Application of MOTS-c polypeptide and analogue thereof in preparation of medicine for preventing and / or treating tumors or cancers

Abstract

The invention discloses application of MOTS-c polypeptide and analogues thereof in preparation of drugs for preventing and / or treating tumors or cancers, and belongs to the technical field of biomedicine.The MOTS-c polypeptide can remarkably induce apoptosis of tumor cells or cancer cells and effectively delay progression of the tumors or the cancers in vivo, has ideal anti-tumor and anti-cancer effects and can be used for preparing drugs for preventing and / or treating the tumors or the cancers. Therefore, a new thought is provided for prevention and treatment of tumors or cancers, and the MOTS-c polypeptide has great application prospects and market value when being applied to related health care products for preventing the tumors or the cancers and the development process of drugs for treating the tumors or the cancers.

Description

Technical Field

[0001] The invention discloses the use of MOTS-c polypeptide and its analogs in preparing drugs for preventing and / or treating tumors or cancers, belonging to the technical field of biomedicine. Background Art

[0002] Hepatocellular carcinoma (HCC) is mainly caused by hepatitis B and C virus infection and persistent liver damage caused by fungal toxins and alcohol. HCC patients are usually treated with surgery or transplantation, but due to the high metastasis rate, the 5-year survival rate is less than 20%.

[0003] The discovered mitochondrial 12S rRNA type c (MOTS-c) is a mitochondrial-derived peptide that exists in various tissues as a systemic hormone. The expression of MOTS-c can be affected by a variety of factors, including oxidative stress, DNA damage, and metabolic changes in mitochondria. In recent years, MOTS-c has received extensive attention due to its multiple biological functions in the field of tumor biology. Studies have shown that MOTS-c can promote mitochondrial biogenesis, inhibit microglial activation, and reduce the production of inflammatory factors during cancer progression. In addition, studies have also found that exogenous MOTS-c can effectively inhibit the proliferation, migration, and invasion of ovarian cancer cells, while inducing cell cycle arrest and apoptosis. However, there is no clear conclusion on the specific effect of MOTS-c on HCC cell apoptosis. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention provides the use of MOTS-c polypeptide and its analogs in the preparation of drugs for preventing and / or treating tumors or cancers. MOTS-c reduces the resistance of HCC cells to TRAIL-induced apoptosis under hypoxic conditions by upregulating MEF2A expression.

[0005] The technical solution of the present invention to solve the technical problem is as follows:

[0006] A use of a MOTS-c polypeptide and its analogs, the use being for preparing a drug for preventing and / or treating tumors or cancer, wherein the amino acid sequence of the polypeptide is:

[0007] The MOTS-c polypeptide is a composition, which contains the amino acid sequence of the MOTS-c polypeptide MRWQEMGYIFYPRKLR (SEQ ID NO.1); it is abbreviated as MOTS-c.

[0008] Preferably, the dosage form of the drug is an oral dosage form.

[0009] Preferably, the dosage form of the drug is a non-oral dosage form.

[0010] Preferably, the tumor includes at least liver cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, cervical cancer, leukemia and lymphoma, melanoma, glioblastoma, neuroblastoma, sarcoma and gastric cancer.

[0011] Preferably, the MOTS-c polypeptide derivative is a polypeptide derivative or analogue having the same activity as the amino acid sequence of SEQ ID NO.1 after substitution, deletion or addition of one or several amino acid residues.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention can significantly induce apoptosis of tumor cells or cancer cells through MOTS-c polypeptide, and can effectively delay the progression of tumors or cancers in the body, has ideal anti-tumor and anti-cancer effects, and provides new ideas for the prevention and treatment of tumors or cancers. The MOTS-c polypeptide is applied to the development of related health products for preventing tumors or cancers and drugs for treating tumors or cancers, which has great application prospects and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a comparison chart of MOTS-c levels in peripheral blood of healthy control group and HCC patients in Example 2.

[0015] Figure 2 This is a diagram showing that MOTS-c promotes apoptosis of HCC cells under hypoxic conditions in Example 3.

[0016] Figure 3 This is a diagram showing that MOTS-c promotes apoptosis of HCC cells under hypoxic conditions in Example 3.

[0017] Figure 4 This is a diagram showing that MOTS-c promotes apoptosis of HCC cells under hypoxic conditions in Example 3.

[0018] Figure 5 This is a graph showing that Example 3 MOTS-c effectively reversed the low expression of apoptotic protein caspase-8 under hypoxic conditions.

[0019] Figure 6 This is a graph showing that MOTS-c inhibited hypoxia-induced tumor growth in vivo in Example 4.

[0020] Figure 7 This is a graph showing that MOTS-c inhibited hypoxia-induced tumor growth in vivo in Example 4.

[0021] Figure 8 It is the expression of related proteins in the primary tumors of Huh7 and HCCLM3 cells in Example 4. MOTS-c effectively reversed the low expression of apoptotic protein caspase-8 under hypoxic conditions. DETAILED DESCRIPTION

[0022] The following is based on the attached Figure 1-8 The present invention is further described:

[0023] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] In the description of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc. indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, they cannot be understood as limitations on the present invention.

[0025] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0026] The first embodiment of the present invention provides a use of a MOTS-c polypeptide and its analogs based on the prior art, which is used to prepare a drug for preventing and / or treating tumors or cancer. The amino acid sequence of the polypeptide is:

[0027] The MOTS-c polypeptide is a composition, which contains the amino acid sequence of the MOTS-c polypeptide MRWQEMGYIFYPRKLR (SEQ ID NO.1); it is abbreviated as MOTS-c.

[0028] In one embodiment of the present application, the dosage form of the drug is an oral dosage form.

[0029] In one embodiment of the present application, the dosage form of the drug is a non-oral dosage form.

[0030] In one embodiment of the present application, the tumor includes at least liver cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, cervical cancer, leukemia and lymphoma, melanoma, glioblastoma, neuroblastoma, sarcoma and gastric cancer.

[0031] In one embodiment of the present application, the MOTS-c polypeptide derivative is a polypeptide derivative or analogue having the same activity as the amino acid sequence of SEQ ID NO.1 after substitution, deletion or addition of one or several amino acid residues.

[0032] The second embodiment of the present invention is a comparative analysis of the levels of MOTS-c in the peripheral blood of a healthy control group and HCC patients. Figure 1 shown.

[0033] Experimental methods:

[0034] First, the levels of MOTS-c in the peripheral blood of healthy controls and HCC patients were compared. Enzyme-linked immunosorbent assay (ELISA) was used according to the Human MOTS-c ELISA kit (EH5056, Fine The MOTS-c levels in the peripheral blood of healthy individuals and HCC patients were analyzed according to the instructions of the company (Wuhan, China). The samples were detected using an enzyme immunoassay analyzer (Thermo Fisher), and the results were expressed as the optical density at 450 nm.

[0035] Experimental results: The results showed that the peripheral blood MOTS-c level of HCC patients was significantly lower than that of the healthy control group. Figure 1 shown.

[0036] The third embodiment of the present invention is that MOTS-c promotes apoptosis of HCC cells under hypoxic conditions, such as Figure 2-5 shown.

[0037] Experimental methods: HCC cell lines Huh7 and HCCLM3 were provided by EK-Bioscience, Shanghai, China. Huh7, HepG2, and HCCLM3 cells were cultured in Dulbecco's modified Eagle's medium provided by Biosun (Shanghai). All cells were cultured in medium containing 10% fetal bovine serum (Biosun) and 1% penicillin / streptomycin (Cytiva, Shanghai) at 37°C and 5% CO2. The medium was changed every 1 to 2 days. To simulate the hypoxic environment, cells were exposed to a gas environment containing 94% nitrogen, 5% carbon dioxide, and 1% oxygen for a certain time point (4, 8, 12, 16, and 20 hours), and the control group was cells cultured under normal conditions containing 95% air and 5% carbon dioxide. In addition, Huh7 and HCCLM3 cells were treated with different concentrations of MOTS-c (China Peptide Co., Ltd., Shanghai) for 24 hours (the control group was treated with normal saline) and then subjected to hypoxia.

[0038] The effect of MOTS-c on HCC cell apoptosis under hypoxia was analyzed. Huh7 and HCCLM3 cells were exposed to hypoxia for 4, 8, 12, 16, and 20 h, respectively. Under hypoxia, Huh7 and HCCLM3 cells were treated with MOTS-c (0, 2.5, 5, 10, 20, and 40 μM), respectively, for 12 h. Cell viability was analyzed by MTT assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Solerbo, Beijing, China) was added to each well of Huh7 and HCCLM3 cells seeded in 96-well plates and incubated for 4 h. Then, the cells were incubated with 110 μL of color development solution for 10 min. Finally, the optical density at 490 nm was measured using an enzyme-linked immunosorbent assay (Thermo Fisher). Flow cytometry analysis. Cell apoptosis was detected using a flow cytometer (Thermo Fisher Scientific). Huh7 and HCCLM3 cells were washed with cold phosphate buffer and then suspended in binding buffer. According to the instructions of the Solarbio commercial kit (#CA1020), cells were incubated with Annexin V-FITC and propidium iodide in binding buffer for 15 minutes. Tunel staining was used to detect cell apoptosis. The culture medium of Huh7 and HCCLM3 cells was removed, and Tunel working solution was added to the culture wells, incubated for 0.5 hours, and then fixed for Tunel staining according to the specific protocol of TUNEL apoptosis detection kit (#T2130, Solarbio). Finally, the cells were analyzed for fluorescence intensity using a fluorescence microscope (Thermo Fisher Scientific) under the conditions of excitation wavelength / emission wavelength of 490 / 525 nm. For the detection of apoptotic protein caspase-8 by western blotting, total protein lysates were obtained from Huh7 and HCCLM3 cell samples and tumor tissues using RIPA buffer (Thermo Fisher Scientific). 20 μg of lysate was electrophoresed on polyacrylamide gel (Thermo Fisher Scientific) and then transferred to PVDF membrane. Subsequently, the membranes were incubated with specific antibodies against caspase-8 (Thermo Fisher Scientific, #MA1-41280) and β-actin (Thermo Fisher Scientific, #PA1-16889). After incubation with secondary antibodies (Thermo Fisher Scientific), protein bands were visualized using the EasyBlot ECL kit from Songon Biotech.

[0039] Experimental results: Huh7 and HCCLM3 cells were exposed to hypoxia for 4, 8, 12, 16 and 20 h, respectively. The results showed that the survival ability of HCC cells was the highest after 12 h of hypoxia treatment compared with other time points. Figure 2As shown. Under hypoxic conditions, Huh7 and HCCLM3 cells were treated with MOTS-c (0, 2.5, 5, 10, 20 and 40 μM) for 12 h. After treatment with 20 μM MOTS-c, the survival rate of Huh7 and HCCLM3 cells was 50%, which was significantly higher than that of the untreated group, as shown in Figure 3 As shown. On this basis, Huh7 and HCCLM3 cells were treated with 20μM MOTS-c under hypoxic conditions for 12h for further analysis. After treatment with 20μM MOTS-c, the survival rate of Huh7 and HCCLM3 cells was 50%, which was significantly higher than that of the untreated group. Figure 4 As shown. On this basis, Huh7 and HCCLM3 cells were treated with 20 μM MOTS-c under hypoxia for 12 h for further analysis. Figure 2 As shown in Figure 2, under hypoxic conditions, MOTS-c significantly promoted the apoptosis of hepatocellular carcinoma cells (HCC). In addition, hypoxia reduced the expression level of the apoptotic protein caspase-8, while MOTS-c effectively reversed these effects, as shown in Figure 2. Figure 5 shown.

[0040] Fourth embodiment of the present invention Experimental method: In order to further explore the effect of MOTS-c on tumor growth, a tumor-bearing nude mouse model was further established, and Huh-7 and HCCLM3 cells were used to study the effect of MOTS-c on tumor growth. 30 male BALB / c nude mice aged 5-6 weeks were purchased from Hunan Shengyi Jingda Experimental Animal Co., Ltd. (Changsha, China). The nude mice were placed in a sterile environment and were free to eat and drink. Huh7 and HCCLM3 cells (5×106) were made into a cell suspension with phosphate buffered saline (PBS) and injected into the axilla of nude mice by subcutaneous injection. The mice injected with HCC cells were divided into three groups: a normoxic group (maintained normal conditions and treated with PBS before cell transplantation, n=5), a MOTS-c group (maintained hypoxic conditions before cell transplantation, and then 5 mg / kg MOTS-c was injected into the mice by intraperitoneal injection every day for 20 days, n=5) and a control group (maintained hypoxic conditions before cell transplantation, n=5). Next, PBS was injected intraperitoneally every day (n=5), and the control group (hypoxic conditions were maintained before cell transplantation). Tumor volume was measured every two days after injection. Mice were killed by cervical dislocation after 20 days, and the formed nodules were collected for further analysis. Immunohistochemistry (IHC) analysis was then performed. The primary tumors of HCC cells were embedded in paraffin and cut into 4-micron thick sections. The slides were heated in a microwave oven and immunohistochemically stained using p-AMPK (#PA5-17831, Thermo Fisher), MEF2A (#PA5-27380, Thermo Fisher), DR4 (#PA1-30795, Thermo Fisher) and DR5 (#PA5-19895, Thermo Fisher) antibodies. The slides were counterstained with hematoxylin (Solarbio) and images were captured with an optical microscope (ThermoFisher).

[0041] Experimental results: Figure 6 and 7 As shown in the figure, hypoxia treatment significantly increased tumor volume and weight, while MOTS-c treatment effectively offset the effects of hypoxia treatment. In addition, the expression of related proteins in primary tumors of Huh7 and HCCLM3 cells was analyzed. The results showed that hypoxia treatment inhibited the protein expression of apoptotic protein caspase-8, while MOTS-c treatment reversed this trend, as shown in the figure. Figure 8 shown.

[0042] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the illustrations shown and described here.

Claims

1. A use of a MOTS-c polypeptide and its analogs, characterized in that: The use is to prepare drugs for preventing and / or treating tumors or cancers. The amino acid sequence of the polypeptide is: The MOTS-c polypeptide is a composition, which contains the amino acid sequence of the MOTS-c polypeptide MRWQEMGYIFYPRKLR (SEQ ID NO.1); it is abbreviated as MOTS-c.

2. The use according to claim 1, characterized in that The dosage form of the drug is an oral dosage form.

3. The use according to claim 1, characterized in that The dosage form of the drug is a non-oral dosage form.

4. The use according to any one of claims 1 to 3, characterized in that The tumors include at least liver cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, cervical cancer, leukemia and lymphoma, melanoma, glioblastoma, neuroblastoma, sarcoma and gastric cancer.

5. The use according to claim 4, characterized in that The MOTS-c polypeptide derivative is a polypeptide derivative or analogue that has undergone substitution, deletion or addition of one or several amino acid residues and has the same activity as the amino acid sequence of SEQ ID NO.1.

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