Application of lonidamine combined with apigenin to treatment of colorectal cancer

Abstract

The invention relates to the technical field of biology, in particular to application of lonidamine combined with apigenin to treatment of colorectal cancer. The invention provides a novel pharmaceutical composition for treating colorectal cancer. LND and AP form the pharmaceutical composition according to different molar concentrations or different weight ratios, the anti-tumor activity of the pharmaceutical composition is respectively tested on a colon cancer cell line MC38 culture and a tumor-bearing mouse, and results prove that compared with the effect of a single drug, the combined drug obviously improves the activity of anti-tumor cells in vitro or in vivo.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the use of lonidamine combined with apigenin in treating rectal cancer. Background Art

[0002] Colorectal cancer (CRC) is the second most common cancer worldwide. Traditional treatments include surgical resection, chemotherapy, radiotherapy, and immunotherapy, but there are problems with serious side effects and drug resistance. Therefore, the development of new treatment strategies is of great significance.

[0003] Rapidly proliferating tumor cells often require more energy than normal cells. This increased energy demand drives metabolic reprogramming of cancer cells, in which they exhibit a clear preference for glycolysis, even under aerobic conditions. This phenomenon is known as the Warburg effect and is considered a hallmark of cancer. By relying on glycolysis and altering mitochondrial function, cancer cells gain a metabolic advantage that promotes their rapid growth and survival. Given the important role of metabolic reprogramming in cancer cell proliferation, targeting these altered pathways has emerged as a promising therapeutic strategy.

[0004] Lonidamine (LND), an indazole-3-carboxylic acid derivative that was initially investigated as a male contraceptive for its antispermatogenic properties, has attracted attention in this context for its ability to modulate tumor metabolism. LND exerts its antitumor effects primarily by inhibiting key glycolytic enzymes, such as hexokinase II, leading to disruption of energy metabolism in cancer cells. In addition, LND interferes with mitochondrial function, leading to loss of mitochondrial membrane potential, increased oxidative stress, and ultimately apoptosis. Although LND shows promise as an anticancer agent, its use as a monotherapy is limited by limited efficacy and dose-dependent toxicity.

[0005] Apigenin (AP) is a flavonoid compound that is widely found in various fruits and vegetables, such as celery, thyme, parsnip, chamomile, onion, orange and tea. It exhibits a wide range of biological activities, including antioxidant, anti-inflammatory, antibacterial, antiviral and anticancer effects. Many studies have demonstrated the anti-tumor effects of AP on various cancers, such as colorectal cancer, lung cancer, prostate cancer, pancreatic cancer and breast cancer. AP has many targets. Studies have shown that AP can effectively inhibit the expression of GLUT1, thereby inhibiting the glycolysis pathway. In addition, AP can inhibit the proliferation of tumor cells by upregulating cell cycle inhibitors such as p21 and p27, and downregulating Cyclin D1 and Cyclin E. The combination of AP and multiple chemotherapeutic drugs can increase the efficacy of chemotherapeutic drugs and reduce the side effects of chemotherapy. Summary of the invention

[0006] The purpose of the present invention is to provide a combined drug composition for treating colorectal cancer. The drug composition can effectively inhibit tumor growth by synergistically inhibiting the proliferation and migration of colon cancer cells. Compared with the use of two drugs alone, it greatly enhances the killing of tumor cells and more significantly inhibits the proliferation of tumor cells, with unexpected technical effects. The drug composition has potential clinical application value and provides a new treatment strategy for CRC patients.

[0007] The present invention discloses the use of a pharmaceutical composition in preparing a drug for preventing and / or treating colorectal cancer; The active ingredients of the pharmaceutical composition are lonidamine and apigenin.

[0008] The molar ratio of lonidamine to apigenin is 2.5-40:1.

[0009] Preferably, the molar ratio of lonidamine to apigenin is 1.68-40:1.

[0010] Preferably, the molar ratio of lonidamine to apigenin is 5-20:1.

[0011] More preferably, the molar ratio of lonidamine to apigenin is preferably 1.68:1 or 10:1.

[0012] The pharmaceutical composition is used to inhibit the proliferation and migration of colon cancer cells.

[0013] In the present invention, the dosage form of the drug can be any pharmaceutically acceptable dosage form, including one or more of tablets, capsules, granules, powders, suspensions, syrups, oral solutions and injections. The administration routes of the drug include intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection and sublingual administration.

[0014] Compared with the prior art, the present invention has the following significant effects: The present invention provides a new pharmaceutical composition for treating colorectal cancer, comprising lonidamine and apigenin; the pharmaceutical composition exerts its effects through the following mechanisms: 1. Synergistically inhibiting the proliferation and migration of colon cancer cells; 2. Inducing cell cycle arrest at the G2 / M phase; 3. Promoting apoptosis by downregulating Bcl-2 and upregulating Bax; 4. Disrupting the glycolytic pathway, reducing the expression of HK2 and Glut1, reducing glucose consumption and lactate production; 5. Causing nucleotide depletion and NAD+ metabolic disorder. LND and AP were combined into a pharmaceutical composition at different molar concentrations or different weight ratios, and the anti-tumor activity of the pharmaceutical composition was tested on colon cancer tumor cell line MC38 culture and tumor-bearing mice, respectively. The results showed that compared with the effect of a single drug, the combination drug significantly improved the anti-tumor cell activity both in vitro and in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : (A) MC38 cells were cultured in the presence of different concentrations and ratios of LND and AP alone or in combination for 48 hours, and cell proliferation was measured; (B) Combination index (CI) graph. CI values ​​were calculated using CompuSyn software, where CI values ​​<1, = 1, and >1 indicate synergistic, additive, and antagonistic effects, respectively; (CE): (C): Cell migration of MC38 cells after combined treatment with LND (140 µM) and AP (14 µM); (D, E): Quantitative analysis of migration rate at different time points. All data were obtained from at least three independent experiments. Results are expressed as mean ± standard deviation (SD). Data were analyzed by one-way ANOVA. ****p ≤ 0.0001, **p ≤ 0.01, *p ≤ 0.05, ns, not significant.

[0016] Figure 2 :(A) Body weight changes of mice treated with LND combined with AP. (B) Tumor volume change curves of mice in different treatment groups. (C) Tumor weight (n = 5). (D) Mouse tumor. (E, F) Immunohistochemistry (IHC) was used to detect the expression of Ki67 in tumor tissues (E), and the expression level was quantified (F). Data were analyzed by one-way ANOVA. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, ns indicates no significant difference. DETAILED DESCRIPTION

[0017] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The raw materials and equipment used in the examples are well known to those skilled in the art, and are all available on the market or easily obtained or prepared.

[0018] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0019] Example 1: The combined use of LND and AP can significantly inhibit the proliferation and migration of MC38 tumor cells Cell viability and proliferation assays: Cells (6 × 10^3 cells / mL) were seeded into 96-well plates with 100 μL per well and cultured overnight. Subsequently, cells were treated with medium containing LND, AP, or a combination of both for 48 h. Cell viability was assessed using a CCK-8 kit, following the manufacturer’s instructions. Cell proliferation was quantified by measuring absorbance at 450 nm on a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

[0020] Cell migration assay: Wound healing assay was used to assess cell migration. Cells were seeded into 6-well plates and cultured to 80%-90% confluence. A scratch was introduced into the cell monolayer using a 200 μL pipette tip, followed by two washes with serum-free medium to remove debris. Subsequently, cells were cultured in serum-free medium containing LND (40-140 μM) and AP (4-14 μM) for 12 and 24 h, alone and in combination. Wound closure images were captured using an inverted microscope, maintaining consistent magnification and position. Cell migration rates at 12 and 24 h were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

[0021] The results showed that the use of LND or AP alone resulted in a dose-dependent decrease in cell activity; when the LND:AP ratio was 5-20, the combined administration of LND and AP could increase tumor killing, especially 140 µM LND and 14 µM AP significantly inhibited cell proliferation, which was better than the two single drug treatments and had a significant synergistic effect (CI = 0.637) (e.g. Figure 1 The results of the wound healing experiment showed that the combined administration of LND and AP effectively inhibited cell migration at 12 and 24 hours (as shown in Figure 2A and B). Figure 1 CE shown in Figure 1).

[0022] Example 2: The combined use of LND and AP can significantly inhibit the growth of tumors in MC38 tumor-bearing mice Adult female C57BL / 6 mice were subcutaneously injected with MC38 cells (1 × 10^6 cells per mouse) to establish a colorectal cancer mouse model. The model mice were randomly divided into four groups (5 mice per group), and the average tumor volume in each group was 50-100 mm 3 The groups were as follows: i) vehicle control (methylcellulose / Tween 80, intraperitoneal injection [ip], once every 2 days); ii) LND (30 mg / kg, ip, once every 2 days); iii) AP (15 mg / kg, ip, once every 2 days); and iv) combined treatment of LND (30 mg / kg) and AP (15 mg / kg) (ip, once every 2 days). Tumor size was measured using a caliper and the formula: volume = length × width 2 × 0.5 to calculate the tumor volume. After the treatment endpoint, the mice were killed, and the tumor tissues were collected and weighed. After the tumor tissues were fixed in 4% formaldehyde, the tissues were dehydrated, embedded in paraffin, and then cut into 5 μm thick sections. Immunohistochemical staining was performed according to standard methods, including the following steps: paraffin sections were hydrated to water, antigen retrieval, blocking endogenous peroxidase, serum blocking, incubation with primary antibody (anti-Ki67), incubation with secondary antibody, DAB color development, nuclear counterstaining, dehydration and sealing. The sections were observed using an optical microscope (Olympus, Japan).

[0023] The results showed that the weight of mice treated with LND or AP alone, as well as mice treated with LND and AP combined, decreased in the early stage, but gradually increased slowly, and at the end of the treatment, the weight was the same as that of the control group, indicating that the toxicity of both single-drug and combined-drug therapy was relatively low ( Figure 2 There was no significant difference in tumor growth and final tumor weight between the LND and AP monotherapy groups and the non-treated control group, indicating that monotherapy had no significant therapeutic effect at a given dose, while tumor growth and final tumor weight were significantly inhibited in the LND and AP combination therapy group ( Figure 2 To further investigate the effect of LND and AP combination on tumor growth, Ki67 expression levels were evaluated by immunohistochemistry. The results showed that Ki67 expression was significantly decreased in tumor tissues of the combined treatment group ( Figure 2 E and F), indicating that the combined treatment effectively inhibited cell proliferation in vivo.

[0024] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating colorectal cancer; The active ingredients of the pharmaceutical composition are lonidamine and apigenin.

2. The use according to claim 1, characterized in that: The molar ratio of lonidamine to apigenin is 1-40:

1.

3. The use according to claim 1, characterized in that: The molar ratio of lonidamine to apigenin is 1.68-40:

1.

4. The use according to claim 1, characterized in that: The molar ratio of lonidamine to apigenin is 5-20:

1.

5. The use according to claim 3, characterized in that: The molar ratio of lonidamine to apigenin is preferably 1.68:1 or 10:

1.

6. The use according to claim 1, characterized in that: The pharmaceutical composition is used for inhibiting the proliferation and migration of colon cancer cells.

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