CN116585452A - Application of Drp1 protein in preparation of antitumor drugs - Google Patents

Abstract

The invention discloses the application of Drp1 protein in the preparation of anti-tumor drugs, and relates to the technical field of biomedicine. The present invention provides the application of Drp1 protein in the preparation of anti-tumor drugs, the Drp1 protein is: a) the amino acid sequence shown in SEQ ID NO: 1; or b) the amino acid sequence shown in SEQ ID NO: 1 lacks A polypeptide fragment of at least one amino acid; or c) a polypeptide obtained by phosphorylation or acetylation modification of the polypeptide of the amino acid sequence shown in SEQ ID NO:1. The inventors of the present application have verified through experiments that the Drp1 protein has significant anti-tumor activity, especially anti-hepatic ascites tumor activity. The invention not only provides a new source for the preparation of anti-tumor drugs, but also discovers the new medicinal value of the Drp1 protein.

Description

Application of Drp1 protein in the preparation of anti-tumor drugs

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to the application of Drp1 protein in the preparation of antitumor drugs.

Background technique

Malignant tumor, also known as cancer, is a malignant disease caused by abnormal cell differentiation and proliferation and uncontrolled tissue growth leading to death. In addition, malignant tumors are highly infiltrating and metastatic, making conventional clinical treatment difficult , poor prognosis.

With the intensification of human industrialization, aging, and globalization, as well as changes in the ecological environment and the influence of biological and genetic factors, the incidence and mortality of malignant tumors are also increasing year by year, which has become a threat to human health and affects society. important questions of life. In recent years, the incidence of cancer in my country has also increased year by year. According to reports, since the 1970s, the incidence of cancer in my country has increased from 10.13% to 22.32%, and the death rate has increased to 82.11%, ranking first in urban death factors and second in rural areas. Currently, the top ten cancers in my country are: lung cancer, gastric cancer, colorectal cancer, liver cancer, esophageal cancer, breast cancer, pancreatic cancer, lymphoma cancer, bladder cancer and thyroid cancer.

At present, the clinical treatment methods for malignant tumors are mainly surgical removal of lesions, radiotherapy, etc. Conventional treatment brings physical and mental pain to patients and also has great side effects. Therefore, it is extremely urgent to seek new and effective therapeutic drugs, reduce the pain of patients, and treat malignant tumors in a targeted manner.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of Drp1 protein in the preparation of anti-tumor drugs.

In order to achieve the above object, the technical solution adopted by the present invention is: the application of Drp1 protein in the preparation of anti-tumor drugs, and the Drp1 protein is: a) the amino acid sequence shown in SEQ ID NO: 1; or b) SEQ ID NO : a polypeptide fragment lacking at least one amino acid in the amino acid sequence shown in 1; or c) a polypeptide obtained by phosphorylation or acetylation modification of the polypeptide of the amino acid sequence shown in SEQ ID NO: 1.

The inventors of the present application have found through a large number of experiments that the Drp1 protein of the present invention can significantly inhibit the tumor volume of the liver cancer ascites tumor model, therefore, the Drp1 protein of the present invention has good anti-tumor activity.

As a preferred embodiment of the application of the present invention, the tumor is a tumor with RAS mutation.

Ras protein is a key molecule involved in cell signal transduction, regulation of cell proliferation and differentiation. After the Ras gene mutation, the Ras protein is continuously activated, and the signal transduction is disordered, resulting in the continuous proliferation of cells and the occurrence of tumors. The inventors of the present application have found through a large number of experiments that Drp1 protein has a significant inhibitory effect on the expression of Ras protein in tumors.

As a preferred embodiment of the application of the present invention, the tumor is liver cancer.

As a preferred embodiment of the application of the present invention, the tumor is liver cancer ascites tumor.

As a preferred embodiment of the application of the present invention, the drug is in liquid dosage form.

As a preferred embodiment of the application of the present invention, the solvent of the liquid dosage form includes at least one of water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid or aminoethanol.

As a preferred embodiment of the application of the present invention, the drug is administered by subcutaneous injection, intravenous injection, intramuscular injection or nasal administration.

The present invention also provides an anti-tumor pharmaceutical composition, which is characterized in that the pharmaceutical composition includes the Drp1 protein.

As a preferred embodiment of the pharmaceutical composition of the present invention, the Drp1 protein is the only or main active ingredient in the pharmaceutical composition. In order to achieve a better therapeutic effect, the Drp1 protein of the present invention can be used in combination with antitumor drugs commonly used in the prior art.

As a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition is in liquid dosage form.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides the application of the Drp1 protein in the preparation of anti-tumor drugs. The inventors of the present application have verified through experiments that the Drp1 protein has significant anti-tumor activity, especially anti-hepatic ascites tumor activity. The invention not only provides a new source for the preparation of anti-tumor drugs, but also discovers the new medicinal value of the Drp1 protein.

Description of drawings

Figure 1 shows the length and width of the tumor measured with a vernier caliper on the first day and the ninth day after the administration of Drp1 protein in Example 2, and the tumor volume was calculated by the formula; ***: p<0.001.

Fig. 2 is the dissected tumor body at the end of the experiment in Example 2.

Fig. 3 shows that the tumor body was dissected at the end of the experiment of Example 2, the surface body fluid was cleaned, and the weight of the tumor body was weighed by an electronic scale; ***: p<0.001.

Figure 4 shows the protein bands of Drp1 in mouse tumor tissue and contralateral tissue after Drp1 intervention, ****, p<0.0001.

Figure 5 shows that the tumor was dissected at the end of the experiment in Example 2, and the tumor tissue protein was obtained by lysing the tissue, and the expression of Ras protein and β-actin protein was detected by Western blotting; *: p<0.05.

Detailed ways

The above-mentioned content of the present invention will be further described in detail below through specific implementation methods in the form of examples. However, it should not be construed that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials and reagents used are commercially available unless otherwise specified.

Expression of embodiment 1Drp1 protein

1. Construction of monoclonal bacteria

The amino acid sequence of the Drp1 protein of the present invention is shown in SEQ ID NO.1. After being digested with BamHI and XhoI, it was inserted into the pET28a vector to obtain a recombinant plasmid. After the sequencing was correct, the recombinant plasmid was transformed into the host bacterium Escherichia coli to obtain a monoclonal bacterium.

2. Expression of Drp1 protein

Prepare aseptic kanamycin (KA) solid LB medium, spread the monoclonal bacteria obtained in step 1, overnight; pick the monoclonal colony to add KA (50ug/ml) LB liquid medium 10ml, 250rpm37℃ Shake bacteria overnight; transfer 1% overnight bacteria to 1000ml of the above liquid medium, expand the shake bacteria at 37°C and 250rpm for 3h to logarithmic phase, add IPTG (0.5mM) to induce shake bacteria at 20°C and 250rpm for 12h. Centrifuge at 7000rpm for 20min to collect the bacteria, wash once with PBS, resuspend the bacteria solution, sonicate at 60%-70% frequency, 10s ultrasonic interval, about 30min, until the bacteria solution is clear; centrifuge at 12000rpm for 20min, dissolve the precipitate with urea, and go through Ni chromatography The column was eluted with 50-200 mM imidazole; SDS-PAGE (15%) to verify the size of Drp1; 7k dialysis bag PBS dialysate was desalted for 16 hours, BCA was used to measure the protein concentration, freeze-dried overnight, and stored at -80°C.

Example 2 Inhibitory effect of Drp1 protein on liver cancer ascites tumor cell model

1. Establishment of abdominal xenograft tumor model of mouse liver cancer

After the Drp1 protein was freeze-dried, it was formulated with distilled water into solutions of different concentrations for administration. Male C57BL/6 mice, 6-8 weeks old, weighing 20±2g, were subcutaneously inoculated with H22 liver cancer cell line suspension (containing about 1× ¹⁰¹⁰ cells) in the abdominal cavity and then observed continuously, and injected intraperitoneally every day from the eighth day 4 mg/kg was given to Drp1 protein aqueous solution or normal saline, and the tumor volume of each group was detected at the same time.

After intraperitoneal subcutaneous inoculation of H22 liver cancer cells, pay attention to observe the life and mental state of the mice, diet and body weight changes. After successful inoculation, the mice have obvious irregular protrusions in the abdomen, poor mental state, reduced diet, and weight loss or gain. Slower, less active.

A test cycle is seventeen days, and the treatment starts on the eighth day after inoculation of tumor cells. They were divided into two groups: the control group (administered with normal saline), and the treatment group (administered with 4mg/kgDrp1). Inject every other day. On the first day and the ninth day, the length (L) and width (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated. On the seventeenth day, the mice were sacrificed by myelotomy, the tumor body was removed, the surface fat tissue was removed, the tumor body was weighed, and placed on a plate to measure the length (L) and width (W) of the tumor with a vernier caliper to calculate the tumor volume . Tumor volume measurement formula: V=(length×width2)/2.

Western blotting experiment: The obtained tumor tissue was lysed with RIPA lysate at 4° C. for 30 minutes, and centrifuged at 12,000 g to obtain the supernatant (protein fraction) to obtain tumor tissue protein. The concentration of each protein was measured by BCA method, separated by polypropylene gel electrophoresis according to the loading amount of 30 μg, and then the protein was transferred from the gel to the nitrocellulose membrane, sealed with 5% skimmed milk powder at room temperature for 1 hour, and rabbit Ras protein-specific antibody (1:1000, CST, 3965) and rabbit-derived Drp1 protein-specific antibody (1:1000, CST, 8570) were incubated overnight at 4°C, rinsed with TBST washing buffer, and washed 3 times, 5 Every minute, goat anti-rabbit fluorescent secondary antibody (1:10000, Licor, 926-32211) was incubated at room temperature for 1 hour, rinsed with TBST washing buffer, rinsed 3 times, 5 minutes each time, and scanned under the ODYSSEY imager Obtain protein bands. Subsequently, a mouse-derived β-actin specific antibody (1:10000, Affinity, AF7018) was used to recognize the internal reference β-actin protein, rinsed with TBST membrane washing buffer, and rinsed 3 times for 5 minutes each time, and then goat anti-mouse fluorescence II Antibody (1:10000, Licor, 926-68070) was incubated at room temperature for 1 hour, rinsed with TBST washing buffer, rinsed 3 times, 5 minutes each time, and scanned under ODYSSEY imager to obtain protein bands.

The experimental results are shown in Figures 1-5. It can be seen from Figure 1 that there was a significant difference in the tumor size between the control group and the treatment group on the ninth day after administration, and the tumor size of the mice in the Drp1 treatment group was significantly smaller than that in the control group. It can be seen from FIG. 2 that at the end of one test period (ie seventeen days), the tumor volume of the mice in the Drp1 treatment group was significantly smaller than that in the mice in the control group. It can be seen from Figure 3 that after the end of the administration, the tumor was dissected and weighed on the seventeenth day, and it was found that 4 mg/kg Drp1 protein could inhibit the tumor growth in the mouse model, and the Drp1 protein could reduce the tumor weight. It can be seen from Figure 4 that the Drp1 protein bands in the mouse tumor tissue (Drp1) after Drp1 intervention and the mouse tumor tissue (NS) after normal saline injection, compared with the mouse tumor tissue after normal saline injection, after Drp1 intervention The expression of Drp1 in tumor tissue was significantly increased, ***, p<0.001. It can be known from FIG. 5 that 4 mg/kg Drp1 protein has obvious inhibitory effect on the expression of Ras protein in tumor.

Finally, it should be noted that the above-mentioned embodiments are only illustrative to illustrate the principles and effects of the present invention, and are not intended to limit the present invention. Anyone skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by persons with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims (10)

1. The application of the Drp1 protein in preparing an anti-tumor medicament is characterized in that the Drp protein is as follows:

   a) An amino acid sequence shown in SEQ ID NO. 1; or (b)

   b) A polypeptide fragment lacking at least one amino acid in the amino acid sequence shown in SEQ ID NO. 1; or (b)

   c) The polypeptide with the amino acid sequence shown in SEQ ID NO.1 is obtained by phosphorylation or acetylation modification.
2. 2. The use according to claim 1, wherein the tumor is a tumor in which a RAS mutation is present.
3. 3. The use according to claim 2, wherein the tumour is liver cancer.
4. 4. The use according to claim 2, wherein the tumor is liver cancer ascites tumor.
5. 5. The use according to claim 1, wherein the medicament is in a liquid dosage form.
6. 6. The use of claim 5, wherein the solvent of the liquid dosage form comprises at least one of water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, or aminoethanol.
7. 7. The use according to claim 1, wherein the medicament is administered subcutaneously, intravenously, intramuscularly or nasally.
8. 8. An anti-tumor pharmaceutical composition comprising the Drp protein of claim 1.
9. 9. The pharmaceutical composition of claim 8, wherein the Drp protein is the only or major active ingredient in the pharmaceutical composition.
10. 10. The pharmaceutical composition according to claim 8 or 9, wherein the pharmaceutical composition is in a liquid dosage form.