CN117982626A

CN117982626A - Application of F1/F3 polypeptide in preparation of medicines for inducing tumor cell apoptosis

Info

Publication number: CN117982626A
Application number: CN202410288383.0A
Authority: CN
Inventors: 倪国颖; 罗远东; 刘晓松; 王天放
Original assignee: Zhongao Biomedical Technology Guangdong Co ltd
Current assignee: Zhongao Biomedical Technology Guangdong Co ltd
Priority date: 2024-03-14
Filing date: 2024-03-14
Publication date: 2024-05-07

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of F1/F3 polypeptide in preparation of medicines for inducing tumor cell apoptosis. The invention proves for the first time that the natural polypeptide F1 polypeptide, F3 polypeptide or modified F1 polypeptide can induce the scorching of tumor cells by activating the Caspase 3/GSDME pathway: when the tumor cells die, the cell membrane swells into a bubble, which then ruptures, secreting large amounts of interleukin 18 and LDH. And when F1 and F3 are combined, the effect is optimal. Experiments prove that the secretion amount of IL18 is in a time-dose dependent relationship. And the tumor cells are epithelial tumor cells, including cervical cancer cell line Hela cells, lung cancer A549 cells, liver cancer Huh-7 cells and the like. The invention further explores the mechanism of the natural polypeptide for preparing the tumor treating medicine, and lays a solid foundation for further expanding the application range of the natural polypeptide.

Description

Application of F1/F3 polypeptide in preparation of medicines for inducing tumor cell apoptosis

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of F1/F3 polypeptide in preparation of medicines for inducing tumor cell apoptosis.

Background

Caerin polypeptides are a class of natural antimicrobial peptides found in the skin secretions of the Australian tree frog (Litoria). Studies have shown that in vitro, caerin1.1/1.9 is cytotoxic to a variety of cancer cell lines. In an in vivo mouse model, caninin 1.1/1.9 can inhibit the growth of various tumors; and attracts T cells, NK cells into the tumor, and enhances the adaptive T cell immune response. Furthermore, calerin 1.1/1.9 may enhance the efficacy of tumor therapeutic vaccines, immune checkpoint inhibitors, such as anti-PD 1 antibodies, anti-CD 47 antibodies. Importantly, caerin 1.1.1/1.9 peptides were able to significantly enhance Immune Checkpoint Blockade (ICBs) and therapeutic vaccine efficacy; the survival time of the tumor-bearing mice is obviously prolonged, so that 30 to 50 percent of the mice completely remove tumors and can endure the second tumor inoculation to continue to survive.

The detection of the intratumoral cells shows that the Caninin 1.1/1.9 can promote the tumor infiltration of macrophages, improve the occupation ratio of inflammatory macrophages in the tumor, improve the secretion level of interleukin 12 secreted by the macrophages, reduce the secretion of interleukin 10, enable the intratumoral macrophages to convert from the immunosuppressive M2 type to the M1 type with the immunostimulation function, and the antitumor effect of the Caninin 1.1/1.9 depends on the existence of the intratumoral macrophages. Further analysis showed that macrophages, T cells, NK cells, dendritic cells, etc. within the tumor are all in an immunocompetent state.

Programmed cell death (Programed CELL DEATH) plays an important physiological role in the growth, survival, homeostasis and innate immunity of all multicellular organisms. Programmed cell death includes apoptosis, pyro-death, iron death, and the like. Apoptosis (Apoptosis) is generally divided into exogenous and endogenous Apoptosis. The extrinsic apoptosis pathway is activated by binding of death receptor ligands to their cognate receptors on the cell membrane, activating Caspase7, thereby triggering a series of apoptosis events. Endogenous apoptosis pathways are mediated by mitochondrial damage, leading to increased mitochondrial outer membrane permeability by activation of pro-apoptotic proteins Bax and Bak, release of cytochrome C and binding to APAF-1 to form apoptotic bodies, recruit and activate Caspase 9, followed by Caspase3 and Caspase7, ultimately leading to apoptosis.

Cell death is a regulated cell death that relies on the GASDERMIN family of proteins to form plasma membrane pores, unlike apoptosis, which is characterized by cell membrane rupture, where many cytokines and danger signals (e.g., HMGB1, ATP, HSP, etc.) are released, activating the immune system, leading to an inflammatory response. Cell apoptosis is a very important natural immune response of the body and plays an important role in antagonizing pathogenic infection.

Therefore, further intensive research on the death mode of tumor cells is of great significance for expanding the application range of the polypeptide.

Disclosure of Invention

Aiming at the common problems in the prior art, the invention deeply explores the new application of the natural polypeptide in preparing the medicines for inducing the scorching and the death of tumor cells, and lays a solid foundation for further expanding the application range of the natural polypeptide.

In order to achieve the above purpose, the invention adopts the following technical scheme:

an application of F1/F3 polypeptide in preparing medicine for inducing tumor cell pyrosis.

Preferably, the amino acid sequence of the F1 polypeptide is shown as SEQ ID NO. 1; or the F1 polypeptide is an improved F1 polypeptide, and the amino acid sequence of the F1 polypeptide is shown as SEQ ID NO. 2.

Caerin1.1 is designated as F1, GLLSVLGSVAKHVLPHVVPVIAEHL-NH2 (SEQ ID NO. 1);

caerin 1.1.1 after modification: GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2 (SEQ ID NO. 2);

Preferably, the medicine for inducing tumor cell apoptosis also comprises F3 polypeptide, and the amino acid sequence of the F3 polypeptide is shown as SEQ ID NO. 3.

Caerin 1.9.9 is designated F3, GLFGVLGSIAKHVLPHVVPVIAEKL-NH2 (SEQ ID NO. 3);

preferably, the adding ratio of the F1 polypeptide to the F3 polypeptide in the medicine for inducing tumor cell apoptosis is 1:1, wherein the concentration of the F1 and F3 polypeptides is 5 mug-20 mug/mL.

Preferably, the tumor cells are cells of an epithelial cell tumor.

Preferably, the cells of the epithelial cell tumor comprise cervical cancer cell line Hela cells, lung cancer A549 cells and liver cancer Huh-7h cells.

Preferably, the medicament further comprises physiological saline and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier comprises a pharmaceutical composition comprising an oral formulation adjuvant or an adjuvant for parenteral administration.

Preferably, the auxiliary materials comprise starch glucose, mannitol, xylitol, polyethylene glycol, isopropanol, tween-80, glycerol, propylene glycol, microcrystalline cellulose sodium, sodium chloride, vitamin C, cysteine, citric acid, stearate and gelatin.

Preferably, the drug delivery system includes, but is not limited to, bacteria, viruses, plasmids, nano-delivery systems, mRNA, and the like.

Pyro-death (Pyroptosis) is programmed cell death initiated by pro-inflammatory bodies, which activates Caspase-1/4/5/11 cleavage of the pneumolysin D (GASDERMIN D, GSDMD), and the N-terminus of GSDMD forms small pores in the cell membrane, causing swelling and rupture of the cell, releasing lactate dehydrogenase and interleukin 18, interleukin 1 beta. Unlike GSDMD, GSDME can be cleaved by Caspase-3, releasing the N-terminal fragment, which can result in perforation of the cell membrane. Caspase-3 may be activated by tumor necrosis factor-alpha (TNFa) or chemotherapeutic agents. Inducing tumor cell apoptosis can activate the inherent and adaptive anti-tumor immune response of the organism.

Compared with the prior art, the invention has the following advantages:

(1) The invention proves that the F1/F3 polypeptide can induce cell apoptosis, and the apoptosis process occurs before apoptosis, thereby realizing the application of the F1/F3 polypeptide in the preparation of new drugs and expanding the application range of natural polypeptides;

(2) The invention proves that F1 and F3 polypeptides can induce cell apoptosis, and further optimizes the amino acid sequence of F1, and the result shows that compared with the sequence before optimization, the optimized F1 polypeptide can better induce tumor cells to cause apoptosis.

Drawings

FIG. 1 is a real-time image of a microscope taken of F1/F3 and control P3 treated Hela cells;

FIG. 2 is a graph showing the results of F1/F3 peptides promoting the release of LDH from Hela cells;

FIG. 3 shows the promotion of IL-18 secretion by Hela cells by different polypeptides;

FIG. 4 is a graph showing the effect of F1/F3 concentration on IL-18 secretion by HeLa cells;

FIG. 5 is a graph showing the results of stimulating IL-18 secretion by HeLa cells with F1/F3 peptides for various periods of time;

FIG. 6 is a graph showing the results of the F1/F3 polypeptide not promoting IL-6 secretion from HeLa cells, IL-1β;

FIG. 7 is a real-time photographed microscope image of F1/F3 and control P3 treated different cells;

FIG. 8 is a graph showing the effect of F1/F3 polypeptide on A549 cell activity and LDH release therefrom;

FIG. 9 is a graph showing the effect of F1/F3 polypeptides on Huh-7 cell activity and LDH release therefrom;

FIG. 10 is a graph showing the results of stimulation of IL-18 secretion by A549 cells by F1/F3 polypeptides over various time periods;

FIG. 11 is a graph showing the results that the F1/F3 polypeptide does not promote IL-1β secretion by A549 cells;

FIGS. 12-13 are graphs showing the results of Caninin 1.1/1.9 mediated apoptosis of Hela cells via the caspase-3/GSDME signaling pathway;

FIG. 14 is a graph showing the results of comparison of the ability of F1 and engineered F1 to induce interleukin 18 secretion by Hela cells;

FIGS. 15-16 are graphs of the results of Caer1.1/1.9 activation of mitochondrial/Cytochrome c/Apaf-1 signaling pathways to mediate apoptosis.

Detailed Description

The present invention will be further explained with reference to specific examples, but it should be noted that the following examples are only for explaining the present invention, and are not intended to limit the present invention, and all technical solutions identical or similar to the present invention are within the scope of the present invention. The specific techniques or conditions are not noted in this example and are practiced according to methods and apparatus conventional in the art; the reagents or instruments used are not noted to manufacturers and are all conventional products which can be obtained through commercial purchase; in the test, the mass ratio of caerin 1.1.1 to caerin1.9 in Caninin 1.1/1.9 was 1:1, which is not specifically described.

Example 1 Caninin 1.1/1.9 promotes the Coke's death of Hela

1. Test sample: F1/F3 polypeptide, hela cells;

2. the test method comprises the following steps:

Cell membrane changes were observed: heLa cells of 5X 10 ⁵ were inoculated in 24-well plates and cultured overnight, and cells were treated according to CELL PLASMA MembraneStainingKit (cat# S0033S, company name: biyun day) the following day, and then 10. Mu.g/mL of Canerin 1.1/1.9 or P3 was added, or not treated, immediately after time-lapse imaging using an Olympus microscope (AP X100), and cell membrane changes after drug addition were observed.

Lactate dehydrogenase detection: heLa cells of 4X 10 ⁴ were seeded overnight in 96-well plates, 10. Mu.g/mL of Caninin 1.1/1.9 or P3 was added the next day, or not treated, the cells were cultured for 1h, culture supernatants were collected, centrifuged at 600g for 10min, 10. Mu.L of the different supernatant was transferred to new 96-well plates, and LDH assay was performed. LDH detection kit was purchased from Eimer (cat# ab 65393). Detection was performed using an enzyme-labeled instrument at OD450nm wavelength.

The calculated formula of LDH activity (LDH _{Sample of}− LDH_{low control group}）/（LDH_{High control group}−LDH_{low control group}). Times.100%. Each sample was tested in triplicate and averaged.

Wherein the sample group is F1/F3 treated group or P3 group, the low control group is untreated group, and the high control group is cell supernatant collected by 0.5h of Lysis reagent treatment provided according to the kit.

ELISA method for detecting interleukin 18, interleukin 1 beta:

HeLa cells of 5X 10 ⁵ were inoculated in 24-well plates and cultured overnight, F1/F3 polypeptide or control polypeptide P3 (GTELPSPPSVWFEAEFK-OH) was added at various concentrations (0-20. Mu.g/mL), or not treated, for various periods (1, 2,4,6 hours or overnight), and after harvesting the supernatant, interleukin 18 was detected using the ELISA kit (cat# VAL 131) from R & D Systems, interleukin 1. Beta. Was detected using the ELISA kit (cat# VAL 101) from R & D Systems, and interleukin 6 was detected using the ELISA kit (cat# 430504) from BIOLEGEND.

3. Test results: the specific test results are shown in fig. 1-5: in the figure, interleukin 6 is IL-6, and interleukin 1 is IL-1β. FIG. 1 is a real-time microscopic image of treatment of Hela cells with F1/F3 polypeptide (10. Mu.g/mL) and control polypeptide P3 (10. Mu.g/mL). From this, the F1/F3 polypeptide induced Hela cell swelling, and produced a large number of bubbles (the apoptotic cells are indicated by dark and light arrows). As can be seen from FIG. 2, the F1/F3 polypeptide promotes the release of large amounts of LDH by Hela cells; as can be seen from FIG. 3, F1 and F3 can promote IL-18 secretion of Hela cells by both F1 and F3 in combination (10. Mu.g/mL), and the combination of F1 and F3 has the best effect; as can be seen from FIG. 4, F1/F3 gradually increased with increasing concentration, and IL-18 secreted by HeLa cells was gradually increased to become concentration-dependent; as can be seen from FIG. 5, the F1/F3 polypeptide (10. Mu.g/mL) stimulated Hela cells for various periods of time, with IL-18 secretion at a maximum of 1 h; as can be seen from FIG. 6, the F1/F3 polypeptide (10. Mu.g/mL) did not promote IL-6 secretion by HeLa cells, nor did it promote IL-1β secretion by HeLa cells.

Example 2 Caninin 1.1/1.9 promotes the apoptosis of A549, huh-7

1. Test sample: F1/F3 polypeptides, P3 polypeptides, A549 cells, huh-7 cells;

2. the test method comprises the following steps:

Cell membrane changes were observed: a549 cells of 5×10 ⁵ were seeded in 24 well plates and cultured overnight, the following day cells were treated according to CELL PLASMA MembraneStainingKit (cat No. S0033S, company name: bi yun), DAPI (cat No. 10236276001, company name: merck) instructions, then 10 μg/mL of calin 1.1/1.9 or P3 was added, or no treatment was performed, immediately delayed imaging photographing was performed using an olympus microscope (ap×100), and cell membrane changes after drug addition were observed.

Huh-7 cells of 5X 10 ⁵ were seeded in 24-well plates and cultured overnight, 10. Mu.g/mL of Caninin 1.1/1.9 or P3 was added the next day, or no treatment was performed, and immediately delayed imaging photographing was performed using an Olympus microscope (AP X100) to observe cell membrane changes after drug addition.

Lactate dehydrogenase detection: a549 cells or Huh-7 cells of 4X10 ⁴ were seeded in 96-well plates, then 10. Mu.g/mL of Caninin 1.1/1.9 or P3 was added, or alternatively, cells were cultured for 1h, the high control group was treated with Lysis reagent according to kit for 0.5h, culture supernatants were collected, centrifuged at 600g for 10min, 10. Mu.L of the supernatant of the different group was transferred to a new 96-well plate, and LDH assay was performed. LDH kit was purchased from Eimer (cat# ab 65393). Detection was performed using an enzyme-labeled instrument at OD450nm wavelength.

ELISA method for measuring interleukin 18, interleukin 1 beta:

HeLa cells of 5X 10 ⁵ were inoculated in a 24-well plate and cultured overnight, and then various concentrations (0-20. Mu.g/mL) of F1/F3 polypeptide or control polypeptide P3 (GTELPSPPSVWFEAEFK-OH) were added, or alternatively, the supernatant was collected for various periods (1, 2,4,6 hours or overnight), and then interleukin 18 and interleukin 1. Beta. Were detected using an ELISA kit (product number: VAL 101) from R & D Systems.

3. Test results: the specific test results are shown in fig. 7-11. From FIG. 7, which shows that F1/F3 polypeptide (10. Mu.g/mL) and control polypeptide P3 (10. Mu.g/mL) treat A549 cells and Huh-7 cells for real-time microscopic image, F1/F3 polypeptide induces A549 cells to swell and generate a large amount of bubbles (scorched cells are indicated by light arrows); the F1/F3 polypeptide induces Huh-7 cells to swell and generate a large amount of bubbles (the scorched cells are represented by dark arrows); as can be seen from fig. 8, the F1/F3 polypeptide reduces a549 cell activity while promoting a549 cell release of a large amount of LDH; as can be seen from FIG. 9, the F1/F3 polypeptide reduced Huh-7 cell activity while promoting release of large amounts of LDH by Huh-7 cells. As can be seen from FIG. 10, the F1/F3 polypeptide (10. Mu.g/mL) stimulated A549 cells for various periods of time, with IL-18 secretion being maximized for 1 h; as can be seen from FIG. 11, the F1/F3 polypeptide (10. Mu.g/mL) did not promote IL-1β secretion by A549 cells.

EXAMPLE 3 Caninin 1.1/1.9 mediation of Hela cell apoptosis by caspase-3/GSDME Signal pathway

1. Test sample: F1/F3 polypeptide, hela cells;

2. the test method comprises the following steps:

1X 10 ⁶ HeLa cells were incubated overnight in 6 well plates, then 10. Mu.g/mL of Caerin1.1/1.9 or P3 was added the next day, or after 1 hour of no treatment, the cells were all scraped off with a cell scraper, and after 1 hour, the supernatant was collected using RIPA Buffer (FD 009, fdbio science, china) protein lysate (in lysate, RIPPA Buffer: PMSF: protein phosphatase inhibitor mixture = 100:1:1) containing PMSF (FD 0100, fdbio science, china) and protein phosphatase inhibitor mixture (FD 1002, fdbio science, china) was centrifuged for 10 min.

Total protein concentration was measured by BCA protein assay kit (ab 287853, abcam) and then 5 x SDS (P0285, fdbio science, china) loading buffer (total protein volume: 5 x SDS loading buffer = 4:1) was added and denatured by boiling for 10min at 100 ℃. Samples were separated by SDS-PAGE and transferred to PVDF (FFP 39, beyotime) membranes, which were blocked with Quick Block Western blocking solution (P0018M-2, beyotime) for 1h at room temperature. The membrane was then incubated overnight with the target protein antibody (i.e., primary antibody: primary antibody diluent = 1:1000), followed by incubation with HRP-conjugated secondary antibody ((secondary antibody: secondary antibody diluent = 1:1000)) for 1h, then visualized using Enhanced ChemiLuminescence substrate solution (T014-500, beyotime, china) and photographed by image scanner MINICHEMI910 (Beijing Sage Creation Science Co, LTD), protein grey values were analyzed at ImageJ and the results counted.

The primary antibodies used were:

Anti-cleaved N-terminal DFNA5/GSDME （ab222408）,anti-GSDMD antibody（AB210070）,anti-GSDMB antibody（AB215729）,Anti-Caspase-1 antibody（AB207802）,Anti-Caspase-3 antibody（AB32351）,Anti-Cleaved Caspase-3 antibody（AB32042）,anti-GAPDH antibody（AB181602） These antibodies were purchased from eimeric corporation. Caspase-5 (D3G 4W), caspase-4 Antibody (# 4450) was purchased from cell signalingTechnology company. Cleaved-Caspase 4 (AF 5373) was purchased from Affinity Bioscience. The dilution and amount of each antibody are referred to the instructions.

The secondary antibodies used were:

anti-rabit lgG HRP-linked Anti-body (# 7074) is purchased from cell signalingTechnology company. Dilution and amount of antibody are referred to the instructions.

3. Test results: the test results are shown in FIGS. 12-13, wherein the left graph A shows the expression level of GSDMD in Hela cells detected by western blotting, and the right graph shows the average result of three tests of GSDMD protein; b is western blot image detection GSDMB protein expression level in Hela cells; c is western blot image detection of caspase-1 protein expression level in Hela cells; d is western blot image detection of CLEAVED CASPASE-4 protein expression level in Hela cells; e is western blot image detection of caspase-5 protein expression level in Hela cells; the results showed that the expression of F1/F3 treated HeLa cells, GSDMD, GSDMB, caspase-1,cleaved caspase-4, caspase-5 was not significantly altered compared to P3 polypeptide treated or untreated HeLa cells. In FIG. 13, A is Western blot image of CLEAVED GSDME-N protein expression levels in Hela cells; b is western blot image detection of caspase-3 protein expression level in Hela cells; c is western blot image detection of CLEAVED CASPASE-3 protein expression level in Hela cells; d is western blot image to detect the expression level of caspase-4 protein in Hela cells. The results showed that F1/F3 treated HeLa cells, CLEAVED GSDME-N, caspase-3,cleaved caspase-3 expression was significantly up-regulated compared to P3 polypeptide treated or untreated HeLa cells. caspase-4 expression levels were significantly down-regulated. The results in connection with FIGS. 12-13 show that F1/F3 induces apoptosis of Hela cells via the Caspase 3/GSDME pathway.

Example 4 comparison of F1 with modification F1

1. Test sample: f1 polypeptides, engineered F1 polypeptides, hela cells;

2. The test method comprises the following steps: heLa cells of 5X 10 ⁵ were inoculated into a 24-well plate and cultured overnight, and old F1 (SEQ ID NO. 1), F1 (SEQ ID NO. 2) polypeptide or control polypeptide P3 (GTELPSPPSVWFEAEFK-OH) was added at 10. Mu.g/mL, respectively, or alternatively, without treatment, cultured for 1 hour, and 600g were centrifuged for 10min, and after collecting the supernatant, interleukin 18 and interleukin 1. Beta. Were detected using ELISA kit (product number: VAL 101) of R & D Systems Co.

3. Test results: the results show that, as shown in FIG. 14, F1 (i.e., old F1) was found to induce IL18 production by HeLa cells from F1 (i.e., old F1) and that the altered F1 induced greater secretion of interleukin 18 by HeLa cells.

Example 5 demonstrates that apoptosis by treatment of Hela cells with Caninin 1.1/1.9 occurs after focal death

1. Test sample: F1/F3 polypeptide, hela cells;

2. the test method comprises the following steps:

In order to compare the relation between apoptosis and pyroapoptosis, 1× ⁶ HeLa cells were cultured in 6-well plates, and protein was extracted by two different treatments, the first was adding 10 μg/mL of Canerin 1.1/1.9 or P3, or not, for 1 hour; the second was to add different concentrations of Caninin 1.1/1.9 (0, 5, 10, 15, 20. Mu.g/mL) to treat overnight, using RIPA Buffer (FD 009, fdbio science, china) protein lysate (RIPPA Buffer: PMSF: protein phosphatase inhibitor mixture=100:1:1) containing PMSF (FD 0100, fdbio science, china) and protein phosphatase inhibitor mixture (FD 1002, fdbio science, china), lyse cells, scrape all cells with a cell scraper, centrifuge 12500g for 10min, collect the supernatant, and obtain the protein.

Total protein concentration was measured by BCA protein assay kit (ab 287853, abcam) and then 5 x SDS (P0285, fdbio science, china) loading buffer (total protein volume: 5 x SDS loading buffer = 4:1) was added and denatured by boiling for 10min at 100 ℃. Samples were separated by SDS-PAGE and transferred to PVDF (FFP 39, beyotime) membranes, which were blocked with Quick Block Western blocking solution (P0018M-2, beyotime) for 1h at room temperature. The membrane was then incubated overnight with the target protein antibody, followed by incubation with HRP-conjugated secondary antibody for 1h, then visualized using Enhanced ChemiLuminescence substrate solution (T014-500, beyotidme, china) and photographed by image scanner MINICHEMI (Beijing Sage Creation Science Co, LTD), protein grey values were analyzed at ImageJ and the results were counted.

The primary antibodies used were:

anti-GAPDH anti-body (AB 181602) and anti-clAP anti-body (AB 32059) such antibodies were purchased from Eimer .The Apaf-1（D5C3）Rabbit mAb（#8969）,Cytochrome c antibody（#4272）,Bax（D2E11）Rabbit mAb（#5023）,β-Actin（E4D9Z）Mouse mAb（#58169）) ,Smac/Diablo（79-1-83）Mouse mAb（#2954）, Noxa（D8L7U）Rabbit mAb（#14766）,Cleaved Caspase-7（Asp 198）（D6G1）Rabbit mAb（#8438）,Caspase-7 Antibody（#9492）,Caspase-8（1C12）Antibody（#9746）,Cleaved Caspase-8（Asp 374）（18C8）Rabbit mAb（#9496） and from cell signalingTechnology. BCL-2 Antibody (Bcl-2-100) (13-8800) was purchased from Sieimer. The dilution and amount of each antibody are referred to the instructions.

The secondary antibodies used were:

Goat anti-Mouse lgG Light-Chain Specific Antibody（HRP Conjugate）（#91196）,Anti-rabbit lgG HRP-linked antibody（#7074） Purchased from cell signalingTechnology company. The dilution and amount of each antibody are referred to the instructions.

3. Test results: as shown in FIGS. 15 and 16, it was found from FIG. 15 that the protein expression levels of Bax, cytochrome c, apaf-1, caspase-8, caspase-7, CLEAVED CASPASE-8 and CLEAVED CASPASE-7 were not changed after F1/F3 was treated for Hela 1h by Western blotting and quantifying the protein levels of Bax, cytochrome c, apaf-1, caspase-8, caspase-7, CLEAVED CASPASE-8 and CLEAVED CASPASE-7 after F1/F3 was stimulated for Hela 1 h.

As can be seen from FIG. 16, F1/F3 stimulated Hela overnight and Bax, bcl-2, cytochrome c, apaf-1, noxa, smac, caspase-7, CLEAVED CASPASE-7 protein levels were analyzed and quantified by Western blotting. A, C, D, H, I results indicated that after F1/F3 stimulation of Hela overnight, bax, cytochrome C, APaf-1, noxa, CLEAVED CASPASE-7 protein expression levels were up-regulated. B results show that Bcl-2 protein expression levels are down-regulated. E-G results showed that F1/F3 stimulation of Hela overnight did not alter Smac, clap2, caspase-7 protein expression levels. FIGS. 15 and 16 show that F1/F3 promotes apoptosis of Hela cells after focal apoptosis.

Finally, it should be noted that the above-mentioned embodiments are merely illustrative of the principles, performances and effects of the present invention, and are not meant to limit the invention. 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 invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. Application of F1/F3 polypeptide in preparing medicine for inducing tumor cell apoptosis; the F1 polypeptide is characterized in that the amino acid sequence of the F1 polypeptide is one of SEQ ID NO.1 and SEQ ID NO. 2; the amino acid sequence of the F3 polypeptide is shown as SEQ ID NO. 3.

2. The use according to claim 1, wherein the mass ratio of the F1 polypeptide to the F3 polypeptide in the medicament for inducing the apoptosis of tumor cells is 1:1, and the concentration of the F1 and F3 polypeptides is 5 μg/mL to 20 μg/mL.

3. The use of claim 1, wherein the tumor cell is a cell of an epithelial cell tumor.

4. The use according to claim 3, wherein the cells of the epithelial cell tumor comprise cervical cancer cell line Hela cells, lung cancer a549 cells, liver cancer Huh-7 cells.

5. The use of claim 1, wherein the medicament further comprises physiological saline and a pharmaceutically acceptable carrier.

6. The use according to claim 5, wherein the pharmaceutically acceptable carrier comprises an oral formulation adjuvant or an adjuvant for parenteral administration.

7. The use according to claim 6, wherein the excipients comprise starch glucose, mannitol, xylitol, polyethylene glycol, isopropanol, tween-80, glycerol, propylene glycol, microcrystalline cellulose sodium, sodium chloride, vitamin C, cysteine, citric acid, stearates and gelatin.

8. The use of claim 1, wherein the drug delivery system comprises bacteria, viruses, plasmids, nanodelivery systems, mRNA.