WO2024221959A1 - Oxidative stress inducer for triple-negative breast cancer cells and pharmaceutical composition for synergistically resisting triple-negative breast cancer - Google Patents

Abstract

Provided are an oxidative stress inducer for triple-negative breast cancer cells and a pharmaceutical composition for synergistically resisting triple-negative breast cancer. The active ingredients of the oxidative stress inducer for triple-negative breast cancer cells include brusatol and polydatin. The active ingredients of the pharmaceutical composition for synergistically resisting triple-negative breast cancer include brusatol and polydatin. Polydatin and brusatol feature good treatment efficacy and low toxicity in synergistically resisting triple-negative breast cancer.

Description

An oxidative stress inducer for triple-negative breast cancer cells and a pharmaceutical composition for synergistically combating triple-negative breast cancer Technical Field

The present invention belongs to the field of biomedicine technology, and specifically relates to an oxidative stress inducer for triple-negative breast cancer cells and a synergistic anti-triple-negative breast cancer pharmaceutical composition.

Background Art

Breast cancer is the most common cancer in women worldwide, accounting for 24.3% of all female cancers, ranking fifth in the world's tumor-related mortality rate, and is the leading cause of cancer death in women. Triple-negative breast cancer (TNBC) is a breast cancer that lacks the expression of estrogen receptors, progesterone receptors, and human epidermal growth factor 2 receptors, accounting for 15% to 20% of all breast cancer cases, and is characterized by invasiveness and high mortality. At present, surgical resection, radiotherapy, and chemotherapy are still the main options for the treatment of TNBC. Commonly used drugs include platinum drugs, poly ADP-ribose polymerase inhibitors, targeted PI3K/AKT/mTOR inhibitors, etc. However, they have problems such as systemic toxicity, easy to develop drug resistance, and poor prognosis. Therefore, more efficient and low-toxic methods still need to be developed to treat TNBC.

In recent years, natural products have great application prospects in cancer treatment due to their easy availability, low cost, low toxicity and side effects, low resistance to drug resistance, and high efficacy. Polydatin (PD) is a component of Polygonum cuspidatum and a glycoside of resveratrol. It has multiple biological functions, including anti-platelet aggregation, antioxidant, heart protection, anti-inflammation, anti-tumor and immunomodulation. The PI3K/AKT/mTOR signaling axis is often abnormally activated in TNBC and is related to the occurrence and development of tumors and the development of chemotherapy resistance. Studies have shown that PD can inhibit the PI3K/AKT/mTOR pathway of cervical cancer cells. PD can also inhibit the proliferation of laryngeal cancer and cervical cancer cells and induce tumor cell apoptosis by regulating the PDGF/AKT signaling pathway. Among them, the treatment of laryngeal cancer and cervical cancer is to give 50mg/kg of PD three times a week for three consecutive weeks; in the study of Yaodong Chen et al., PD was given at a dose of 50mg/kg every day to treat glioblastoma multiforme; Tao Zhang et al. combined PD with 2-deoxyglucose to treat breast cancer, giving each at a dose of 100mg/kg every other day for three consecutive weeks. However, these treatment regimens use large doses of PD, The frequent dosing leads to high treatment costs and drug toxicity, so there is still a need to develop lower-cost and less toxic TNBC treatment options.

Brucea brucea is a commonly used anti-tumor herbal medicine in traditional Chinese medicine. Brusatol (BRU) is a quassinol compound extracted and purified from it, which has anti-tumor, anti-malarial and anti-inflammatory activities; its anti-cancer effect and mechanism have been studied in many studies. Many studies have found that the anti-cancer effect of BRU is to inhibit the growth of tumor cells by promoting the degradation of Nrf2 and inhibiting the expression of downstream antioxidant genes. BRU reverses chemotherapy resistance by inhibiting Nrf2. Combining with cisplatin and gemcitabine can make a variety of tumor cells sensitive to chemotherapy drugs and enhance the effect of chemotherapy. In existing treatment studies, Ruifan Yea et al. used 2 mg/kg of BRU every other day for four consecutive weeks to treat liver cancer; Yun Yang et al. also used 2 mg/kg of BRU in combination with trastuzumab once a day for one week to treat breast cancer; Yang Liu et al. used 2 mg/kg of BRU every other day for three consecutive weeks to treat non-small cell lung cancer. The above treatment methods have the disadvantages of general effect, high cost and high toxicity. Therefore, more effective, low-cost, and low-toxic treatment options are needed for the treatment of TNBC.

Summary of the invention

In order to solve the deficiencies in the prior art, the object of the present invention is to provide an oxidative stress inducer for triple-negative breast cancer cells and a pharmaceutical composition for synergistically fighting triple-negative breast cancer.

The invention provides an oxidative stress inducer for triple-negative breast cancer cells. The active ingredients of the oxidative stress inducer include brucein and resveratrol glycosides.

Furthermore, in the oxidative stress inducer, the molar ratio of brucein to resveratrol glycoside is 5:1.

Furthermore, the oxidative stress inducer increases the oxidative stress pressure in triple-negative breast cancer cells.

The present invention also provides a pharmaceutical composition for synergistically combating triple-negative breast cancer, wherein the active ingredients of the pharmaceutical composition include brucea bruceiol and resveratrol glycosides.

Furthermore, in the pharmaceutical composition, the mass ratio of brucea javanica to resveratrol glycoside is 25:1.

Furthermore, the pharmaceutical composition also includes other pharmaceutically acceptable excipients.

The present invention also provides the use of the pharmaceutical composition in preparing a synergistic anti-triple-negative breast cancer drug.

Beneficial effects of the present invention:

The present invention uses resveratrol glycosides and brucein to synergistically fight triple-negative breast cancer, solving the problem of poor therapeutic effect and strong toxicity when resveratrol glycosides and brucein are used alone, and provides a new treatment plan for efficient, low-cost, and low-toxic treatment of triple-negative breast cancer. Resveratrol glycosides synergize with brucein to inhibit the proliferation of triple-negative breast cancer cells by increasing the intracellular ROS level.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the changes in cell proliferation ability and intracellular ROS levels in human breast cancer cells MDA-MB-231 after treatment with BRU and PD, as well as the determination of their potential regulatory mechanisms. (A) After treating MDA-MB-231 cells for 24h, 48h, and 72h, CCK-8 was used to determine cell viability. (B) Results of the synergistic effect experiment of BRU and PD. (C and D) After treating MDA-MB-231 cells for 24h, flow cytometry was used to detect changes in intracellular ROS levels. The presented data represent mean ± SD, and significant differences were determined using Two-way ANOVA. *, P < 0.05; ***, P < 0.001. (E) After treating MDA-MB-231 cells for 24h, WB was used to detect changes in intracellular Nrf2 expression levels.

Figure 2 shows the determination and comparison of the growth and weight of Balb/c tumors in nude mice. (A) Schematic diagram of the experimental scheme. (B-D) Determination of the growth of Balb/c subcutaneous tumors in nude mice. The tumor volume of mice was measured every 7 days; after the mice were killed, the tumor tissues were weighed and the tumor weights of each group were compared. The data presented represent the mean ± SD, and the significant differences were determined by two-sided T test analysis and non-parametric test analysis. *, P < 0.05; **, P < 0.01. (E) The curve of mouse weight changes during the experiment. There was no significant difference between the drug-treated group and the control group, and the body weight remained relatively stable, indicating that the combined drug toxicity was low.

DETAILED DESCRIPTION

In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

Example 1

This example explores the effects of PD in combination with BRU on TNBC cell proliferation and intracellular oxidative stress.

The experiment on the change of cell proliferation ability was divided into Control (1‰ DMSO), BRU (20nM), PD (100nM), and BRU (20nM) + PD (100nM). The human breast cancer MDA-MB-231 cells were treated with each group of drugs, and the cell viability was measured using CCK-8 at 24h, 48h, and 72h. The results are shown in Figure 1A.

Calcusyn software was used to analyze whether PD and BRU had synergistic effects. Five concentrations were set, namely BRU 20, 40, 60, 80, 100 nM; PD 100, 200, 300, 400, 500 nM, according to the drug combination quantitative method widely used in anti-tumor combination therapy jointly established by Chou and Talalay: Chou-Talalay (median pharmacodynamic method), and the formula for the combination index of the two compounds is: in:

(D)1——the concentration required for compound 1 to produce effect X when it works in combination with other compounds;

(D)2——the concentration required for compound 2 to produce effect X when it works in combination with other compounds;

(Dx)1——the concentration of compound 1 required to produce effect X when used alone;

(Dx)2 – the concentration of compound 2 required to produce effect X when used alone.

The IC50 value of the drug's half effective dose (representing the inhibition rate, i.e., the effect value entered in the software) is calculated based on the OD value measured when the drug is used alone or in combination at different concentrations. The corresponding inhibition rate value is entered into the software to calculate the corresponding CI value. The software automatically generates an inhibition rate (Fa)-drug synergy index (CI value) graph. When the CI value is <1, it indicates that the compounds have a synergistic effect. The results are shown in Figure 1B , with a CI value of <1, indicating that PD and BRU have a synergistic effect.

The experiment on the change of intracellular ROS level was divided into Control, BRU (20nM), PD (100nM), and BRU (20nM) + PD (100nM). The human breast cancer MDA-MB-231 cells were treated with each group of drugs. After 24 hours, the change of intracellular ROS level was detected by flow cytometry. The results are shown in Figure 1C and D.

The experimental groups for changes in intracellular Nrf2 expression levels were Control, BRU (20nM), PD (100nM), and BRU (20nM) + PD (100nM). After treating MDA-MB-231 cells for 24 hours, the changes in intracellular Nrf2 expression were detected by WB. The results are shown in Figure E.

The experimental results showed that PD could cooperate with BRU to increase the level of ROS in TNBC cells by inhibiting the expression of intracellular antioxidant gene Nrf2, thereby inhibiting cell proliferation to a certain extent and thus inhibiting tumor growth.

Example 2

In this example, human breast cancer cells MDA-MB-231 (5*10^6) were first injected subcutaneously into nude mice Balb/c to establish a subcutaneous tumor model, and then intraperitoneal injection was performed to divide the mice into a control group (Ctrl) and an experimental group. The control group was PBS containing 1% DMSO, and the experimental group was PD (50 mg/kg); PD (25 mg/kg) combined with BRU (1 mg/kg), administered at intervals of seven days, and administered a total of three times (administered at 45 days, 52 days, and 59 days in Figure 2A) according to the tumor growth. The tumor growth of the mice was monitored, the tumor volume was measured at intervals of seven days, and the mice were finally killed and the tumor tissue was weighed. The results are shown in Figure 2.

The experimental results showed that PD could synergize with BRU to inhibit the growth of TNBC by improving drug efficacy and reducing drug toxicity, and the combined drug toxicity was low.

Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims (7)

An oxidative stress inducer for triple-negative breast cancer cells, characterized in that the active ingredients of the oxidative stress inducer include brucea bruceiol and resveratrol glycosides. The oxidative stress inducer according to claim 1, characterized in that the molar ratio of brucea bruceiol to resveratrol glycoside is 5:1. The oxidative stress inducer according to claim 1, characterized in that the oxidative stress inducer increases the oxidative stress pressure in triple-negative breast cancer cells. A pharmaceutical composition for synergistically combating triple-negative breast cancer, characterized in that the active ingredients of the pharmaceutical composition include brucea bruceiol and resveratrol glycosides. The pharmaceutical composition according to claim 4, characterized in that the mass ratio of brucea bruceiol to resveratrol glycoside is 25:1. The pharmaceutical composition according to claim 4, characterized in that the pharmaceutical composition further comprises other pharmaceutically acceptable excipients. Use of the pharmaceutical composition according to claim 4 in the preparation of a synergistic anti-triple-negative breast cancer drug.