferation and migration of various TNBC cell lines, but also effectively inhibits tumor growth in tumor-bearing mice. More importantly, it exhibits no significant toxicity to normal mammary epithelial cells, demonstrating good safety and overcoming the drawbacks of existing chemotherapy drugs with significant toxic side effects. This invention not only verifies the direct anti-tumor effect of I3A, but also reveals for the first time that it exerts its anti-TNBC efficacy by reshaping the intestinal flora structure of tumor-bearing mice, upregulating beneficial bacteria and downregulating harmful bacteria, and correcting breast cancer-related flora imbalances. This dual mechanism of action of "direct killing + microecological regulation" provides a completely new approach to TNBC treatment. Furthermore, since I3A is a natural product and intestinal flora metabolite, it possesses a good foundation for drug development and has high clinical translational value as a drug candidate for treating TNBC. Attached Figure Description

[0022] Figure 1Effects of I3A treatment for 24 h on the viability of 4T1 cells (A), MDA-MB-231 cells (B), and BT549 cells (C).

[0023] Figure 2 Effects of I3A treatment for 48 h on the viability of 4T1 cells (A), MDA-MB-231 cells (B), and BT549 cells (C).

[0024] Figure 3 Effects of I3A treatment for 24 h (A) and 48 h (B) on the viability of MCF-10A cells.

[0025] Figure 4 The effect of I3A on the migration ability of 4T1 cells (A), MDA-MB-231 cells (B) and BT549 cells (C).

[0026] Figure 5 This is a technical roadmap for in vivo experiments.

[0027] Figure 6 Photographs of tumor tissue in each group of mice (A) and a statistical graph of tumor weight in each group (B).

[0028] Figure 7 The curves showing the dynamic changes in tumor volume over the number of days since inoculation are shown in Figure (A), and the curves showing the changes in tumor volume in individual mice in the model group (B) and the I3A treatment group (C).

[0029] Figure 8 The effect of I3A on Ki67 expression in mouse tumor tissue (A: Ki67 positive staining image; B: Ki67 expression level).

[0030] Figure 9 The effects of I3A on the α-diversity and community structure of mouse gut microbiota (A: Shannon index analysis; B: Simpson index analysis; C: PCA analysis based on OTU levels).

[0031] Figure 10 The effects of I3A on the species composition and differentially expressed bacteria in the mouse gut microbiota (A: phylum-level species composition; B: genus-level species composition; C: Bacillota / Bacteroides ratio statistics; D: LEfSe analysis of differentially expressed species between groups). Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0033] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0034] The reagents used in the examples were as follows: indole-3-carboxaldehyde (I3A) was purchased from Maclean's; cell culture grade DSMO was purchased from MPBiomedicals; fetal bovine serum was purchased from Gibco Life Technologies and NEWZERUM; DMEM medium was purchased from Gibco Life Technologies; MCF-10A special medium was purchased from Pronosai; penicillin-streptomycin solution was purchased from Gibco Life Technologies; and CCK-8 kit was purchased from Meilun Biotechnology.

[0035] The mouse triple-negative breast cancer cell line 4T1, the human triple-negative breast cancer cell line MDA-MB-231, and BT549 used in the examples were all purchased from the Chinese Academy of Sciences Type Culture Collection Committee and cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. The human normal mammary epithelial cell line MCF-10A was purchased from the Chinese Academy of Sciences Type Culture Collection Committee and cultured in MCF-10A-specific medium. All culture conditions were 37°C and 5% carbon dioxide.

[0036] Example 1: Detection of the effect of I3A on cell viability using the CCK-8 assay Logarithmically growing 4T1, MDA-MB-231, BT549, and MCF-10A cells were collected and seeded at a density of 5000 cells / well in 96-well plates. After cell attachment, indole-3-carboxaldehyde (I3A) at different concentration gradients (50, 100, 500, and 1000 μM) was added for treatment, followed by further culture for 24 h and 48 h. After culture, cell morphology changes were observed, and cell viability was measured and calculated using a CCK-8 assay kit.

[0037] The measurement results are as follows Figures 1-3 As shown, after treatment with different concentration gradients of I3A for 24 h and 48 h, the viability of three triple-negative breast cancer cell lines (4T1, MDA-MB-231, and BT549) decreased in a concentration-dependent manner with increasing I3A concentration, and the inhibitory effect increased with prolonged treatment time, showing a highly significant difference compared with the control group. In contrast, under the same treatment conditions, the viability of normal human breast epithelial MCF-10A cells was not significantly different from that of the control group, and no obvious cytotoxicity was observed.

[0038] Example 2: Scratch assay to detect the effect of I3A on cell migration 4T1, MDA-MB-231 and BT549 cells were respectively loaded at 8×10 4Cells were seeded at a density of 10 cells / mL in 96-well plates. After culturing for 24 hours until the cells were fully adherent, the plates were streaked vertically with a sterile pipette tip, and the cells were gently washed with PBS to remove floating cells. Different concentration gradients of I3A (10, 50, 100, and 500 μM) were added, and scratch images were taken under a microscope at 0 h and 24 h. The scratch healing process was recorded and calculated.

[0039] The results are as follows Figure 4 As shown, after treatment with different concentration gradients of I3A for 24 h, the scratch healing ability of the three TNBC cell lines was significantly inhibited, and the relative migration area decreased significantly with increasing I3A concentration. The difference was extremely significant compared with the control group, indicating that I3A has an inhibitory effect on the migration ability of TNBC cells.

[0040] Example 3: Animal experiments to verify the in vivo antitumor activity of I3A Log-phase 4T1 cells were collected and adjusted to a concentration of 5 × 10⁻⁶ cells using a mixture of PBS and Matrigel. 6 A tumor-bearing mouse model was established by subcutaneous injection of 0.1 mL of the drug at a dose of 1 / mL into BALB / c mice. One week after tumor inoculation, mice were randomly divided into a model group and an I3A treatment group (50 mg / kg, administered by gavage daily), with 5 mice in each group. The drug was administered continuously for 16 days, following the treatment protocol as follows: Figure 5 As shown, the tumor volume was measured every 2 days during the period, and the tumor tissue was dissected and weighed after the patient was euthanized.

[0041] The measurement results are as follows Figures 6-7 As shown, compared with the model group, the tumor growth of mice in the I3A-treated group was significantly inhibited, the tumor volume growth rate was significantly slowed down, and the tumor weight was significantly reduced, indicating that I3A can effectively inhibit the growth of 4T1 xenografts in mice.

[0042] Example 4: Immunohistochemical detection of the effect of I3A on Ki67 expression in tumor tissue Tumor tissue specimens from mice in the model group and the I3A drug administration group were taken, fixed, embedded, and sectioned, and then subjected to antigen retrieval and blocking. Ki67 primary antibody was added and incubated, followed by secondary antibody incubation, color development, counterstaining, and image acquisition using a slide scanner.

[0043] The results are as follows Figure 8 As shown, the model group tumor tissue showed strong and widespread Ki67 positive staining signals, while the I3A-treated group showed significantly weakened positive staining signals and a substantial reduction in the number of positive cells. This indicates that I3A can inhibit the in vivo proliferation of triple-negative breast cancer cells by significantly downregulating the expression level of Ki67 in tumor tissue.

[0044] Example 5: Detection of the effect of I3A on the structure of mouse gut microbiota by 16S rRNA sequencing Colonic contents were collected from mice in the model group and the I3A-treated group. Total microbial DNA was extracted, and the V3-V4 variable region of the bacterial 16S rRNA gene was amplified by PCR and sequenced using high-throughput sequencing. Alpha diversity, β diversity, species composition analysis, and differential bacterial community analysis between groups were performed on the sequencing data.

[0045] The results are as follows Figures 9-10 As shown, compared with the model group, the Shannon index of the gut microbiota in the I3A-treated group was significantly decreased, while the Simpson index showed an increasing trend. PCA analysis revealed significant differences in community structure between the two groups. Species composition analysis showed that the Bacillota / Bacteroidota ratio was significantly increased in the I3A-treated group. Ligilactobacillus The abundance of beneficial bacteria was significantly increased. Prevotellaceae The abundance of potentially harmful bacterial genera such as _UCG-001 was downregulated. This indicates that I3A can effectively improve and precisely regulate the intestinal microecology of tumor-bearing mice by modulating gut microbiota diversity, reshaping the community structure, and regulating the abundance of key bacterial genera.

[0046] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of indole-3-carboxaldehyde in the preparation of drugs for treating triple-negative breast cancer.

2. Application of indole-3-carboxaldehyde in the preparation of products that inhibit triple-negative breast cancer cells in vitro.

3. The application according to claim 1 or 2, characterized in that, The drug or product can inhibit the growth of triple-negative breast cancer cells.

4. The application according to claim 1 or 2, characterized in that, The drug or product can reduce tumor volume and tumor weight.

5. The application according to claim 1, characterized in that, The drug can inhibit the proliferation and migration of triple-negative breast cancer cells in the body.

6. The application according to claim 1, characterized in that, The drug can reduce the expression of the tumor marker Ki67.

7. The application according to claim 1, characterized in that, The drug can regulate the diversity of intestinal flora, reshape the community structure of the flora, and regulate the abundance of key bacterial genera.

8. The application according to claim 7, characterized in that, The drug can upregulate beneficial bacteria. Ligilactobacillus The abundance of.

9. The application according to claim 1, characterized in that, The dosage form of the drug is capsule, tablet, pill, granule, oral liquid preparation or injection.

10. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

Images