Application of astragaloside in preparation of medicine for treating obesity-related triple negative breast cancer

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of astragaloside in preparation of a medicine for treating obesity-related triple negative breast cancer. It is found that astragaloside can inhibit growth of cancer cells of the obesity-related triple negative breast cancer, reverse cell viability and lipid accumulation of the obesity-related triple negative breast cancer and regulate a lipid metabolism network of the obesity-related triple negative breast cancer, and particularly has the most obvious regulation effect on GalCer sphingolipid in the obesity-related triple negative breast cancer. The compound has a very good prospect when being developed and applied as a medicine for resisting obesity-related triple negative breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of astragaloside IV in the preparation of a drug for treating obesity-related triple-negative breast cancer. Background Art

[0002] Breast cancer is the most common malignant tumor in women worldwide and one of the most common malignant tumors causing death and disability in women. In recent years, it has shown a trend of younger patients. Breast cancer is divided into many subtypes, including luminal A, luminal B, HER-2 overexpression and triple-negative breast cancer (TNBC). TNBC is named because it lacks estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). "Research Progress in the Treatment of Triple-Negative Breast Cancer" (Sun Yafei et al., Advances in Modern General Surgery in China, 2024, 27(09):714-719) points out that triple-negative breast cancer is insensitive to endocrine therapy or HER2-targeted therapy. Its clinical characteristics include high invasiveness, high metastatic potential, high tendency to relapse and poor prognosis. Currently, there is no standardized TNBC treatment plan.

[0003] Obesity has become a major public health problem. What is more worrying is that obesity significantly increases the incidence of malignant tumors and the risk of malignant tumor-related death. Among all deaths caused by malignant tumors, 14% and 20% of men and women, respectively, are caused by obesity. Obesity caused by a high-fat diet is common in the population and promotes the occurrence and development of various cancers, including breast cancer (especially TNBC). It is reported that about 30% of breast cancer patients in my country are diagnosed as overweight or obese, and the proportion of obese breast cancer patients in Europe and the United States is as high as 70%. A survey of 1,106 female patients with TNBC found that their overall survival rate was closely related to obesity, suggesting that obese women have a potentially higher risk of TNBC. In addition, some animal experiments have also shown that obesity can promote the development of breast cancer. However, there is currently a serious lack of therapeutic drugs for obesity-related TNBC in clinical practice. It is urgent to develop new strategies to intervene in the occurrence and development of TNBC, which is exacerbated by the obesity epidemic, and to seek better prevention and treatment methods. Summary of the invention

[0004] In order to overcome the problem of lack of clinical drugs for treating obesity-related triple-negative breast cancer, the present invention provides the use of astragaloside IV in preparing drugs for treating obesity-related triple-negative breast cancer.

[0005] Astragaloside IV (ASIV) is the main monomer active substance extracted from Astragalus. The structural formula of Astragaloside IV is as follows:

[0006]

[0007] Currently, there are no reports on the anti-obesity-related TNBC effect of astragaloside IV by remodeling tumor sphingolipid metabolism.

[0008] The purpose of the present invention is to provide the use of astragaloside IV in the preparation of a drug for treating obesity-related triple-negative breast cancer.

[0009] According to the above application of a specific embodiment of the present invention, the drug reduces the viability of cancer cells, and / or the drug reduces the lipid accumulation of cancer cells.

[0010] According to the above application of a specific embodiment of the present invention, the drug reduces the NEFA content in cancer cells; and / or, the drug reduces the TC content in cancer cells; and / or, the TG content in cancer cells.

[0011] The present invention found through experiments that 60 μM ASIV intervention can significantly reduce the contents of NEFA, TC and TG in human breast cancer cells, wherein the content of NEFA is reduced by 153%, the content of TC is reduced by 62%, and the content of TG is reduced by 47%.

[0012] According to the above application of a specific embodiment of the present invention, the drug regulates the sphingolipid metabolic pathway in cancer cells. The present invention uses UHPLC-QTOF-MS / MS lipidomics technology to find that astragaloside IV can effectively regulate the metabolites of TNBC induced by a high-fat diet, screen out differential metabolites, and combine the chromatographic and mass spectrometric information of the differential metabolites to find that the differential metabolites are mainly enriched in sphingolipid metabolism, pyruvate metabolism and selenium complex metabolic pathways.

[0013] According to the above application of the specific embodiment of the present invention, the drug reduces GalCer sphingolipids in cancer cells. The present invention uses UHPLC-QTOF-MS / MS lipidomics technology to find that astragaloside IV can reverse the changes in GalCer, Lactosyceramide and Sphingomyelin induced by a high-fat diet. Among them, GalCer was reduced by 49%, Lactosyceramide was reduced by 24%, and Sphingomyelin was reduced by 22%. The most obvious regulatory effect was on GalCer sphingolipids.

[0014] According to a specific embodiment of the present invention, the drug for treating obesity-related triple-negative breast cancer has astragaloside IV as its main component.

[0015] Preferably, the drug also includes pharmaceutically acceptable excipients. The drug can be prepared into oral preparations or injections; the oral preparations include but are not limited to capsules, tablets, granules, oral liquids; the injections include but are not limited to sterile powder for injection, water injection, sodium chloride or glucose intravenous infusion.

[0016] In the above-mentioned medicine, the oral preparation includes additives, and the additives are selected from at least one of fillers, diluents, disintegrants, binders, lubricants, glidants, surfactants, solvents, flavoring agents, stabilizers, colorants, and preservatives.

[0017] The fillers or diluents include sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, α-starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, medicinal calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0018] The lubricant, glidant or anti-adhesive agent includes stearic acid; metal stearate such as calcium stearate or magnesium stearate; talc; colloidal silica; micro powder silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicate such as silicic anhydride or silicate hydrate, etc.

[0019] The adhesive comprises distilled water, ethanol of different concentrations, starch slurry, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone, polyethylene glycol and compounds similar to the above excipients.

[0020] The disintegrants include cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or cross-linked carboxymethyl cellulose sodium; cross-linked polyvinyl pyrrolidone; and chemically modified starch / cellulose, such as carboxymethyl starch or carboxymethyl starch sodium.

[0021] The surfactant includes Tween-80, sodium lauryl sulfate, sodium stearate sulfonate and the like.

[0022] The antioxidants include sodium bisulfite, sodium pyrosulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine (methionine), thiourea, phosphoric acid, citric acid and the like.

[0023] The preservatives or antibacterial agents include benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens (parabens), benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethyl alcohol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil and peppermint oil.

[0024] The flavoring agents include sweeteners such as saccharin sodium, aspartame, syrup, stevioside, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, etc.; sour flavoring agents such as citric acid, malic acid or tartaric acid; and aromatics such as fennel oil, peppermint oil, menthol, peppermint water, cinnamon oil, lemon essence, lemon oil and spices of various flavors.

[0025] According to a specific embodiment of the present invention, the drug reduces the viability of cancer cells, and / or the drug reduces the lipid accumulation effect of cancer cells.

[0026] The method for treating obesity-related triple-negative breast cancer comprises administering an effective amount of the above-mentioned drug to a subject, and the administration method can be oral, intravenous injection or transdermal permeation, and is applied to a patient in need of treatment.

[0027] Pharmaceutically effective amount refers to a reasonable benefit / risk ratio that can be obtained by applying drug treatment, and a quantity sufficient to treat the disease. The level of effective dose can be determined depending on some factors, including the patient's disease type, severity, activity of the drug, sensitivity to the drug, application time, route of administration, excretion rate, treatment cycle, drugs used simultaneously, and other factors known in the medical field. The drug of the present invention can be used as an independent therapeutic agent, or combined with other therapeutic agents. In addition, the composition of the present invention can be added with typical therapeutic agents continuously or simultaneously, and the composition can be applied once or multiple times. It is important to consider all the above factors and apply the minimum dose that can produce the maximum effect without side effects, and the dosage can be determined by the physician according to the patient's condition, age, etc.

[0028] Beneficial effects of the present invention:

[0029] The present invention finds that astragaloside IV can inhibit the growth of obesity-related triple-negative breast cancer cells, reverse the cell viability and lipid accumulation of obesity-related triple-negative breast cancer, and regulate the lipid metabolism of obesity-related triple-negative breast cancer, especially the most obvious regulatory effect on GalCer sphingolipids in obesity-related triple-negative breast cancer. Astragaloside IV has very good prospects for development and application as an anti-obesity-related TNBC drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The SCID obese mouse model was established. A. Body shape changes of mice after 8 weeks of high-fat diet induction; B. Changes in food intake; C. Body weight changes; D. Changes in BMI index; E. Changes in LEE'S index. The data of each group are expressed as x±SD, n=6 (in vivo). * P<0.05, ** P<0.01, *** P<0.001vs NC group.

[0032] Figure 2 ASIV showed an inhibitory effect on obesity-related TNBC in vivo, among which,

[0033] A. Effect of ASIV on the size of transplanted tumors in SCID obese mice bearing human TNBC;

[0034] B. Effect of ASIV on the volume of transplanted tumors in SCID obese mice bearing human TNBC;

[0035] C. Effect of ASIV on the weight of transplanted tumors in SCID obese mice bearing human TNBC;

[0036] D. Effects of ASIV on the pathological status of liver, kidney and spleen in SCID obese mice bearing human TNBC;

[0037] Effects of EF.ASIV on serum ALT and AST in SCID obese mice bearing human TNBC; data of each group are expressed as x±SD, n=6 (in vivo). ## P < 0.01 vs CON group; *** P<0.001vs HF group.

[0038] Figure 3 It was shown that FFA induction could significantly promote TNBC cell viability and lipid accumulation, among which,

[0039] A. MTT assay was used to detect the effect of FFA induction on the viability of MDA-MB-231 cells;

[0040] BD. Kit to detect the effect of FFA induction on lipid accumulation in MDA-MB-231 cells;

[0041] E.MTT assay was used to detect the effect of FFA induction on the viability of MDA-MB-468 cells;

[0042] FH. Kit was used to detect the effect of FFA induction on lipid accumulation in MDA-MB-468 cells; the data of each group were expressed as x±SD, n=3 (in vitro). * P<0.05, ** P<0.01, ***P<0.001vs 0 mM FFA concentration group.

[0043] Figure 4 ASIV can significantly reverse the effects of FFA on TNBC cell viability and lipid accumulation;

[0044] A. MTT assay was used to detect the effect of ASIV on FFA-induced MDA-MB-231 cell viability;

[0045] BD. The kit was used to detect the effect of ASIV on FFA-induced lipid accumulation in MDA-MB-231 cells;

[0046] E.MTT assay was used to detect the effect of ASIV on FFA-induced MDA-MB-468 cell viability;

[0047] FH. The kit was used to detect the effect of ASIV on FFA-induced lipid accumulation in MDA-MB-468 cells; the data of each group were expressed as x±SD, n=3 (in vitro). ## P<0.01, ### P<0.001vs 0mM FFA+0mM ASIV concentration group; * P<0.05, ** P<0.01, *** P<0.001vs 0.25mM FFA+0mM ASIV concentration group.

[0048] Figure 5 Show the regulatory effect of ASIV on the lipid metabolism network of obesity-related TNBC in vitro;

[0049] A. OPLS-DA scatter plot in positive ion mode;

[0050] B. OPLS-DA scatter plot in negative ion mode;

[0051] C. S-plot scatter diagram in positive ion mode;

[0052] D. S-plot scatter diagram in negative ion mode;

[0053] E. ASIV regulates metabolic pathways of obesity-related TNBC in vitro.

[0054] Figure 6 Showing the regulatory effect of ASIV on the lipid metabolism network of obesity-related TNBC in vivo;

[0055] A. OPLS-DA scatter plot in positive ion mode;

[0056] B. OPLS-DA scatter plot in negative ion mode;

[0057] C. S-plot scatter diagram in positive ion mode;

[0058] D. S-plot scatter diagram in negative ion mode;

[0059] E. ASIV regulates metabolic pathways of obesity-related TNBC in vivo.

[0060] Figure 7 Show the regulatory effect of ASIV on differential intracellular sphingolipids in obesity-related TNBC;

[0061] A. Effect of ASIV on the level of differential sphingolipid GalCer in MDA-MB-231 cells after FFA induction;

[0062] B. Effect of ASIV on the level of differential sphingolipid lactosyceramide in MDA-MB-231 cells after FFA induction;

[0063] C. Effects of ASIV on the level of differential sphingomyelin in MDA-MB-231 cells after FFA induction; data of each group are expressed as x±SD, n=3 (in vitro). # P<0.05, ## P < 0.01 vs CON group; * P<0.05, *** P<0.001vs FFA group. DETAILED DESCRIPTION

[0064] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0065] In the present invention, TNBC cell lines MDA-MB-231 and MDA-MB-468 are derived from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0066] Cell viability refers to the percentage of living cells among total cells.

[0067] Lipid accumulation refers to the abnormal accumulation of lipids in the body. General evaluation indicators include non-esterified fatty acids (NEFA), total cholesterol (TC) and triglyceride (TG) content.

[0068] Example 1 Investigating the effect of astragaloside IV on inhibiting the growth of obesity-related TNBC

[0069] A female severe combined immunodeficiency (SCID) obese mouse model was established using a high-fat diet, and mice in the normal diet group were used as controls (NC group). After 8 weeks of high-fat diet, the body shape of the mice changed significantly ( Figure 1 A), there was no significant change in food intake between the high-fat diet group and the normal diet group ( Figure 1 B). Compared with the normal diet group, the high-fat diet group had a significant increase in body weight ( Figure 1 C), BMI and LEE'S index increased significantly ( Figure 1 D and E).

[0070] After the obesity model was established, the human TNBC cell line MDA-MB-231 was used to perform orthotopic tumor inoculation surgery on SCID mice. 3 The tumor tissue particles were orthotopically transplanted into the fourth mammary gland of the right side of SCID mice. 3 When, group experiments were conducted.

[0071] Blank control group (CON): provided with normal diet and intraperitoneal injection of the solvent 0.5% sodium carboxymethylcellulose solution;

[0072] High-fat diet model group (HF): provided with high-fat diet and intraperitoneal injection of solvent 0.5% sodium carboxymethylcellulose solution;

[0073] 50 mg / kg ASIV group (ASIVL): provided with high-fat diet and intraperitoneal injection of 50 mg / kg ASIV;

[0074] 100 mg / kg ASIV group (ASIVH): provided with high-fat diet and intraperitoneal injection of 50 mg / kg ASIV;

[0075] 2 mg / kg doxorubicin positive control group (DOX): provided with high-fat diet and intraperitoneal injection of 2 mg / kg doxorubicin;

[0076] The experiment was carried out for 30 consecutive days.

[0077] The results are as follows Figure 2 The results showed that the tumor growth in obese mice was significantly higher than that in lean mice, and ASIV could significantly inhibit the growth of TNBC ( Figure 2 AC).

[0078] The liver, kidney and spleen of SCID tumor-bearing mice were stained with hematoxylin-eosin. The results showed that ASIV had no obvious damage to the liver, kidney and spleen of SCID tumor-bearing mice ( Figure 2 D).

[0079] The results of biochemical kits showed that ASIV had no significant effect on the levels of ALT and AST in the serum of SCID tumor-bearing mice ( Figure 2 EF).

[0080] Example 2 Investigating the effect of astragaloside IV on reversing obesity-related TNBC cell viability and lipid accumulation

[0081] Human breast cancer cell lines MDA-MB-231 and MDA-MB-468 were selected as the research objects. MDA-MB-231 and MDA-MB-468 cells in logarithmic growth phase were added with DMEM / F12 medium containing 10% fetal bovine serum, inoculated in 96-well plates, and the cell concentration was adjusted. 3×10 3 cells, with a total volume of 100 μL, and set up a blank group and different concentration groups of free fatty acids (FFA) (0.125, 0.25, 0.5, 1, 2 mM). After 48 hours, add 20 μL of 5 mg / ml MTT working solution to each well, mix well and culture in a 37°C, 5% CO2 saturated humidity incubator for 4 hours. Add 100 μL of triple solution (10% sodium dodecyl sulfate-5% isobutanol-0.012M hydrochloric acid) to completely dissolve the purple-blue formazan precipitate. Place in an enzyme reader and detect the absorbance value with an enzyme reader (570 nm).

[0082] The results showed that in human breast cancer cell lines MDA-MB-231 and MDA-MB-468, FFA at low concentrations could promote cell viability, while high concentrations could inhibit cell viability. When the FFA concentration was 0.25 mM, it significantly promoted the viability of TNBC cells ( Figure 3 A, E).

[0083] The kit was used to detect the effect of FFA induction on lipid accumulation in TNBC cell lines MDA-MB-231 and MDA-MB-468.

[0084] The results showed that 0.25 mM FFA induction could significantly increase the content of NEFA, TC and TG in cells ( Figure 3 BD, FH), indicating that FFA induction could significantly increase intracellular lipid accumulation.

[0085] TNBC cell suspension with a single cell rate of more than 95% was inoculated into a 96-well plate at 3000 cells per well, and culture medium containing 0.25mM FFA or ordinary culture medium was added. Then, different concentrations of ASIV (0, 40, 60) were added for intervention for 48 hours. The MTT method was used to detect the effect of ASIV on FFA-induced promotion of TNBC cell line MDA-MB-231 and MDA-MB-468 cell viability.

[0086] The results showed that 40 and 60 μM ASIV intervention could significantly reverse the viability of FFA-induced cells ( Figure 4 A, E).

[0087] A kit was used to detect the effect of ASIV on FFA-induced lipid accumulation in TNBC cell lines MDA-MB-231 and MDA-MB-468.

[0088] The results showed that 60 μM ASIV intervention could significantly reduce the contents of NEFA, TC and TG in cells ( Figure 4 BD, FH).

[0089] Example 3 Investigating the role of astragaloside IV in regulating the lipid metabolism network of obesity-related TNBC

[0090] The UHPLC-QTOF-MS / MS lipidomics technology platform was used to analyze the TNBC cell line MDA-MB-231 after 48 hours of intervention with 0.25mM FFA and 60μM ASIV. The mass spectrometry information showed that ASIV can effectively regulate the metabolites of TNBC cells after FFA induction. Figure 5 As shown in the figure, OPLS-DA and S-plot analysis were performed on the LC-MS data, and differential metabolites were screened according to VIP ≥ 1 and |P(corr)| ≥ 0.58. Combining the chromatographic and mass spectrometric information of differential metabolites, a total of 103 differential metabolites were identified, including 29 in positive ion mode and 74 in negative ion mode. These differential metabolites were mainly enriched in the sphingolipid metabolic pathway and the glycerophospholipid metabolic pathway.

[0091] The UHPLC-QTOF-MS / MS lipidomics technology platform was used to analyze the tumor tissues of SCID obese mice, and the mass spectrometry information showed that ASIV could effectively regulate the metabolites of TNBC induced by high-fat diet. Figure 6As shown in the figure, OPLS-DA and S-plot analysis were performed on the LC-MS data, and differential metabolites were screened according to VIP ≥ 1 and |P(corr)| ≥ 0.58. Combining the chromatographic and mass spectrometric information of differential metabolites, a total of 470 differential metabolites were identified, including 265 in positive ion mode and 205 in negative ion mode. These differential metabolites were mainly enriched in sphingolipid metabolism, pyruvate metabolism and selenium complex metabolism pathways. The above results confirm that astragaloside IV can significantly correct the lipid metabolite disorder of obesity-related TNBC, especially it can treat obesity-related TNBC by reshaping the tumor sphingolipid metabolism pathway.

[0092] Example 4 Investigating the role of astragaloside IV in regulating sphingolipid GalCer in obesity-related TNBC

[0093] The UHPLC-QTOF-MS / MS lipidomics technology platform was used to detect the changes in lipid metabolites in the TNBC cell line MDA-MB-231 after 48 hours of intervention with 0.25 mM FFA and 60 μM ASIV.

[0094] Galactosylceramide (GalCer), lactosylceramide and sphingomyelin are three important active sphingolipids, which are associated with cell growth, prolonged tumor cell survival time, enhanced tumor invasiveness, etc.

[0095] The results showed that the levels of three sphingolipids, GalCer, Lactosyceramide and Sphingomyelin, in the TNBC cell line MDA-MB-231 were significantly changed after FFA induction ( Figure 7 AC), which was significantly reversed after ASIV administration ( Figure 7 AC). Moreover, by comparison, it can be seen that ASIV has the most obvious regulatory effect on GalCer sphingolipids in obesity-related TNBC.

[0096] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Application of astragaloside IV in the preparation of drugs for the treatment of obesity-related triple-negative breast cancer.

2. The use according to claim 1, characterized in that: The drug reduces cancer cell viability, and / or, the drug reduces lipid accumulation in cancer cells.

3. The use according to claim 2, characterized in that: The drug reduces the NEFA content in cancer cells; and / or, the drug reduces the TC content in cancer cells; and / or, the TG content in cancer cells.

4. The use according to claim 1, characterized in that: The drug modulates sphingolipid metabolism pathways in cancer cells.

5. The use according to claim 3, characterized in that: The drug reduces GalCer sphingolipids in cancer cells.

6. A drug for treating obesity-related triple-negative breast cancer, characterized in that: The main component of the medicine is astragaloside IV.

7. The drug according to claim 6, characterized in that The drug also includes pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that The pharmaceutically acceptable excipients include any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, and stabilizers.

9. The drug according to claim 6, characterized in that The drug reduces cancer cell viability, and / or, the drug reduces lipid accumulation in cancer cells.

Images