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

Abstract

The application discloses an application of Drp protein in preparing an anti-tumor medicament, and relates to the technical field of biological medicines. The application provides an application of Drp protein in preparing an anti-tumor medicament, wherein the Drp protein is as follows: a) An amino acid sequence shown in SEQ ID NO. 1; or b) a polypeptide fragment lacking at least one amino acid in the amino acid sequence shown in SEQ ID NO. 1; or c) polypeptide obtained by phosphorylation or acetylation modification of the polypeptide with the amino acid sequence shown in SEQ ID NO. 1. Experiments prove that the Drp1 protein has remarkable anti-tumor activity, in particular to anti-liver cancer ascites tumor activity. The application not only provides a new source for preparing anti-tumor medicines, but also discovers a new medicinal value of Drp protein.

Description

Application of Drp1 protein in preparation of antitumor drugs

Technical Field

The application belongs to the technical field of biological medicines, and particularly relates to application of Drp protein in preparation of antitumor drugs.

Background

Malignant tumor, which is commonly known as cancer, is a malignant disease that causes death of human body due to abnormal cell differentiation and proliferation and loss of control of tissue growth, and has strong infiltration and metastasis, which results in great difficulty in conventional clinical treatment and poor prognosis.

With the progress of human industrialization, aging, globalization, and changes in ecological environment, biological and genetic factors, the incidence and mortality of malignant tumors have been increasing year by year, which has become an important problem threatening human health and affecting social life. In recent years, the incidence of cancer in China is also increasing year by year. It has been reported that from the 70 s of the 20 th century, the incidence of cancer in China increased from 10.13% to 22.32%, and the increase rate of death increased to 82.11%, which was ranked first in urban death factor and second in rural areas. The ten previous cancers in China are: lung cancer, stomach cancer, colorectal cancer, liver cancer, esophageal cancer, breast cancer, pancreatic cancer, lymphatic cancer, bladder cancer and thyroid cancer.

At present, the treatment means aiming at malignant tumors in clinic mainly comprise operation elimination focus, radiotherapy and the like, and the conventional treatment brings great side effects to patients while bringing physical and psychological pains. Therefore, new effective therapeutic drugs are sought, the pain of patients is reduced, and targeted treatment of malignant tumors is becoming particularly urgent.

Disclosure of Invention

The application aims to overcome the defects of the prior art and provides application of Drp1 protein in preparing antitumor drugs.

In order to achieve the above purpose, the technical scheme adopted by the application is as follows: an application of Drp protein in preparing an anti-tumor medicament, wherein the Drp protein is as follows: a) An amino acid sequence shown in SEQ ID NO. 1; or b) a polypeptide fragment lacking at least one amino acid in the amino acid sequence shown in SEQ ID NO. 1; or c) polypeptide obtained by phosphorylation or acetylation modification of the polypeptide with the amino acid sequence shown in SEQ ID NO. 1.

Through a great number of experiments, the inventor discovers that the Drp1 protein can obviously inhibit the volume of tumors of a liver cancer ascites tumor model, so that the Drp protein has good anti-tumor activity.

As a preferred embodiment of the use according to the application, the tumor is a tumor in which a RAS mutation is present.

Ras proteins are key molecules involved in cell signaling, regulating cell proliferation and differentiation. After mutation of the Ras gene, the Ras protein is continuously in an activated state, and signal transduction is disturbed, resulting in continuous proliferation of cells and tumor development. The present inventors have found through a great deal of experiments that Drp1 protein has an obvious inhibition effect on the expression of Ras protein in tumors.

As a preferred embodiment of the use according to the application, the tumor is liver cancer.

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

As a preferred embodiment of the use according to the application, the medicament is in a liquid dosage form.

As a preferred embodiment of the use according to the application, the solvent of the liquid dosage form comprises at least one of water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid or amino ethanol.

As a preferred embodiment of the use according to the application, the medicament is administered subcutaneously, intravenously, intramuscularly or nasally.

The application also provides an anti-tumor pharmaceutical composition, which is characterized by comprising the Drp protein.

As a preferred embodiment of the pharmaceutical composition of the present application, the Drp protein is the only or major active ingredient in the pharmaceutical composition. In order to achieve better therapeutic effects, the Drp1 protein of the application can be combined with antitumor drugs commonly used in the prior art.

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

Compared with the prior art, the application has the following beneficial effects:

the application provides application of Drp protein in preparing antitumor drugs, and the inventor verifies that Drp protein has remarkable antitumor activity, in particular to liver cancer ascites tumor resisting activity through experiments. The application not only provides a new source for preparing anti-tumor medicines, but also discovers a new medicinal value of Drp protein.

Drawings

FIG. 1 shows the tumor volume calculated by measuring the length and width of the tumor with a vernier caliper on the first and ninth days after administration of Drp1 protein in example 2, respectively; * **: p <0.001.

FIG. 2 shows a tumor body dissected at the end of the experiment in example 2.

FIG. 3 shows the dissection of the tumor body at the end of the experiment in example 2, the cleaning of the surface body fluid, and the weighing of the tumor body by the electronic scale; * **: p <0.001.

Fig. 4 shows protein bands of tumor tissue and contralateral tissue Drp1 of mice with a dry prognosis of Drp, p < 0.0001.

FIG. 5 shows the dissection of tumor mass at the end of the experiment in example 2, tumor mass tissue protein was obtained by tissue lysis, and Ras protein and beta-actin expression were detected by western blot experiment; * : p <0.05.

Detailed Description

The above-described aspects of the present application will be described in further detail by way of specific embodiments of the present application. It should not be construed that the scope of the above subject matter of the present application is limited to the following examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, are commercially available.

Example 1 expression of Drp protein

1. Construction of monoclonal bacteria

The amino acid sequence of the Drp protein is shown as SEQ ID NO. 1. After being digested by BamHI and XhoI, the recombinant plasmid is inserted into a pET28a vector to obtain a recombinant plasmid, and after the sequence is correct, the recombinant plasmid is transformed into host bacterium escherichia coli to obtain monoclonal bacteria.

2. Expression of Drp1 protein

Preparing a sterile Kanamycin (KA) solid LB medium, and coating the monoclonal bacteria obtained in the step 1 overnight; selecting monoclonal colonies, adding 10ml of LB liquid medium added with KA (50 ug/ml), shaking overnight at 250rpm37 ℃; 1% overnight bacteria were transferred to 1000ml of the above liquid medium, shaking was expanded at 37℃and 250rpm for 3 hours to logarithmic phase, and IPTG (0.5 mM) was added to induce shaking at 20℃and 250rpm for 12 hours. Centrifuging at 7000rpm for 20min to collect thalli, washing with PBS for one time, re-suspending the bacteria breaking liquid, ultrasonically crushing at 60% -70% frequency, and ultrasonically spacing for 10s for about 30min until the bacteria liquid is clarified; centrifuging at 12000rpm for 20min, dissolving urea, precipitating, and eluting with 50-200mM imidazole via Ni chromatographic column; SDS-PAGE (15%), verifying the size of Drp 1; 7k dialysis bag PBS dialysate for 16h desalting, BCA protein concentration measurement, freeze drying overnight, and storage at-80deg.C.

Example 2 inhibition of the ascites tumor cell model by the Drp protein

1. Establishment of mouse liver cancer abdomen transplantation tumor model

The Drp protein is freeze-dried and then is prepared into solutions with different concentrations with distilled water for administration. Male C57BL/6 mice, 6-8 weeks old, weight 20+ -2 g, were inoculated subcutaneously with H22 hepatoma cell line suspension (containing about 1×10 cells) ¹⁰ Individual) and was continuously observed, and each group of tumor volumes was simultaneously examined by intraperitoneal injection of a Drp1 protein aqueous solution or physiological saline at a dose of 4mg/kg per day from the eighth day, respectively.

After the H22 liver cancer cells are inoculated subcutaneously in the abdominal cavity, the living and mental states, diet conditions and weight changes of the mice are observed, and obvious irregular protrusions appear on the abdomen of the mice after successful inoculation, so that the mental states are poor, the diet is reduced, the weight is reduced or increased slowly, and the activity is reduced.

One trial period was ten and seven days, with treatment beginning on the eighth day after tumor cell inoculation. A total of two groups: control group (physiological saline administration), treatment group (4 mg/kg Drp1 administration). The injection is administered once every other day. On the first and ninth days, the length (L) and width (W) of the tumor were measured with a vernier caliper and the tumor volume was calculated. At the tenth seven days, mice were sacrificed at a pulp-breaking, tumor bodies were removed, surface adipose tissues were removed, the tumor bodies were weighed, and the length (L) and width (W) of the tumor were measured with a vernier caliper on a plate, and the tumor volume was calculated. Tumor volume measurement formula: v= (length×width 2)/2.

Western blotting experiments: the obtained tumor tissue was lysed with RIPA lysate at 4℃for 30 minutes, and the supernatant (protein fraction) was obtained by centrifugation at 12000g to obtain tumor tissue protein. The protein concentration was measured by BCA method, polypropylene gel electrophoresis was performed according to a loading amount of 30. Mu.g, then the protein was transferred from the gel onto nitrocellulose membrane, rabbit source Ras protein specific antibody (1:1000, CST, 3965) and rabbit source Drp protein specific antibody (1:1000, CST, 8570) were incubated overnight at 4℃respectively with 5% nonfat dry milk for 1 hour at room temperature, washed with TBST membrane wash buffer for 3 times each for 5 minutes, incubated with goat anti-rabbit fluorescent secondary antibody (1:10000, licor, 926-32211) for 1 hour at room temperature, washed with TBST membrane wash buffer for 3 times each for 5 minutes, and scanned under an ODYSSEY imager to obtain protein bands. Then, the murine beta-actin specific antibody (1:10000, affinity, AF7018) was used for the identification of the reference beta-actin protein, the washing with TBST washing buffer was performed 3 times for 5 minutes each, the goat anti-mouse fluorescent secondary antibody (1:10000, licor, 926-68070) was incubated at room temperature for 1 hour, the washing with TBST washing buffer was performed 3 times for 5 minutes each, and the protein bands were obtained by scanning under an ODYSSEY imager.

The experimental results are shown in FIGS. 1 to 5. As can be seen from fig. 1, the tumor sizes of the control group and the treated group were significantly different on the ninth day after administration, and the tumor sizes of the mice in the Drp treatment group were significantly reduced as compared with the control group. As can be seen from fig. 2, at the end of one test period (i.e., seventeen days), the tumor volume of the mice in the Drp1 treatment group was significantly reduced as compared with the mice in the control group. As can be seen from fig. 3, after the end of the administration, tumor weights were weighed after dissection on the seventeenth day, and it was found that 4mg/kgDrp1 protein could inhibit tumor growth in the mouse model, and Drp1 protein could reduce tumor weights. As can be seen from fig. 4, the protein Drp1 band of the mouse tumor tissue (Drp 1) after Drp1 intervention and the mouse tumor tissue (NS) after physiological saline injection significantly increased Drp1 expression in the tumor tissue after Drp1 intervention compared to the mouse tumor tissue after physiological saline injection, p <0.001. As can be seen from FIG. 5, 4mg/kg Drp1 protein has a significant inhibitory effect on the expression of Ras protein in tumors.

Finally, it should be noted that the above-mentioned embodiments are merely illustrative of the principles of the present application and its efficacy, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the application. Therefore, it is intended that all equivalent modifications and changes which a person having ordinary skill in the art can accomplish without departing from the spirit and technical spirit of the present application shall be covered by the claims of the present application.

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.