Application of telelactone in preparation of medicine for inhibiting tumor angiogenesis

Abstract

The invention relates to the technical field of biological medicines, in particular to application of telelactone in preparation of a medicine for inhibiting tumor angiogenesis. Research finds that the telelactone can inhibit proliferation, migration and lumen formation of human umbilical vein vascular endothelial cells and inhibit angiogenesis induced by a tumor conditioned medium. A further research shows that the telelactone can reduce angiogenesis in a tumor microenvironment by inhibiting a vascular endothelial growth factor signal channel, so that the growth and metastasis of tumors are prevented. In in-vivo experiments, the telelactone can reduce the number of chick embryo allantoic membrane vascular intersections, inhibit the growth of blood vessels in a chick embryo allantoic membrane model, and reduce the formation of internode blood vessels in a zebra fish embryo model, so that the anti-angiogenesis effect of the telelactone is further verified. Research results of the invention show that the telelactone can be used as a natural candidate compound for anti-tumor angiogenesis, and has high application value and development potential.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of terylene lactone in the preparation of a medicine for inhibiting tumor angiogenesis. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Tumor angiogenesis refers to the formation of new blood vessels in tumor tissues, a process that is crucial for tumor growth, invasion, and metastasis. Tumor angiogenesis involves endothelial cell proliferation, migration, lumen formation, and extracellular matrix remodeling, and its level is closely related to the malignancy of the tumor. Studies have found that the VEGF signaling pathway plays a key role in regulating tumor angiogenesis. Signal transduction mediated by VEGF receptors (VEGFRs) promotes the growth and migration of endothelial cells and ultimately forms new tumor blood vessels.

[0004] Existing anti-angiogenesis therapies mainly include VEGF inhibitors (such as bevacizumab (Avastin)), but such therapies have problems such as drug resistance, high tumor recurrence rate, and severe side effects. Therefore, it is still necessary to find new drugs that inhibit tumor angiogenesis to improve the effectiveness of anti-tumor treatment.

[0005] In this context, it is of great significance to explore new natural products and their potential to inhibit tumor angiogenesis. Summary of the invention

[0006] The present invention provides a new use of Telekin as an anti-angiogenic drug, especially in inhibiting tumor angiogenesis. Through systematic research, it is found that Telekin can exert anti-angiogenic effects by inhibiting the VEGF signaling pathway, providing a new treatment option for the treatment of angiogenesis-dependent tumors.

[0007] Specifically, the present invention provides the use of Telekin in the preparation of a drug for inhibiting tumor angiogenesis. The Telekin described in the present invention is a eucalyptus sesquiterpene lactone compound isolated from the plant Carpesium divaricatum. Telekin can activate mitochondrial-mediated apoptosis and has anti-cancer activity. The present invention first discovered that the compound has significant anti-angiogenic activity. Among them, the CAS number of Telekin is 6752-90-5, and the structure is shown below:

[0008]

[0009] The present invention has been studied in depth through the following aspects:

[0010] Through the study of the mechanism of action, it was found that Telekin achieves its anti-angiogenic effect by inhibiting the vascular endothelial growth factor (VEGF) signaling pathway. Therefore, in one embodiment of the present invention, the drug for inhibiting tumor angiogenesis inhibits tumor angiogenesis by inhibiting the vascular endothelial growth factor (VEGF) signaling pathway.

[0011] Further studies have shown that Telekin inhibits VEGF-mediated signal transduction by downregulating the expression levels of VEGFA and VEGFR2, thereby exerting an anti-angiogenic effect. Western blot experimental results confirmed this mechanism. Therefore, in one embodiment of the present invention, the VEGF signaling pathway includes signal transduction mediated by VEGFA and VEGFR2.

[0012] The present invention also identifies the use of telomerase in the preparation of drugs for the treatment of angiogenesis-dependent tumors, including but not limited to triple-negative breast cancer, lung cancer, liver cancer and colorectal cancer. In experimental models represented by triple-negative breast cancer, telomerase exhibited significant anti-angiogenesis effects.

[0013] In the angiogenesis model induced by tumor conditioned medium, teryl lactone still showed a significant inhibitory effect. In the study of tumor microenvironment, the present invention uses the conditioned medium of tumor cells to induce angiogenesis model for verification. For example, in one embodiment, triple-negative breast cancer MDA-MB-231 is used as an example for illustration. The results show that compared with the control group, the luminal structure formed under the induction of conditioned medium is more complete and the number of vascular nodes is more. However, as the concentration of teryl lactone increases, the number of tubes is significantly reduced, the lumen becomes shorter, and even tubule formation is not observed at higher concentrations, indicating that teryl lactone can effectively inhibit angiogenesis induced by tumor microenvironment. Therefore, the present invention also determines the use of teryl lactone in the preparation of drugs for inhibiting angiogenesis induced by tumor microenvironment.

[0014] In addition, the present invention systematically verifies the anti-angiogenesis effect of telolactone through in vitro and in vivo experiments.

[0015] The present invention confirms the effect of Tellerolactone on vascular endothelial cells, especially on human umbilical vein endothelial cells (HUVEC) through multiple in vitro experiments: this includes but is not limited to significantly inhibiting the proliferation of human umbilical vein endothelial cells, inhibiting the horizontal and vertical migration ability of human umbilical vein endothelial cells, and inhibiting the lumen formation ability of human umbilical vein endothelial cells. Therefore, the present invention also determines the use of Tellerolactone in the preparation of a drug for inhibiting the proliferation of human umbilical vein endothelial cells, and / or its migration, and / or its lumen formation ability. Specifically, experimental verification is provided in some embodiments.

[0016] For example, in the cell scratch experiment, the scratch healing rates of HUVEC cells at drug concentrations of 10μmol / L, 20μmol / L and 30μmol / L were 75.11%, 47.54% and 24.39% at 12 hours, and 77.17%, 48.57% and 29.48% at 24 hours, which were significantly lower than those of the control group.

[0017] For example, the Transwell migration assay showed that after 24 hours of treatment with telomerase, the number of cells crossing from the upper chamber to the lower chamber was significantly reduced.

[0018] For example, in the matrigel experiment, after 12 h of telomerase treatment, the number of HUVEC tubes gradually decreased, the length of the formed tubes shortened, the number of vascular nodes was significantly reduced, and the lumen morphology was incomplete.

[0019] In addition, the present invention also proves the anti-angiogenic effect of telolactone in in vivo experimental verification. In the in vivo model, the present invention adopts the chicken embryo chorioallantoic membrane (CAM) experiment for verification. The results show that a relatively complete vascular network is developed in the control group. With the increase of the dosage of telolactone, the number of angiogenesis is significantly reduced, the growth of small and medium blood vessels and small capillaries is significantly inhibited, and the number of vascular nodes is significantly reduced. This shows that telolactone significantly reduces the number of vascular intersections and inhibits the formation of vascular networks.

[0020] In the zebrafish model, the experimental results showed that Tellerolide can significantly inhibit the growth of intersegmental vessels (ISV) and exhibit a concentration-dependent effect. As the concentration of Tellerolide increases (25-200μmol / L), the length of the intersegmental vessels gradually shortens, accompanied by the loss of some blood vessels. In the higher concentration group (100-200μmol / L), the intersegmental vessels almost completely disappeared, and some embryos showed developmental abnormalities. This result further indicates that Tellerolide can significantly inhibit the development of embryonic blood vessels and reduce the formation of intersegmental vessels, verifying its inhibitory effect on the angiogenesis process.

[0021] The present invention also determines through systematic research that the effective dosage range of the tolerolactone in the present invention is 10 μM to 30 μM. Within this concentration range, tolerolactone exhibits significant anti-angiogenic activity and has good safety.

[0022] The drug of the present invention can be administered by a variety of ways, including oral administration, injection or local delivery, providing flexible administration options for clinical applications.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention is the first to discover that telolactone has an anti-angiogenic effect, expanding its pharmacological application. Through in vivo and in vitro experiments, it is verified that telolactone can significantly inhibit VEGF-mediated signal transduction, reduce angiogenesis, and inhibit tumor growth, and the mechanism of action is clear. In addition, the present invention provides a natural, safe and efficient anti-angiogenic candidate drug, which has the following advantages compared with existing VEGF targeted therapeutic drugs: (1) strong innovation, for the first time, it is found that teryl lactone can inhibit angiogenesis through the VEGF signaling pathway, which expands the application scope of the compound; (2) clear mechanism of action, studies have shown that it can reduce angiogenesis and affect blood vessel formation in the tumor microenvironment by inhibiting the signaling pathway mediated by VEGFA and VEGFR2; (3) in vivo and in vitro experimental support, HUVEC cell experiments, CAM models and zebrafish embryo experiments have verified its anti-angiogenic effect; (4) wide application range, can be used for angiogenesis-dependent tumors such as triple-negative breast cancer, lung cancer, liver cancer, etc., and has good clinical application value; (5) natural source, high safety, as a natural product, teryl lactone has good biocompatibility and lower toxicity than synthetic VEGF inhibitors; (6) multiple modes of administration, including oral, injection and local delivery, to meet different clinical needs and improve the feasibility of treatment. The research results of the present invention provide a new research direction for the development of anti-angiogenic drugs and have high clinical transformation value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein:

[0026] Figure 1 The figure shows that CCK8 detected the cell viability of HUVEC cells after treating them with Telekin at different concentrations for 12 hours, 24 hours, 48 ​​hours and 72 hours, indicating that Telekin inhibited the proliferation of HUVEC cells in a time- and dose-dependent manner.

[0027] Figure 2 :The wound healing experiment found that Telekin inhibited the horizontal migration ability of HUVEC cells at 12h and 24h, scale bar = 100μm.

[0028] Figure 3 : Quantitative analysis of migration ability in the horizontal direction in a 12h scratch test.

[0029] Figure 4 : Quantitative analysis of horizontal migration ability in 24h scratch assay.

[0030] Figure 5The figure shows the effect of Telekin on the vertical migration ability of HUVEC cells detected by Transwell assay. Scale bar = 100 μm.

[0031] Figure 6 :Quantitative analysis of the effect of Telekin on the vertical migration ability of HUVEC cells detected by Transwell assay.

[0032] Figure 7 The figure shows the effect of different concentrations of Telekin on HUVEC cell angiogenesis in vitro, scale bar = 200 μm.

[0033] Figure 8 :Quantitative analysis of the number of vascular nodes (Nbjunctions) in the in vitro angiogenesis experiment of HUVEC cells treated with different concentrations of Telekin.

[0034] Fig. 9 Quantitative analysis of the total vessel length (Tot.VesselLength) in the in vitro angiogenesis experiment of HUVEC cells treated with different concentrations of Telekin.

[0035] Fig.10 : The results of Telekin inhibiting the angiogenesis of HUVEC induced by the tumor-conditioned medium of triple-negative breast cancer MDA-MB-231. Scale bar = 200 μm.

[0036] Fig.11 : Quantitative analysis of the number of vascular nodes in HUVEC angiogenesis experiment induced by tumor conditioned medium of triple-negative breast cancer MDA-MB-231 by different concentrations of Telekin.

[0037] Fig.12 : Quantitative analysis of the total length of blood vessels in HUVEC angiogenesis experiment induced by tumor conditioned medium of triple-negative breast cancer MDA-MB-231 by different concentrations of Telekin.

[0038] Fig.13 The ability of Telekin to inhibit blood vessel formation in chick embryo chorioallantoic membrane was shown.

[0039] Fig.14 : Quantitative analysis of vessel length (vascular growth) in chick embryo chorioallantoic membrane angiogenesis assay using Telekin.

[0040] Fig.15 :Quantitative analysis of the number of vascular nodes in chick embryo chorioallantoic membrane angiogenesis assay using Telekin.

[0041] Fig.16The experimental structure of Telekin inhibiting vascular development in zebrafish embryos is shown. The figure shows the development of intersegmental blood vessels in zebrafish, and intuitively shows the number and length of intersegmental blood vessels in zebrafish.

[0042] Fig.17 : Expression of VEGFR2 and VEGFA proteins in HUVEC cells treated with different concentrations of Telekin for 48 hours.

[0043] Fig.18 : Quantitative analysis of VEGFR2 protein expression in HUVEC cells treated with different concentrations of Telekin for 48 hours.

[0044] Fig.19 : Quantitative analysis of VEGFA protein expression in HUVEC cells treated with different concentrations of Telekin for 48 h. DETAILED DESCRIPTION

[0045] The present invention is further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope thereof. Experimental methods for which specific conditions are not specified in the examples are generally carried out under conventional conditions or conditions recommended by the manufacturer.

[0046] Unless otherwise defined, all technical terms and scientific terms used in the present invention shall have the meanings familiar to those skilled in the art. Unless otherwise specified, the reagents or raw materials used in the present invention can be obtained by conventional means and used according to conventional methods or product instructions in the art. In addition, any content similar or equivalent to the methods or materials described can be applied to the methods of the present invention. The preferred embodiments and materials described in the present invention are for illustrative purposes only.

[0047] Example 1 Experimental study on the effect of Telekin on HUVEC cell proliferation and migration

[0048] 1. HUVEC cell proliferation ability test method: Digest the cells in the logarithmic growth phase and adjust to the appropriate density with complete culture medium. Add the cell suspension evenly to a 96-well plate, 100 μL per well, 37°C, 5% CO 2The cells were cultured in a culture incubator for 12-24 hours to allow the cells to adhere to the wall. The culture medium was removed by suction, and 200 μL of culture medium containing different concentrations of drugs was added, and the cells were cultured continuously in a cell culture incubator for 12 h, 24 h, 48 h, and 72 h. After the drug action time was up, CCK8 was mixed with the culture medium at 10 μL / well and the culture medium at 90 μL / well. After mixing evenly, the mixture was added to a 96-well plate (100 uL / well) and placed in a cell culture incubator for incubation for 45 min. Finally, the absorbance was measured at a wavelength of 450 nm using a multifunctional microplate reader to calculate the survival rate of cells in different drug treatment groups. Finally, the proliferation ability of HUVEC was detected at 12 h, 24 h, 48 h, and 72 h.

[0049] The experimental results show that Telekin inhibits HUVEC cell proliferation in a concentration-dependent and time-dependent manner, and the inhibitory effect is particularly significant under high concentrations (≥30μmol / L) and long-term (≥48h) action. Under short-term (12h) and low concentrations (≤10μmol / L), HUVEC cell viability is less affected, but when the action time is extended to 72h, even low concentrations of Telekin can significantly inhibit cell proliferation. Telekin at a concentration of 50μmol / L almost completely inhibits HUVEC cell viability after 72h of action, indicating that Telekin may significantly inhibit HUVEC cell proliferation by interfering with angiogenesis-related signaling pathways. Results are shown in Figure 1 .

[0050] 2. Cell scratch test method: Inoculate an appropriate amount of HUVEC cells in a 6-well plate and grow overnight to 90% confluence. Then use a 10μL pipette tip to scratch each well in the vertical direction with uniform force. After washing three times with PBS, add 2mL of fresh serum-free DMEM and (10μmol / L, 20μmol / L, 30μmol / L) concentrations of Telekin to each well, and then immediately take pictures of each group on an inverted microscope. After the photo processing is completed, continue to culture in a normal culture environment. At 12 and 24 hours of growth, use an inverted microscope to observe and take pictures, and the results are quantified and analyzed using imageJ software.

[0051] The results showed that at 12 hours, the scratch healing rates of HUVEC cells were 75.11%, 47.54% and 24.39% at drug concentrations of 10μmol / L, 20μmol / L and 30μmol / L, respectively. At 24 hours, the scratch healing rates of HUVEC cells were 77.17%, 48.57% and 29.48% at drug concentrations of 10μmol / L, 20μmol / L and 30μmol / L, respectively, which were significantly lower than those of the control group, indicating that Telekin can significantly inhibit the horizontal migration ability of HUVEC. Figure 2 , Figure 3 and Figure 4 .

[0052] 3. Transwell cell migration assay method: Prepare a 24-well cell culture plate and test cell migration in a Transwell chamber with a pore size of 8.0 μm. First, place each Transwell chamber in a 24-well cell culture plate, and then add 200 μL of 2.5×10 5 / mL of serum-free medium cell suspension and corresponding concentrations (10μmol / L, 20μmol / L, 30μmol / L) of drugs; then add 20% FBS cell culture medium to the lower chamber of the Transwell chamber. After 24 hours of treatment, take out the 24-well plate with the Transwell chamber from the cell culture incubator, wash it with PBS three times, fix the chamber with 4% paraformaldehyde fixative for 15 minutes, then stain it with 0.1% crystal violet for 10 minutes, and then gently wipe the upper surface of the inner membrane of the Transwell chamber with a cotton swab or cleaning cotton, wash it with PBS again for three times, and then put the chamber on a cover glass, and observe and take images using an inverted microscope. The results were counted and analyzed using imageJ.

[0053] The results showed that after 24 hours of exposure to Telekin in the Transwell migration assay, the number of cells crossing from the upper chamber to the lower chamber was significantly reduced compared with the control group, indicating that Telekin can significantly inhibit the vertical migration ability of HUVEC cells. Figure 5 and Figure 6 .

[0054] Example 2 Effects of Telekin on HUVECs angiogenesis in vitro

[0055] Experimental method: One night before the experiment, take out the matrix gel and insert it into ice to slowly melt in a 4℃ refrigerator, then pre-cool the pipette tip and 24-well plate in advance and keep them in a sterile environment, then quickly add 20μL Matrigel to each well of the 24-well plate, spread the Matrigel evenly into a uniform circle, and then quickly put the 24-well plate into a cell culture incubator at 37℃ for 30min to wait for the matrix gel to solidify. During the room temperature pretreatment, HUVECs were digested, centrifuged, resuspended, and 1.5×10 5 / mL HUVEC cells were seeded in a pretreated 24-well plate, and the cells were cultured in a medium containing 10% FBS and 10μmol / L, 20μmol / L and 30μmol / L Telekin; the cells were cultured in an incubator for 12 hours, and the number of angiogenesis was observed under a microscope and photographed. The results were quantified and analyzed using imageJ.

[0056] The results showed that after treating HUVECs cells with 10μmol / L, 20μmol / L and 30μmol / L Telekin concentrations for 12 hours, the control group had a larger number of tubes and a complete lumen. After 12 hours of Telekin treatment, compared with the control group, the number of HUVEC tubes gradually decreased, the length of the formed small tubes also gradually decreased, the number of vascular nodes (Nb junctions) also significantly decreased, and the lumen was incomplete. The experimental results show that Telekin can inhibit the tube-forming ability of HUVEC. Results are shown in Figure 7 , Figure 8 and Fig. 9 .

[0057] Example 3 Telekin affects HUVEC angiogenesis induced by tumor-conditioned medium

[0058] Prepare MDA-MB-231 cells, wait until the MDA-MB-231 cell density reaches about 60%, discard the waste liquid, rinse with 1×PBS three times, add 4 mL of serum-free medium and continue culturing for 24 hours; after 24 hours, aspirate the culture medium and place it in a centrifuge tube, centrifuge at 4°C and 8000 rpm for 10 minutes, and store at -80°C to avoid repeated freezing and thawing. In the HUVEC angiogenesis experiment, add conditioned medium, and the other steps are the same as Example 2.

[0059] To further explore whether Telekin has the ability to inhibit tumor angiogenesis in the tumor microenvironment, this example uses the conditioned medium of triple-negative breast cancer MDA-MB-231 cells to act on HUVEC. Through in vitro HUVEC angiogenesis experiments, it is found that compared with the control group, the tubules formed after the conditioned medium of the cells are more complete, the number of vascular nodes is more, and the length of the blood vessels is more complete. As the drug concentration of Telekin increases, the number of tubes decreases, the lumen becomes shorter, and even no tubules are formed. This shows that Telekin can still significantly inhibit the ability of HUVEC angiogenesis in vitro, and that Telekin can inhibit the ability of tumor angiogenesis in the tumor microenvironment of triple-negative breast cancer cells. Results are shown in Fig.10 , Fig.11 and Fig.12 .

[0060] Example 4 Effect of Telekin on angiogenesis of chick embryo chorioallantoic membrane in vivo

[0061] Experimental method: First, prepare SPF-grade fertilized eggs with uniform appearance, clean the egg shells in a low-temperature environment, wipe and disinfect with alcohol, and place the eggs with the big end facing up and the small end facing down in an incubator, maintain the temperature at 37.8°C, and control the humidity at 60% to 70%. Turn the eggs at least 3 times a day, with an interval of 6-8 hours between each turning of the eggs; (3) After incubation for 3 days, observe the hatching and growth of the eggs with an egg light, remove undeveloped fertilized eggs and damaged eggs, and continue to culture and incubate the remaining eggs, then select chicken embryos that have been incubated for 7 days, randomly group them, and use a surgical operating knife, a small electric drill and surgical forceps to create a hole with a diameter of about 1 cm × 1 cm in the air chamber of the chicken embryo. m small window, and then add a small amount of physiological saline to soften the shell membrane, and use sterile tweezers to gently peel off the outer membrane near the window to avoid puncturing the chorioallantoic membrane; cut the absorbable gelatin sponge into 2×2×2mm cubic particles with scissors, soak the gelatin sponge in drug solutions of different concentrations, fully absorb the drug solution, gently place the soaked gelatin sponge particles on the CAM, and seal with sterile cloth, mark the eggshell, put it back into the incubator and continue incubating for 48h, then take out the egg, carefully open it from the window with tweezers to completely expose the CAM, observe and photograph it under a stereo microscope after complete exposure, and analyze the acquired pictures with ImageJ software.

[0062] Results The effect of Telekin on the angiogenesis of the chick embryo chorioallantoic membrane showed that the control group had little effect on the growth of blood vessels, and a relatively complete vascular network developed on the chick embryo chorioallantoic membrane; with the increase in the dosage of Telekin, compared with the control group, the number of blood vessels in the dandelion polysaccharide group was significantly reduced, the growth of small and medium blood vessels and small capillaries was significantly inhibited, and the number of nodes between blood vessels was significantly less, which shows that Telekin can significantly inhibit the angiogenesis of the chick embryo chorioallantoic membrane in vivo. Fig.13 , Fig.14 and Fig.15 .

[0063] Example 5 Telekin inhibits the generation and development of fluorescently labeled blood vessels in zebrafish

[0064] This example uses wild-type zebrafish AB and transgenic vascular fluorescent zebrafish Tg (kdrl: EGFP). The zebrafish were paired and spawned one day before the experiment, and the fertilized eggs were collected the next day and incubated at a constant temperature in fish farming water; because the present example needs to collect zebrafish fluorescent vascular images, it is selected to add an appropriate amount of PTU at 4hpf (4h after fertilization) to inhibit the formation of embryonic melanin. Select well-developed 24hpf (24h after fertilization) fish embryos under a stereomicroscope, use an insulin injection needle to demembrane the zebrafish embryos, and then randomly group them and put them into a 24-well plate. The experiment sets up DMSO group, 25μmol / L Telekin, 50μmol / L Telekin, 100μmol / L Telekin, and 200μmol / L Telekin marking and then transfer to a light incubator. After the drug administration treatment, it is necessary to frequently observe and promptly remove dead fry to avoid water pollution affecting the statistical results. After 24h of development, the zebrafish are observed and photographed using a fluorescent inverted microscope. After collecting images, the growth of zebrafish intersegmental vessels (ISVs) was counted and the total length of zebrafish intersegmental vessels was measured.

[0065] The results showed that Telekin could significantly inhibit the growth of intersegmental blood vessels. As the concentration of Telekin increased, the length of intersegmental blood vessels gradually shortened and intersegmental blood vessels were missing. In the high-dose group, the zebrafish blood vessels almost disappeared and the embryos were deformed. This indicates that Telekin can inhibit the development of zebrafish embryonic blood vessels in vivo, further demonstrating that Telekin can inhibit tumor angiogenesis. Results are shown in Fig.16 .

[0066] Example 6 Effect of Telekin on the Signaling Pathway of Vascular Endothelial Growth Factor Production in HUVEC

[0067] When the HUVEC cell density reaches about 80%-90%, wash 3 times with pre-cooled PBS; then add 1mL 1×PBS to each well, scrape the cells, put the collected cells into a centrifuge tube, centrifuge at 800r / min for 5min, suck up the waste liquid, keep the cell pellet, take 1mL RIPA protein lysis buffer to melt, add 10μL of PMSF, a protease inhibitor, to each tube, add 100μL of lysis buffer to each tube, lyse on ice for 50min, then centrifuge at 12000r / min, 20min at 4℃ in a low-temperature centrifuge, then absorb the protein supernatant, discard the precipitate, take 2μL from each tube for BCA protein concentration determination; then calculate according to the protein concentration, add lysis buffer and 5×LoadingBuffer to make the protein concentration the same, finally heat denaturation in a 95℃ water bath for 10min, and store at -80℃.

[0068] Take the protein sample to melt, take the same volume of protein and add it to the sample well, slowly add the sample to the bottom of the well to avoid overflow and inconsistent protein amount. Electrophoresis: Turn on the power, run electrophoresis for 30 minutes at 80V; let the protein sample run to the separation gel; then adjust the voltage to 120V until the end of electrophoresis. Cut the appropriate NC membrane and soak it in the transfer buffer for 5 minutes in advance; then stack it on the black surface transfer plate in sequence, positive electrode-sponge pad-filter paper-NC membrane-gel-filter paper-sponge pad-negative electrode, keep the NC membrane flat, and use a glass rod to drive out bubbles, insert it into the transfer tank, and pay attention to the direction of the transfer plate. Place the transfer tank on ice, adjust the power supply, and transfer the membrane at a constant current of 180mA for 2.5h1. After the transfer, take out the NC membrane, put it in the blocking solution, and block it on a shaker at room temperature for 2h. The NC membrane was washed with PBST for 5 times, 5 min / time, and then placed in an incubation box, and diluted primary antibody was added, and shaken at 4°C overnight; after the incubation was completed, the primary antibody was recovered, and the NC membrane was washed with PBST for 5 times, 10 min each time, and the diluted secondary antibody was added, and incubated at room temperature for 60 min. ECL color development solution was prepared, and then evenly dropped on the surface of the NC membrane, and the image was developed in a dark room, and grayscale analysis was performed using Image J.

[0069] The results showed that Telekin could inhibit the protein expression of VEGFR2 and VEGFA in HUVEC cells, indicating that Telekin could significantly inhibit the vascular epidermal growth factor signaling pathway, which plays an important role in the formation and growth of blood vessels. During tumor angiogenesis, VEGF induces gene expression, regulates vascular permeability and promotes cell migration, proliferation and survival. The binding of VEGF ligands to their cognate membrane-bound receptors induces VEGF signaling, thereby activating multiple downstream pathways, suggesting that Telekin may further inhibit tumor angiogenesis by inhibiting this signaling pathway. The results are shown in Fig.17 , Fig.18 and Fig.19 .

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still make various modifications to the technical solution or replace some of the technical features with equivalents after reading this description. Any modification, equivalent replacement or improvement made within the spirit and principle of the present invention shall be deemed to fall within the protection scope of the present invention.

Claims

1. Application of telolactone in the preparation of drugs for inhibiting tumor angiogenesis.

2. The use according to claim 1, characterized in that The drug inhibits tumor angiogenesis by inhibiting the vascular endothelial growth factor signaling pathway.

3. The use according to claim 2, characterized in that The VEGF signaling pathway includes signal transduction mediated by VEGFA and VEGFR2.

4. The use according to claim 1, characterized in that The drug is used to treat angiogenesis-dependent tumors, including but not limited to triple-negative breast cancer, lung cancer, liver cancer and colorectal cancer.

5. The use according to claim 1, characterized in that The drug is used to inhibit angiogenesis induced by tumor microenvironment.

6. The use according to claim 1, characterized in that The drug can inhibit the proliferation, migration, and / or lumen formation ability of human umbilical vein endothelial cells.

7. The use according to claim 1, characterized in that The drug can reduce the number of blood vessel intersections in the chick embryo chorioallantoic membrane and inhibit blood vessel growth in the chick embryo chorioallantoic membrane model.

8. The use according to claim 1, characterized in that The drug can inhibit the vascular development of zebrafish embryos and reduce the formation of intersegmental blood vessels.

9. The use according to claim 1, characterized in that The effective dose of the tolerolactone is 10 μM to 30 μM.

10. The use according to claim 1, characterized in that The drug is administered orally, by injection or topical delivery.

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