Application of a bromophenol compound in inhibiting tumor angiogenesis

Abstract

This invention discloses the application of bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether (BTDE), a compound derived from marine algae, in inhibiting tumor angiogenesis. Experiments have confirmed that BTDE has no effect on the proliferation of human venous endothelial cells (HUVECs) in vitro, but significantly inhibits their migration and invasion. Further experiments demonstrate that BTDE can inhibit vascular mimicry in HUVECs in vitro, but has no effect on existing blood vessels. Furthermore, we found that BTDE inhibits the activity of matrix metalloproteinase 9 (MMP-9) in HUVECs, but does not affect the expression of AKT and ERK proteins or their phosphorylation levels. In vivo zebrafish embryo experiments showed that BTDE inhibits angiogenesis in the dorsal intersegmental region of zebrafish embryos. BTDE can be obtained from marine algae, is easily isolated and extracted, and can be prepared in large quantities via chemical synthesis, making it a very promising lead compound for anti-tumor angiogenesis.

Description

1. Technical Field

[0001] The present invention relates to the application of a novel marine algae-derived compound, bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether (BTDE), in treating tumor neovascularization, and belongs to the field of chemical medicine. 2. Background Technology

[0002] Cancer is the second leading cause of death worldwide and poses a serious threat to human life and health. At present, the number of cancer patients worldwide is increasing at a rate of 3%-5% per year and gradually shifting to younger people, and the cancer mortality rate is still on the rise. The concept that tumor growth depends on angiogenesis was proposed in the early 1970s. In the past decade, with the in-depth study of angiogenesis-related factors and signaling pathways, as well as the continuous exploration of the important effects of angiogenesis on tumor growth, infiltration and metastasis, especially on the early occurrence of tumors, tumor angiogenesis has become a major research hotspot in the treatment of cancer. Currently, the tumor treatment drugs on the market include anticancer drugs with angiogenesis as the main target. Such as epidermal growth factor inhibitor gefitinib, vascular endothelial growth factor, β-fibroblast growth factor inhibitor thalidomide and recombinant human endostatin endorphin. These drugs specifically target angiogenesis to exert anti-cancer effects. However, clinical data show that some angiogenesis inhibitors often lead to endothelial cell dysfunction and drug resistance. Seeking safer and more effective tumor angiogenesis inhibitors is still an important means of cancer treatment.

[0003] At present, many studies have shown that marine bromophenol compounds have good anti-angiogenesis effects in addition to anti-tumor, anti-diabetic, antioxidant and antiviral activities. For example, the bromophenol compounds bis-(2,3-dibromo-4,5-dihydroxyphenyl) methane and bis-(2,3-dibromo-4,5-dihydroxybenzyl) ether derived from the red alga Rhodomela confervoides exert anti-angiogenesis effects by targeting multiple receptor kinases and VEGF signaling pathways. Therefore, searching for lead compounds with anti-angiogenesis activity from marine bromophenols is a promising strategy for the development of anti-tumor angiogenesis agents.

[0004] BTDE is a bromophenol compound isolated from the red alga Symphyocladia latiuscula. Recent studies have shown that BTDE has good antioxidant, anti-diabetic, neurodegenerative disease inhibition and multiple enzyme inhibition effects, but its role in angiogenesis has not been reported. Therefore, we studied the effects of BTDE on the migration, invasion and tubulogenesis of human umbilical vein endothelial cells (HUVECs); and its effects on angiogenesis in the dorsal intersegment of zebrafish embryos, attempting to evaluate the anti-angiogenic effect of BTDE both in vivo and in vitro.

[0005] Before the present invention, there was no application of BTDE in inhibiting tumor angiogenesis. BTDE can be separated and extracted from red algae, is easy to obtain, can inhibit HUVECs migration, invasion and angiogenesis, is easy to synthesize, and is a very promising anti-tumor angiogenesis drug.

[0006] Bis(2,3,6-tribromo-4,5-dihydroxybenzyl)ether(BTDE) chemical structure Figure 1 . III. Summary of the invention

[0007] One of the objects of the present invention is to discover the anti-angiogenic effect of BTDE.

[0008] The second purpose of the present invention is to explore the effects of BTDE on the proliferation, migration and invasion of HUVECs in vitro.

[0009] The third purpose of the present invention is to explore the effects of BTDE on HUVECs vascular mimicry and existing blood vessels in vitro.

[0010] The fourth purpose of the present invention is to explore the effect of BTDE on the activity of matrix metalloproteinases in HUVECs.

[0011] The fifth purpose of the present invention is to explore the effect of BTDE on the angiogenesis of dorsal intersegmental zebrafish embryos in vivo.

[0012] To achieve the above object, the technical solution adopted by the present invention is:

[0013] The MTT technique was used to detect the effect of BTDE on the proliferation of human umbilical vein endothelial cells (HUVECs).

[0014] Flat scratch and Transwell migration techniques were used to detect the effect of BTDE on the migration of HUVECs in vitro.

[0015] Transwell invasion technique was used to detect the effect of BTDE on the invasion of HUVECs.

[0016] Tube formation technology was used to detect the effects of BTDE on HUVECs vascular mimicry and existing blood vessels.

[0017] Gelatin zymography was used to detect the effect of BTDE on the activity of matrix metalloproteinases in HUVECs.

[0018] Tg(flk1:EGFP) transgenic zebrafish were used to detect the effect of BTDE on angiogenesis in the dorsal intersegment of zebrafish embryos in vivo.

[0019] Compared with the prior art, the advantages and technical effects of the present invention are as follows: the present invention proves the anti-angiogenic effect of BTDE in vitro and in vivo, and finds that it has an inhibitory effect on the migration, invasion and vascular mimicry of HUVECs in vitro, and can inhibit the angiogenesis of the dorsal intersegment of zebrafish embryos in vivo. We further found that BTDE can inhibit the activity of matrix metalloproteinase 9 in HUVECs. All these indicate that BTDE has a good inhibitory effect on angiogenesis and has broad application prospects in anti-tumor angiogenesis. IV. Description of the drawings

[0020] Figure 1 The chemical structure of BTDE in the present invention is shown.

[0021] Figure 2 This indicates that BTDE in the present invention can inhibit the migration and invasion of HUVECs in vitro, but does not affect the proliferation of HUVECs.

[0022] Figure 3 This indicates that BTDE in the present invention can inhibit the formation of HUVECs vascular mimicry in vitro, but has no effect on the existing blood vessels of HUVECs.

[0023] Figure 4 This indicates that BTDE in the present invention can inhibit the activity of matrix metalloproteinase-9 in HUVECs, but does not affect the background expression of AKT and ERK protein molecules and their phosphorylation levels.

[0024] Figure 5 This indicates that BTDE in the present invention can inhibit the angiogenesis of dorsal intersegmental zebrafish embryos in vivo. V. Specific implementation methods

[0025] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and test examples.

[0026] Test Example 1

[0027] The effects of BTDE on the proliferation, migration and invasion of human umbilical vein endothelial cells (HUVECs) in vitro were investigated.

[0028] Cells and materials: Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences.

[0029] Reagents: BTDE, MTT, doxorubicin, Matrigel, DMSO, Transwell chamber, 0.1% crystal violet staining solution, and 33% acetic acid.

[0030] Instruments: CO2 incubator; inverted microscope; microplate reader; clean bench.

[0031] Experimental method MTT method: HUVECs in the logarithmic growth phase were inoculated into 96-well plates. After the cells adhered, different concentrations of BTDE were added to the experimental group, and DMSO diluted in equal proportion was added to the control group. Adriamycin was set as the positive control. The plates were placed in an incubator at 37°C and 5% CO2 for 36 hours. After the incubation, 20 μL of MTT solution was added to each well and the plates were incubated in the incubator for another 4 hours. After the incubation, the supernatant was discarded, 150 μL of DMSO was added to each well, and the plates were incubated in a 37°C oven for 10 minutes. The absorbance was measured at a wavelength of 570 nm using an ELISA reader to calculate the cell viability.

[0032] Experimental method: Plate scratch method: HUVECs in logarithmic growth phase were taken, 1×10 4 Cells were inoculated in a 96-well plate and cultured in an incubator at 37°C and 5% CO2. After reaching 90% confluence, a 10μL pipette tip was placed vertically on the bottom of the plate to draw a line in the center of each well. The cells were washed twice with PBS and replaced with a culture medium containing 1% FBS. An inverted biological microscope was used to take photos at 0h (4×). After that, different concentrations of BTDE were added to the experimental group, and the same amount of sterile water was added to the control group. The plates were placed in an incubator at 37°C and 5% CO2 for 36h and then photographed. The images were processed with Image J software to calculate the scratch healing rate.

[0033] Experimental method Transwell migration method: HUVECs in logarithmic growth phase were taken and the cell density was adjusted to 1×10 5 / mL, resuspended in DMEM containing 1% FBS and different concentrations of BTDE, and 100μL volume was seeded in the Transwell chamber. 500μL of DMEM containing 10% FBS and different concentrations of BTDE was added to the 24-well plate. The chamber was gently placed in the 24-well plate to avoid bubbles, and placed in an incubator at 37°C and a CO2 concentration of 5% for 24 hours. After the culture was completed, the culture medium in the chamber was discarded, washed three times with PBS, and the cells were fixed with methanol for 30 minutes. The fixative was discarded, washed three times with PBS, and the chamber was inverted to allow the membrane to air dry properly. 0.1% crystal violet staining was performed for 10 minutes, the staining solution was discarded, and PBS was washed three times. A cotton swab was gently wiped on the upper surface of the chamber without migrating cells, and a random field of view was selected for photography under an inverted biological microscope (10×). After the photography was completed, 33% acetic acid was used for decolorization, and the decolorization solution of each well was aspirated, and the absorbance was measured at a wavelength of 570nm using an enzyme reader.

[0034] Experimental Methods Transwell invasion method: Matrigel (200 μg / mL) was spread in the Transwell chamber and placed in a 37°C incubator for 1 hour to allow it to solidify into a thin layer. Subsequent experimental steps were the same as those of the Transwell migration method.

[0035] according to Figure 2The results showed that BTDE could inhibit HUVECs migration and invasion. 2.5-10μM BTDE treatment of HUVECs cells for 36h had no effect on HUVECs proliferation, but significantly inhibited its migration, with scratch healing rates of 67.7, 59.1 and 55.2%, respectively. At the same time, the Transwell migration experiment also proved that after 24h of action of different concentrations of BTDE, HUVECs migration was significantly inhibited. The results of the Transwell invasion experiment showed that after 24h of BTDE action on HUVECs cells, the number of HUVECs that penetrated the matrix gel and invaded to the lower surface of the membrane decreased, indicating that BTDE inhibited HUVECs invasion in a concentration-dependent manner. The above results show that BTDE does not affect HUVECs proliferation at a concentration of 2.5-10μM, but inhibits HUVECs migration and invasion.

[0036] Test Example 2

[0037] The effects of BTDE on HUVECs cell vascular mimicry and existing blood vessels were investigated.

[0038] Cells and materials: Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences.

[0039] Reagents: BTDE; Matrigel.

[0040] Instruments: CO2 incubator; inverted microscope; clean bench.

[0041] Experimental Methods Tube formation experiment: Pre-cooled 96-well plates were coated with Matrigel (10 mg / mL), 50 μL per well, and placed at 37°C for 45 min to solidify. HUVECs were pre-treated with different concentrations of BTDE for 24 h, and 1×10 4 The cells were seeded in a 96-well plate covered with gel and cultured in a 37°C incubator with a CO2 concentration of 5% for 20 h. Random fields of view were selected for photography using an inverted biological microscope (4×), and the total length of blood vessels was generated by Image J software analysis.

[0042] Experimental Methods: Pre-cooled 96-well plates were plated with Matrigel (10 mg / mL), 50 μL per well, and placed at 37°C for 45 min to solidify. HUVECs were plated at 1×10 4 The cells were seeded in a 96-well plate covered with gel and cultured in an incubator at 37°C and 5% CO2 for 8 hours to allow tubular structures to form. They were then treated with different concentrations of BTDE. After 6 hours, random fields of view were selected for photography under an inverted biological microscope (4×), and the total length of blood vessels was generated by analysis with Image J software.

[0043] according to Figure 3The experimental results showed that BTDE significantly inhibited the formation of HUVECs vascular mimicry, but had no effect on existing blood vessels. At a concentration of 2.5-10μM, the total length of blood vessels generated was 58.1, 36.3 and 4.9% compared with the control group. In addition, the experimental results of the effect of BTDE on the existing blood vessels of HUVECs showed that BTDE did not affect the tubular structure that had been formed by HUVECs. The above results show that BTDE can inhibit the formation of HUVECs vascular mimicry in vitro, but has no effect on existing blood vessels.

[0044] Test Example 3

[0045] The effects of BTDE on the activity of matrix metalloproteinases and the expression of AKT and ERK protein molecules in HUVECs cells were investigated.

[0046] Cells and materials: Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences.

[0047] Reagents: BTDE; protein electrophoresis buffer; transfer buffer; 1× Loading Buffer; 4× upper layer stacking gel buffer; 4× lower layer separation gel buffer; gelatin; 2.5% Triton X-100; 1μM ZnCl2 / 50mM Tris-HCl; 0.05% Coomassie Brilliant Blue R250 staining solution; 30% methanol / 10% acetic acid decolorizing solution; APS; nitrocellulose membrane; protein pre-stained marker; ECL luminescence kit;

[0048] Instruments: CO2 incubator; clean bench; high-speed refrigerated centrifuge; protein electrophoresis instrument; shaker; ST-Ⅱ transfer electrophoresis tank; FluorChemE multifunctional imaging analysis system; Bio-Red gel imaging analysis system.

[0049] Experimental Methods Gelatin zymography: HUVECs cells in the logarithmic growth phase were resuspended in DMEM and seeded in 6-well plates (8.5×10 cells per well). 5Cells) were collected and treated with different concentrations of BTDE for 24h. The supernatant was collected and centrifuged at 1200rpm for 5min at 4℃, then centrifuged at 12000rpm for 5min at 4℃, the cells and debris in the supernatant were removed, and stored at -80℃. Protein samples were collected by Western Blot method for adherent cells to detect internal reference. The protein components in the supernatant were separated by electrophoresis in 8% SDS-PAGE gel containing 1mg / mL gelatin. After the electrophoresis, the protein gel was washed with 2.5% Triton X-100 and placed in 1μM ZnCl2 / 50mM Tris-HCl incubation solution and incubated at 37℃ on a shaker for 48h. Stain with 0.05% Coomassie Brilliant Blue R250 dye solution for 30min, decolorize with 30% methanol / 10% acetic acid decolorizer until obvious negative staining bands appear, and develop and take pictures using the Bio-Red gel imaging analysis system.

[0050] Experimental Method Western Blot Method: HUVECs cells in the logarithmic growth phase were seeded in 6-well plates (1.5×10 5 cells) and treated with different concentrations of BTDE for 24h. Then the cells were collected, washed twice with PBS, and lysed with 1× Loading Buffer at 4℃ for 45min. After lysis, boiled for 15min and stored at -20℃. Protein samples were separated by electrophoresis on 8% SDS-PAGE gel and transferred to nitrocellulose membrane (NC membrane). After cutting the target band, the NC membrane was sealed with 5% skim milk for 1h and incubated with the primary antibody of the target band at 4℃ overnight. The NC membrane was then incubated with the secondary antibody at room temperature for 1h, and chemiluminescence detection was performed by a developer.

[0051] according to Figure 4 The experimental results showed that BTDE inhibited the activity of matrix metalloproteinase 9 in HUVECs cells. Matrix metalloproteinase, as a protease that can degrade the extracellular matrix, plays an important role in endothelial cell migration, invasion and angiogenesis. The results of gelatin zymography experiments showed that BTDE could inhibit the activity of matrix metalloproteinase 9 in HUVECs cells. AKT and ERK protein molecules are key signaling molecules that regulate cell growth, differentiation and angiogenesis. Western Blot experimental results showed that BTDE did not affect the background expression of AKT and ERK protein molecules and their phosphorylation levels in HUVECs cells. The above results show that BTDE inhibits the activity of matrix metalloproteinase 9 in HUVECs cells, but has no effect on the background expression of AKT and ERK protein molecules and their phosphorylation levels.

[0052] Test Example 4

[0053] The effects of BTDE on dorsal intersegmental angiogenesis in zebrafish embryos were investigated in vivo.

[0054] Cells and materials: Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Tg(flk1:EGFP) transgenic zebrafish embryos were donated by Shanghai Jiao Tong University and preserved and passaged by the Laboratory of Molecular Medical Biology, Ocean University of China.

[0055] Reagents: BTDE; DMSO; tricaine solution; 2% methylcellulose gel

[0056] Instruments: fully automatic water circulation system; embryo incubator; clean bench; DM6000 inverted fluorescence microscope.

[0057] Experimental method: Zebrafish embryo method: Take healthy and mature Tg (flk1: EGFP) heterozygous fish and wild-type fish and place them on both sides of the fish box. After waiting for a period of time, harvest the embryos and incubate them at a constant temperature of 28.5 ° C in the embryo incubator. When the embryos develop to 16 hpf, select healthy and full embryos with exactly the same developmental period and transfer them to 24-well culture plates, with 10 embryos per well. Different concentrations of BTDE were added to the drug-treated group, and DMSO was diluted in equal proportion to the control group, and then placed in the embryo incubator for further culture for 24 hours. After the culture, the embryos were aspirated and placed in a 96-well plate, and individuals with vascular fluorescence were selected, the membranes were peeled, and anesthetized with an appropriate amount of tricaine solution. They were placed in a 2% methylcellulose gel and placed in a correct position. They were observed and photographed under an inverted fluorescence microscope. The images were processed with Image J software to calculate the length of the dorsal intersegmental blood vessels of each group of zebrafish embryos.

[0058] according to Figure 5 The results showed that BTDE inhibited the angiogenesis of dorsal intersegments of zebrafish embryos in vivo. The dorsal intersegmental blood vessels of zebrafish embryos in the control group were evenly arranged longitudinally and intact; under the treatment of 2.5-10μΜ BTDE, the dorsal intersegmental blood vessels were obviously missing, and the length of the blood vessels was 90.1, 40.3 and 31.2% compared with the control group, which shows that BTDE can significantly inhibit the angiogenesis of dorsal intersegments of zebrafish embryos.

Claims

1. Use of bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether as a sole ingredient in the preparation of a drug for inhibiting tumor angiogenesis, characterized in that: Its chemical structure is: 。 2. The use according to claim 1, characterized in that: The bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether can effectively inhibit the migration of human umbilical vein endothelial cells in vitro.

3. The use according to claim 1, characterized in that: The bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether can effectively inhibit the invasion of human umbilical vein endothelial cells in vitro.

4. The use according to claim 1, characterized in that: The bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether can effectively inhibit the vascular mimicry generation of human umbilical vein endothelial cells in vitro.

5. The use according to claim 1, characterized in that: The bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether can inhibit the activity of matrix metalloproteinase 9 in human umbilical vein endothelial cells.

6. The use according to claim 1, characterized in that: The bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether can inhibit the angiogenesis of the dorsal intersegment of zebrafish embryos in vivo.

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