Polypeptide of protein Procaspase-8 DED2-alpha2 for inhibiting tumor proliferation and application of polypeptide

Abstract

The invention discloses a protein Procaspase-8DED2-alpha2 polypeptide for inhibiting cancer cells and application thereof.The 10 polypeptides in Procaspase-8DED2 split protein are screened through a molecular docking technology, the polypeptide with the lowest binding energy with c-FLIP DED1 is obtained, the screened polypeptide acts on a human-derived cancer cell line, and it is obtained that the polypeptide has the good inhibiting effect on tumor proliferation and can be used for inhibiting cancer cells. The polypeptide is expected to become an anti-tumor micromolecule polypeptide and is applied to clinical tumor treatment, and a new method is provided for tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polypeptide of protein Procaspase-8 DED2-α2 that inhibits tumor proliferation and an application thereof. Background Art

[0002] Cancer is the biggest disease threatening human health and life. At present, tumor cells can be eliminated through apoptosis, necrosis, and pyroptosis, but necrosis will release the contents of the cell and cause related inflammatory reactions. Therefore, the most ideal means of treating cancer so far is to induce cells to produce strictly ordered programmed cell death, i.e., apoptosis, through signal transduction. Apoptosis is an evolutionarily conserved cell stress response that involves blistering of the cell membrane, exposing phosphatidylserine, and activating the cysteine-aspartic acid protease (caspase) family proteins. Therefore, traditional chemotherapy drugs that induce apoptosis are widely used in the treatment of cancer. A deep understanding of the signal transduction mechanism of the apoptosis pathway will help develop new and highly effective anti-tumor drugs. At present, apoptosis can be divided into two categories based on the source of cell death signals: extrinsic pathway and intrinsic pathway.

[0003] The extrinsic apoptosis pathway involves interactions mediated by death receptors. Death receptors are a group of cell surface markers discovered in recent years and belong to the tumor necrosis factor receptor superfamily. After binding with the corresponding ligands, they can transmit apoptosis signals to the interior of the cell through a series of signal transduction processes; the intrinsic pathway, also known as the mitochondrial apoptosis pathway, mainly executes apoptosis through the mitochondrial pathway when Procaspase-8 is not fully activated; apoptosis is induced by the cleavage of Bid proteins that only contain the BH3 homology region in the Bcl-2 family.

[0004] Based on this, this paper proposes a peptide of protein Procaspase-8 DED2-α2 that inhibits tumor proliferation and its application. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide of protein Procaspase-8 DED2-α2 for inhibiting tumor proliferation and its application.

[0006] In order to achieve the above technical effects, the present invention is implemented by the following technical scheme: A polypeptide for inhibiting tumor proliferation by Procaspase-8DED2-α2, characterized in that the amino acid sequence of the polypeptide is as shown in SEQ ID NO.9:

[0007] SEQ ID NO.9: KFLL

[0008] Furthermore, the Procaspase-8 DED2-α2 split protein polypeptide is used in the preparation of drugs for inhibiting tumor proliferation.

[0009] The beneficial effects of the present invention are:

[0010] The polypeptide of the present invention can be obtained by screening 10 polypeptides in the Procaspase-8 DED2 split protein using molecular docking technology to obtain a polypeptide with the lowest binding energy with c-FLIP DED1, and the screened polypeptide is applied to a human cancer cell line to obtain non-small cell lung cancer cells sensitive to the polypeptide, and it is concluded that the polypeptide has a good inhibitory effect on tumor proliferation. The polypeptide is expected to become an anti-tumor small molecule polypeptide and be applied in clinical tumor treatment, providing a new method for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0012] Figure 1 It is a schematic diagram of cell viability using TRAIL alone and TRAIL in combination with Procaspase-8 DED2-α2 split protein polypeptide 9 of the present invention. DETAILED DESCRIPTION

[0013] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0014] Example 1

[0015] Design and screening of Procaspase-8 DED2 split protein peptides:

[0016] Existing studies have shown that c-FLIP protein is highly expressed in cancer cells. Therefore, based on the existing technology, the present invention studies and analyzes Procaspase-8 DED2 protein, designs polypeptides of Procaspase-8 DED2 split protein, and screens polypeptides that can specifically antagonize c-FLIP and significantly inhibit cancer cells, providing a reference for the treatment of cancer.

[0017] It is known that the Procaspase-8 DED2-α2 split protein polypeptide comprises 10 polypeptides, and the 10 polypeptides are respectively Procaspase-8 DED2-α2 split protein polypeptide 1, Procaspase-8 DED2-α2 split protein polypeptide 2, Procaspase-8 DED2-α2 split protein polypeptide 3, Procaspase-8 DED2-α2 split protein polypeptide 4, Procaspase-8 DED2-α2 split protein polypeptide 5, Procaspase-8 DED2-α2 split protein polypeptide 6, Procaspase-8 DED2-α2 split protein polypeptide 7, Procaspase-8 DED2-α2 split protein polypeptide 8, and Procaspase-8 DED2-α2 split protein polypeptide 9;

[0018] The amino acid sequences of the Procaspase-8 DED2-α2 split protein polypeptide 1, Procaspase-8 DED2-α2 split protein polypeptide 2, Procaspase-8 DED2-α2 split protein polypeptide 3, Procaspase-8 DED2-α2 split protein polypeptide 4, Procaspase-8 DED2-α2 split protein polypeptide 5, Procaspase-8 DED2-α2 split protein polypeptide 6, Procaspase-8 DED2-α2 split protein polypeptide 7, Procaspase-8 DED2-α2 split protein polypeptide 8, and Procaspase-8 DED2-α2 split protein polypeptide 9 are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively.

[0019] The amino acid sequence of the Procaspase-8 DED2-α2 helix is ​​RSELRSFKFLLQEEIS. According to the analysis of the conserved amino acids of Procaspase-8 DED2-α2, it is known from relevant research reports that c-FLIP DED1 can bind to the α2 helix in the Procaspase-8 DED2 domain and split Procaspase-8 DED2α2. The specific sequence is shown in Table 1.

[0020]

[0021]

[0022] Table 1 Procaspase-8 DED2-α2 protein peptide sequence

[0023] Then, molecular docking technology was used to screen the peptide with the lowest binding energy with c-FLIP DED1;

[0024] After screening, it was found that the Procaspase-8DED2-α2 split protein polypeptide with the lowest binding energy to c-FLIP DED1 is Procaspase-8 DED2-α2 split protein polypeptide 9, the amino acid sequence of which is shown in SEQ ID NO.9, and has the potential to antagonize c-FLIP.

[0025] Example 2

[0026] Screening of anticancer activity of Procaspase-8 DED2-α2 split protein peptide 9:

[0027] Procaspase-8 DED2-α2 split protein peptide 9 was used to act on colon cancer cells (HCT116, HT-29), non-small cell lung cancer cells (NCl-H460, A549), cervical cancer cells (Hela), breast cancer cells (MDA-MB-468), ovarian adenocarcinoma cells (SK-OV-3), melanoma cells (A375), and osteosarcoma cells (U2OS) in human cancer cell lines to conduct a tumor inhibition test (CCK8 test).

[0028] The tumor inhibition test (CCK8 test) was performed as follows:

[0029] (I) Colon cancer cells (HCT116, HT-29), non-small cell lung cancer cells (NCl-H460, A549), cervical cancer cells (Hela), breast cancer cells (MDA-MB-468), ovarian adenocarcinoma cells (SK-OV-3), melanoma cells (A375), and osteosarcoma cells (U2OS) in the logarithmic growth phase of human cancer cell lines were respectively taken and digested with 0.25% trypsin at 37°C. 7000 cells were plated in each well of a 96-well cell culture plate in a culture volume of 100 μL / well. No cells were added to 36 wells around the periphery, only culture medium was added, and the plate was cultured in a 37°C, 5% CO2 incubator for 12 hours.

[0030] (ii) Add Procaspase-8 DED2-α2 split protein peptide 9 in a concentration gradient, with 6 parallels for each concentration, incubate in an incubator for 24 h, add 10 μL CCK-8 reagent to each well, incubate at 37°C in the dark for 1 hour and 30 minutes, and measure the absorbance at 450 nm using a microplate reader.

[0031] The experimental results are shown in Table 2. It can be seen that Procaspase-8 DED2-α2 split protein peptide 9 has a good inhibitory effect on colon cancer cells (HCT116) and non-small cell lung cancer cells (NCl-H460). Therefore, Procaspase-8DED2-α2 split protein peptide 9 can be used to study the anti-cancer mechanism for the treatment of non-small cell lung cancer.

[0032]

[0033] Table 2 Results of screening for anticancer activity of Procaspase-8 DED2-α2 split protein polypeptide 9 The optimal concentration of the Procaspase-8 DED2-α2 split protein polypeptide 9 described above was 400 μg / ml.

[0034] Example 3

[0035] Procaspase-8 DED2-α2 split protein peptide 9 combined with drug treatment

[0036] Since non-small cell lung cancer NCl-H460 cells are insensitive to TRAIL, and c-FLIP binds to Procaspase-8 to inhibit cell apoptosis, the Procaspase-8 DED2-α2 split protein polypeptide 9 screened in Example 2 is combined with TRAIL to increase the sensitivity of NCl-H460 cells to TRAIL. The specific CCK8 test method is shown in Example 2. The results showed that the cell viability of TRAIL combined with Procaspase-8 DED2-α2 split protein polypeptide 9 was significantly lower than that of TRAIL alone. When TRAIL alone treated non-small cell lung cancer NCl-H460 cells, the IC50 value was 217.1ng / mL, while when TRAIL was combined with Procaspase-8 DED2-α2 split protein polypeptide 9, the IC50 value dropped to 56.02ng / mL (see Figure 1 ). This indicates that Procaspase-8 DED2-α2 split protein peptide 9 may bind to c-FLIP and antagonize the binding of c-FLIP to Procaspase-8, thereby activating the TRAIL-mediated apoptosis signaling pathway and increasing the sensitivity of NCl-H460 cells to TRAIL. The combined treatment is more effective than single treatment.

[0037] It can be seen that Procaspase-8 DED2-α2 split protein peptide 9 has a good effect in inhibiting tumor proliferation and is expected to become an anti-tumor small molecule peptide and be used in clinical tumor treatment.

[0038] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A polypeptide for inhibiting tumor proliferation by protein Procaspase-8DED2-α2, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

9.

2. Use of the Procaspase-8DED2-α2 split protein polypeptide according to claim 1 in the preparation of drugs for inhibiting tumor proliferation.

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