CN116726021B - Combined medicine of DRP1 inhibitor and iron death inducer and anti-tumor application thereof - Google Patents

Abstract

The present invention provides a combination drug of a DRP1 inhibitor and an ferroptosis inducer and its anti-tumor use, belonging to the field of biomedical technology. The combination drug of the present invention contains a DRP1 inhibitor and an ferroptosis inducer of the same or different specifications, which are administered simultaneously or separately, and a pharmaceutically acceptable carrier. The present invention uses a DRP1 inhibitor and an ferroptosis inducer in combination, and it is found that it has a significant inhibitory effect on tumors, and compared with the use of a DRP1 inhibitor or an ferroptosis inducer alone, it exerts a synergistic anti-tumor effect. The combination drug of the present invention can eliminate tumors in the early stages of tumors, and in the middle and late stages of tumors, it can reduce the volume of tumors and prepare for surgical treatment. The combination drug of the DRP1 inhibitor and the ferroptosis inducer of the present invention can be used to prepare drugs for preventing and/or treating cancer, especially oral squamous cell carcinoma, and retains the patient's maxillofacial anatomical appearance and function as much as possible, which has important clinical significance.

Description

A drug for combining a DRP1 inhibitor and a ferroptosis inducer and its anti-tumor use

Technical Field

The present invention belongs to the technical field of biomedicine, and specifically relates to a combined drug of a DRP1 inhibitor and a ferroptosis inducer and an anti-tumor application thereof.

Background Art

Oral squamous cell carcinoma is the most common malignant tumor in the oral and maxillofacial region, and the current treatment is mainly surgical. However, due to the special anatomical location of oral squamous cell carcinoma, surgical treatment may often lead to defects in the anatomical appearance and functional destruction of the maxillofacial region, such as limited swallowing and language function, physiological pain, and changes in maxillofacial appearance, which may cause physical and psychological harm to patients, seriously reduce their quality of life, and have a negative impact on their psychological state and social behavior. There are limited drug treatments for oral squamous cell carcinoma, and cisplatin is its main chemotherapy drug, but patients have poor responsiveness to cisplatin, a high resistance rate, and general efficacy. At present, there is a lack of effective chemotherapy drugs for oral squamous cell carcinoma. Finding new and effective chemotherapy drugs is of great significance for the treatment of oral squamous cell carcinoma.

Ferroptosis is an iron-dependent, novel type of programmed cell death that is different from apoptosis, necrosis, and autophagy. The main mechanism of ferroptosis is that under the action of ferrous iron or esteroxygenase, unsaturated fatty acids highly expressed on the cell membrane are catalyzed to cause lipid peroxidation, thereby inducing cell death. Ferroptosis inducers can use ferroptosis to eliminate target cells (such as cancer cells, inflammatory cells, activated fibroblasts, etc.) to achieve the purpose of treating diseases. Currently, the use of ferroptosis inducers to treat cancer has become a research hotspot.

Mitochondria are important "energy factories" of cells and play an important role in the occurrence and development of tumors. Normal mitochondrial function is essential for energy production and cell survival of tumor cells. Mitochondria control the growth of tumor cells by regulating their energy metabolism and apoptosis. Mitochondrial fusion and fission play an essential regulatory role in mitochondrial function. Moreover, the fusion and fission process of mitochondria also plays an important role in the occurrence and development of tumors. Mitochondrial dynamin-related protein 1 (DRP1) is one of the important proteins regulating mitochondrial fission. DRP1 can regulate its ability to mediate mitochondrial fission-fusion, which is related to the growth of tumor cells. Many studies have shown that DRP1 inhibitors can be used for cancer treatment.

Although studies have shown that ferroptosis inducers and DRP1 inhibitors can be used for anti-tumor and cancer treatment, there is no evidence that ferroptosis inducers and DRP1 inhibitors are used in combination for anti-tumor treatment, especially for the treatment of oral squamous cell carcinoma. Whether ferroptosis inducers and DRP1 inhibitors can be used in combination to treat cancer requires further study.

Summary of the invention

The purpose of the present invention is to provide a combined drug of a DRP1 inhibitor and a ferroptosis inducer and its anti-tumor use.

The present invention provides a combined drug for preventing and/or treating cancer, which contains a DRP1 inhibitor and a ferroptosis inducer of the same or different specifications, which are administered simultaneously or separately, and a pharmaceutically acceptable carrier.

Furthermore, the molar ratio of the DRP1 inhibitor to the ferroptosis inducer is 1:1-2;

Preferably, the molar ratio of the DRP1 inhibitor to the ferroptosis inducer is 1:1 to 1.5;

More preferably, the molar ratio of the DRP1 inhibitor to the ferroptosis inducer is 1:1.

Furthermore, the DRP1 inhibitor is Mdivi-1, which has the structure The ferroptosis inducing agent is Erastin, which has the structure

Furthermore, the cancer is oral squamous cell carcinoma.

The present invention also provides a pharmaceutical composition for preventing and/or treating cancer, which consists of a DRP1 inhibitor and a ferroptosis inducer.

Furthermore, the molar ratio of the DRP1 inhibitor to the ferroptosis inducer is 1:1-2;

Preferably, the molar ratio of the DRP1 inhibitor to the ferroptosis inducer is 1:1 to 1.5;

More preferably, the molar ratio of the DRP1 inhibitor to the ferroptosis inducer is 1:1.

Furthermore, the DRP1 inhibitor is Mdivi-1, which has the structure The ferroptosis inducing agent is Erastin, which has the structure

Furthermore, the cancer is oral squamous cell carcinoma.

The present invention also provides the use of the aforementioned pharmaceutical composition in preparing a drug for preventing and/or treating cancer;

Preferably, the cancer is oral squamous cell carcinoma.

The present invention also provides a pharmaceutical preparation for preventing and/or treating cancer, which is a preparation prepared by taking the aforementioned pharmaceutical composition as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

The present invention uses a DRP1 inhibitor and a ferroptosis inducer in combination, and it is found that it has a significant inhibitory effect on tumors. Compared with the use of a DRP1 inhibitor or a ferroptosis inducer alone, it exerts a synergistic anti-tumor effect. The combined drug of the present invention can eliminate tumors in the early stages of tumors, and in the middle and late stages of tumors, it can reduce the volume of tumors and prepare for surgical treatment. The combined drug of the DRP1 inhibitor and the ferroptosis inducer of the present invention can be used to prepare a drug for preventing and/or treating cancer, especially oral squamous cell carcinoma, and retains the patient's maxillofacial anatomical appearance and function as much as possible, which has important clinical significance.

Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the production of ROS and MDA in HSC3 and HN12 cells after treatment in different ways.

FIG2 shows the expression levels of ferroptosis-related proteins in HSC3 and HN12 cells after treatment in different ways.

FIG. 3 shows the results of cell activity detection of HSC3 cells after being treated in different ways.

FIG. 4 is a tumor solid image after the 18th day of the OSCC tumor cell xenograft tumor model experiment (5 parallel samples in each group) and the changes in tumor volume over time after different treatments.

Figure 5 shows the tumor entity image after 18 days of OSCC human tumor xenograft model experiment and the changes in tumor volume over time after different treatments; PDX#1, PDX#2 and PDX#3 are three groups of repeated experiments, each with 4 parallel samples.

FIG. 6 shows the activity of oral squamous cell carcinoma cells after different treatments.

FIG. 7 is a 3D model diagram and median value of the synergistic index of Mdivi-1 and Erastin of the present invention.

FIG8 shows the synergistic index of Mdivi-1 and Erastin at different drug concentrations.

DETAILED DESCRIPTION

The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

Example 1: In vitro cell experiments demonstrate the inhibitory effects of DRP1 inhibitor Mdivi-1 and ferroptosis inducer Erastin on oral squamous cell carcinoma cells

1. Experimental methods

The oral squamous cell carcinoma cells used in this example are HSC3 cells and HN12 cells.

About 20,000 cells (HSC3-NC, HSC3-shDRP1#1, HSC3-shDRP1#2, HN12-NC, HN12-shDRP1#1 or HN12-shDRP1#2 cells) were inoculated in each well of a 6-well plate and incubated overnight at 37°C and 5% CO _2. The production of ROS and MDA in oral squamous cell carcinoma cells in each group was then detected using conventional methods. At the same time, the expression of ferroptosis-related proteins GPX4, FTH1, SLC7A11 and DMT1 was detected.

About 2000 cells (HSC3-NC, HSC3-shDRP1#1 or HSC3-shDRP1#2 cells) were seeded in each well of a 96-well plate. After incubation at 37°C and 5% CO2 _overnight , the cells were treated with different doses of ferroptosis inducer Erastin, with the doses of Erastin being 0μM, 5μM and 10μM. After continued incubation for 24h, CCK8 detection was performed according to conventional methods.

About 2000 cells (HSC3-NC, HSC3-shDRP1#1 or HSC3-shDRP1#2 cells) were seeded in each well of a 96-well plate. After incubation at 37°C and 5% _CO2 overnight, the cells were treated with drugs. One group of cells only added the solvent (Vehicle) that dissolved Erastin, one group of cells only added 10μM ferroptosis inducer Erastin, one group of cells only added 5μM N-acetylcysteine (NAC), and one group of cells simultaneously added 5μM N-acetylcysteine (NAC) and 10μM ferroptosis inducer Erastin. After drug administration, the cells were incubated for 24h, and then CCK8 detection was performed according to the conventional method.

Lentivirus infection-drug screening is a common method for constructing stable transgenic cell lines that interfere with gene expression. In this embodiment, two different sequences of lentivirus were used to inhibit the expression of DRP1 in HSC3 cells or HN12 cells, and stable transgenic cell lines were constructed. The cell lines inhibiting DRP1 expression were HSC3-shDRP1#1, HSC3-shDRP1#2, HN12-shDRP1#1 and HN12-shDRP1#2. HSC3-NC and HN12-NC cells are HSC3 cells and HN12 cells constructed with lentivirus that does not inhibit DRP1 expression, and cell DRP1 expression is not inhibited.

The sequences of the lentivirus that inhibits DRP1 expression are:

shDRP1#1：cgAGATTGTGAGGTTATTGAA(SEQ ID NO.1)

shDRP1#2: cgGTGGTGCTAGAATTTGTTA (SEQ ID NO.2)

The sequence of the lentivirus that does not inhibit DRP1 expression is:

NC: TTCTCCGAACGTGTCACGT (SEQ ID NO. 3).

2. Experimental results

FIG1 shows that after inhibiting the mitochondrial dynamin (DRP1) of cells, the production of ROS and MDA in oral squamous cell carcinoma cells increased.

Figure 2 shows that after inhibiting the mitochondrial dynamin (DRP1) of cells, the expression of ferroptosis-related proteins GPX4, FTH1, and SLC7A11 decreased, and the expression of DMT1 increased.

The left figure of Figure 3 shows that the inhibition of DRP1 alone has little effect on the activity of HSC3 cells. After combined use with the ferroptosis inducer Erastin, the activity of HSC3 cells decreased significantly, indicating that the combined use of DRP1 inhibitors and ferroptosis inducers significantly improves the inhibitory effect on the activity of oral squamous cell carcinoma cells. The combined use of DRP1 inhibitors and ferroptosis inducers can play a synergistic role in inhibiting tumor cells. N-acetylcysteine (NAC) is a ferroptosis inhibitor. The right figure of Figure 3 shows that cell death can be rescued after the addition of NAC, indicating that cells do undergo ferroptosis after the addition of Erastin, excluding other modes of death.

Example 2: In vivo tumor inhibition effect of the combined use of DRP1 inhibitor Mdivi-1 and ferroptosis inducer Erastin

1. Experimental methods

The present invention constructs an OSCC tumor cell xenograft model and an OSCC human tumor xenograft model, using BALB/c nude mice and transplanting tumors subcutaneously. The method for constructing the OSCC tumor cell xenograft model is to inject 1×10 ⁶ oral tumor cells HN12 into the subcutaneous part of the back of the nude mouse by subcutaneous injection, and the injection dose is 100 μL. After the injection, the model is successfully established when the tumor grows to 100 mm ^3. The method for constructing the OSCC human tumor xenograft model is to inject fresh tumor tissue from patients with oral squamous cell carcinoma into the subcutaneous part of the back of the nude mouse by subcutaneous injection, and the injection dose is 5-6 tumor tissues of 2 to 3 mm ³ in size. After the injection, the model is successfully established when the tumor grows to 100 mm ³ .

The mice with successful modeling were divided into 4 groups: control group (DMSO solvent group, Vehicle), Mdivi-1 group, Erastin group, and Mdivi-1+Erastin combination group. The Mdivi-1 group was intraperitoneally administered with Mdivi-1 (dissolved in DMSO) once every other day, with a dose of 15 mg/kg each time, for 16 days. The Erastin group was intraperitoneally administered with Erastin (dissolved in DMSO) once every other day, with a dose of 15 mg/kg each time, for 16 days. The Mdivi-1+Erastin combination group was administered with Mdivi-1 and Erastin (dissolved in DMSO) once every other day, with a dose of 15 mg/kg for each time, for 16 days. The control group was intraperitoneally administered with an equal dose of DMSO.

The first day of drug administration was the 0th day of the experiment. The tumor volume of mice in each group was observed before each drug administration on the 0th, 2nd, 4th, 6th, 8th, 10th, 12th, 14th, 16th and 18th days of the experiment. The tumor volume was measured by using a vernier caliper. No drug was administered after the 18th day of the experiment, and the tumor tissue was directly collected for staining.

At the same time, each group detected the tumor proliferation rate and whether ferroptosis occurred in the tumor. The tumor proliferation rate was detected by immunohistochemical KI67 staining, and whether ferroptosis occurred in the tumor was detected by immunohistochemical GPX4 staining.

2. Experimental results

As shown in Figures 4 and 5, the combined use of Mdivi-1 and Erastin has the best inhibitory effect on tumor tissue, which is significantly better than the use of Mdivi-1 and Erastin alone. The results show that the combined use of Mdivi-1 and Erastin in the treatment of oral squamous cell carcinoma can play a synergistic therapeutic role.

Example 3: Cell experiment using the DRP1 inhibitor Mdivi-1 and the ferroptosis inducer Erastin in combination

1. Experimental methods

About 2000 cells (HSC3 and HN12 cells) were seeded in each well of a 96-well plate. After incubation at 37°C and 5% CO ₂ overnight, the cells were divided into 4 groups: control group (DMSO solvent group, Vehicle), Mdivi-1 group (10 μM), Erastin group (10 μM), and Mdivi-1 (10 μM) + Erastin (10 μM) combined group. After administration, the cells were incubated for 24 hours, and then CCK8 was detected according to the conventional method. The online tool SynergyFingder was used to simulate and calculate whether the DRP1 inhibitor Mdivi-1 and the ferroptosis inducer Erastin have a synergistic effect.

2. Experimental results

The experimental results are shown in Figures 6 to 8: The synergistic index analysis of the combined effect of Mdivi-1 and Erastin showed that the combined use of Mdivi-1 and Erastin can exert a synergistic inhibitory effect on oral squamous cell carcinoma cells. When the molar ratio of Mdivi-1 and Erastin is 1:1, the drug synergistic effect is most significant, and the effect of inhibiting oral squamous cell carcinoma cells is best.

In summary, the present invention uses a DRP1 inhibitor and a ferroptosis inducer in combination, and finds that it has a significant inhibitory effect on tumors, and has a synergistic anti-tumor effect compared to the use of a DRP1 inhibitor or a ferroptosis inducer alone. The combined drug of the present invention can eliminate tumors in the early stages of tumors, and can reduce tumor volume in the middle and late stages of tumors to prepare for surgical treatment. The combined drug of the DRP1 inhibitor and the ferroptosis inducer of the present invention can be used to prepare drugs for preventing and/or treating cancer, especially oral squamous cell carcinoma, and retains the patient's maxillofacial anatomical appearance and function as much as possible, which has important clinical significance.

Claims (3)

1. A pharmaceutical composition for treating cancer, characterized in that it is composed of a DRP1 inhibitor and a ferroptosis inducer, wherein the DRP1 inhibitor is Mdivi-1, which has the structure The ferroptosis inducing agent is Erastin, which has the structure ; The molar ratio of the DRP1 inhibitor to the ferroptosis inducer is 1:1; and the cancer is oral squamous cell carcinoma. 2. Use of the pharmaceutical composition according to claim 1 in the preparation of a medicament for treating oral squamous cell carcinoma. 3. A pharmaceutical preparation for treating cancer, characterized in that it is a preparation prepared by taking the pharmaceutical composition according to claim 1 as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.