A polypeptide with anti-tumor function, its application and pharmaceutical composition

Abstract

The present invention relates to the field of biomedicine and, more specifically, to a polypeptide with anti-tumor properties. The polypeptide sequence is shown in SEQ ID NO. 1: PHQYWLTLQYLLKHFFKLSQTSSKNL. The present invention has the beneficial effect of discovering and confirming for the first time that the anti-glioma function of human p85α protein is dependent on a polypeptide located in the amino acid sequence 232-257, suggesting the potential for development into an anti-glioma drug.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polypeptide with anti-tumor function, its application and pharmaceutical composition. Background Art

[0002] Glioma is a tumor that develops in the brain or spinal cord and is the most common primary intracranial tumor. Due to its rapid growth and invasiveness, most gliomas are difficult to completely remove, resulting in high disability and recurrence rates. Currently, combined treatments such as surgery, radiotherapy, and chemotherapy are the main standard methods for treating gliomas, but traditional chemotherapy cannot kill tumor cells without affecting normal cells. Peptide drugs have the characteristics of small molecular weight, weak toxicity, strong targeting, and easy synthesis, and have received widespread attention in anti-tumor research.

[0003] The PI3K / AKT signaling pathway is involved in regulating many important biological processes, and its overactivation can lead to tumorigenesis. p85α is a key regulatory subunit of type IA PI3K. Reduced p85α expression or p85α mutations often lead to abnormal tumor proliferation. p85α is lowly expressed in glioma tissue and is associated with poor patient prognosis. p85α can inhibit glioma cell proliferation, colony formation, and cell migration. U.S. Patent No. 6,518,021B1 discloses the full-length amino acid sequence of the p85α protein described herein. Summary of the Invention

[0004] The purpose of the present invention is to provide a polypeptide with anti-tumor function. Experiments of the present invention show that the polypeptide has good anti-glioma activity and good safety.

[0005] The first objective of the present invention is to provide a polypeptide with anti-tumor activity, the polypeptide sequence of which is shown in SEQ ID NO. 1. SEQ ID NO. 1: PHQYWLTLQYLLKHFFKLSQTSSKNL. The anti-glioma activity of p85α is dependent on a peptide segment (PHQYWLTLQYLLKHFFKLSQTSSKNL) located within the amino acid sequence 232-257 of the protein. This peptide segment is referred to herein as polypeptide P859.

[0006] Preferably, the polypeptide amino acid sequence may undergo substitution or deletion of one or more amino acid residues; substitution or deletion of certain sites does not affect / substantially does not affect its anti-tumor function. The term "multiple amino acid residues" refers to two, three, four, or five amino acid residues.

[0007] Furthermore, the C-terminus and / or N-terminus of the polypeptide having anti-tumor function is connected to a cell-penetrating peptide (CPPs), which can be selected from various commonly used cell-penetrating peptides.

[0008] The second purpose of the present invention is to use the polypeptide P859 in the preparation of anti-tumor drugs.

[0009] The third purpose of the present invention is to use the above-mentioned polypeptide P859 in the preparation of anti-glioma drugs.

[0010] Preferably, the anti-tumor drug is an anti-glioma drug.

[0011] The dosage form of the drug includes oral preparations and injections, and can be prepared into tablets, capsules or injections by conventional methods; in particular, injections.

[0012] The fourth object of the present invention is to provide a pharmaceutical composition comprising a therapeutic amount of the above polypeptide and other pharmaceutically acceptable excipients.

[0013] Furthermore, the above-mentioned pharmaceutical composition comprises a therapeutic amount of the above-mentioned polypeptide, a therapeutic amount of a commercially available anti-tumor drug, and other pharmaceutically acceptable excipients.

[0014] The beneficial effect of the present invention is that it is the first to discover and confirm that the anti-glioma function of human p85α protein depends on a polypeptide in the 232-257 part of its protein amino acid sequence, and the polypeptide has the potential to be developed into an anti-glioma drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a high performance liquid chromatogram of the polypeptide P859 of the present invention;

[0016] Figure 2 is a mass spectrometry data diagram of the polypeptide P859 of the present invention;

[0017] Figure 3a This is a data graph showing that the polypeptide TAT-P859 of an embodiment of the present invention inhibits the proliferation of glioma LN229 cells;

[0018] Figure 3b This is a data graph showing that the polypeptide TAT-P859 of an embodiment of the present invention inhibits the proliferation of glioma LN18 cells;

[0019] Figure 4a This is a data graph showing that the polypeptide TAT-P859 of the embodiment of the present invention promotes apoptosis of glioma LN229 cells;

[0020] Figure 4b This is a data graph showing that the polypeptide TAT-P859 of the embodiment of the present invention promotes apoptosis of glioma LN18 cells;

[0021] Figure 5 This is a data graph showing the inhibitory effect of the polypeptide TAT-P859 of the present invention on the growth of intracranial glioma in nude mice. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below with reference to the examples, but is not limited thereto.

[0023] Example 1: Cell-penetrating peptide binding polypeptide P859

[0024] The polypeptide P859 involved in the following examples has been linked to a cell-penetrating peptide at the N-terminus or C-terminus to facilitate the entry of the polypeptide into cells and to study its function. The cell-penetrating peptide used in this example is TAT ​​(GGRKKRRQRRR).

[0025] Figure 1 The HPLC conditions for peptide P859 are as follows:

[0026] Solvent A: Acetonitrile containing 0.1% trifluoroacetic acid

[0027] Solvent B: 0.1% trifluoroacetic acid aqueous solution

[0028]

[0029] Flow rate: 1.0ml / min

[0030] Detection wavelength: 220nm

[0031] Figure 2 The mass spectrometry detection conditions for peptide P859 are:

[0032] Probe bias: +4.5kV

[0033] Nebulizer air flow: 1.5L / min

[0034] Mobile phase flow rate: 0.2 ml / min

[0035] Mobile phase ratio: 50% water / 50% acetonitrile;

[0036] Example 2: CCK-8 cell activity assay

[0037] (1) Glioma LN229 and LN18 cells were cultured to the logarithmic growth phase, digested with trypsin, seeded into 96-well plates, and cultured in DMEM medium containing 10% calf serum for 24 hours;

[0038] (2) The peptide TAT-P859 was diluted to the following concentrations: 1, 2, 5, 10, 20, and 30 μM using serum-free DMEM medium and added to glioma LN229 and LN18 cells. The PBS group served as the control group and the cells were cultured for 24 hours.

[0039] (3) Add 20 μl of CCK-8 solution and continue incubating in a 37°C, 5% CO2 incubator for 1 hour.

[0040] (4) The absorbance of glioma cells LN229 and LN18 at 450 nm was measured using an enzyme-labeled instrument. Each group of experiments was the average of three experiments.

[0041] Example 3: Cell apoptosis experiment

[0042] (1) Treat cells with peptide TAT-P859 for 24 hours, remove the supernatant, wash twice with PBS, collect cells, centrifuge at 1000 g for 5 minutes, discard the supernatant, collect cells, gently resuspend the cells in PBS and count;

[0043] (2) Take 50,000 to 100,000 resuspended cells, centrifuge at 1000 g for 5 minutes, discard the supernatant, and add 195 μl Annexin V-FITC conjugate solution to gently resuspend the cells;

[0044] (3) Add 5 μl of Annexin V-FITC and mix gently;

[0045] (4) Add 10 μl of propidium iodide staining solution and mix gently;

[0046] (5) Incubate at room temperature in the dark for 10 minutes, then place in an ice bath. Use aluminum foil to protect from light during incubation. Resuspend the cells 2-3 times to improve staining.

[0047] (6) Detection using flow cytometry.

[0048] Example 4: Nude mouse intracranial tumor growth experiment

[0049] (1) Glioma cells LN229 and LN18 in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged, and the supernatant was removed. The cells were washed twice with PBS, and an appropriate amount of PBS was added to prepare a cell suspension with a certain cell concentration for inoculation.

[0050] (2) Nude mice were anesthetized by intraperitoneal injection of 0.8% sodium pentobarbital, and their heads were fixed on a stereotaxic apparatus in a prone position. The inoculation site was the right caudate nucleus of the brain of the nude mice.

[0051] (3) The nude mice's mental state, diet, activity, and body weight were observed daily. After 3 weeks, the size of the intracranial tumors in the nude mice was monitored using an animal in vivo imaging device. The mice were observed every 7 days and the fluorescence values ​​were recorded.

[0052] (4) 1 mg of peptide P859 was dissolved in 1 ml of normal saline and then diluted to the desired concentration. Three weeks after the tumor cells were intracranially inoculated into nude mice, peptide P859 was injected intracranially once every 7 days.

[0053] (5) After 8 weeks, the nude mice were killed and their brain tissues were collected.

[0054] All documents mentioned herein are incorporated by reference into this application to the same extent as if each document were individually incorporated by reference.

Claims

1. A polypeptide with anti-glioma function, characterized in that The polypeptide sequence is shown in SEQ ID NO.

1.

2. A polypeptide with anti-glioma function, characterized in that A cell-penetrating peptide is linked to the C-terminus and / or N-terminus of the polypeptide according to claim 1.

3. Use of the polypeptide according to claim 1 or 2 in the preparation of anti-glioma drugs.

4. A pharmaceutical composition, characterized in that The invention comprises a therapeutic amount of the polypeptide according to claim 1 or 2 and other pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition comprises a therapeutic amount of the polypeptide according to claim 1 or 2, a therapeutic amount of a commercially available anti-tumor drug, and other pharmaceutically acceptable excipients.

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