Application of heparin oligosaccharide in preparation of antitumor drugs

文档序号：706587 发布日期：2021-04-16 浏览：22次中文

阅读说明：本技术 一种肝素寡糖在制备抗肿瘤药物中的应用 (Application of heparin oligosaccharide in preparation of antitumor drugs ) 是由何书英薛金冰张通于 2021-01-04 设计创作，主要内容包括：本发明涉及一种肝素寡糖在制备抗肿瘤药物中的应用,属于生物医药技术领域。本发明研究了肝素寡糖抑制肿瘤发展的潜在价值及机制,结果显示肝素寡糖的抗肿瘤作用是通过其抑制生长因子诱导细胞表面受体而抑制黏附分子的表达从而发挥作用的。具体是通过血管内皮生长因子模拟肿瘤内环境刺激人脐静脉内皮细胞建立细胞异常模型,通过细胞黏附和qPCR等实验研究肝素寡糖抗肿瘤作用及机制,结果显示肝素寡糖抑制乳腺癌细胞对内皮细胞的黏附,且肝素寡糖下调细胞表面受体VEGFR2的mRNA和蛋白表达水平,同时下调细胞表面黏附分子E-selectin和ICAM-1的mRNA和蛋白表达水平,表明VEGFR2是肝素寡糖抗肿瘤活性的重要靶点之一。此研究可为治疗肿瘤生长转移提供新的途径。(The invention relates to application of heparin oligosaccharide in preparation of antitumor drugs, and belongs to the technical field of biological medicines. The invention researches the potential value and mechanism of inhibiting tumor development of heparin oligosaccharide, and the result shows that the anti-tumor effect of heparin oligosaccharide is realized by inhibiting the expression of adhesion molecules through inhibiting growth factor to induce cell surface receptors. Specifically, a vascular endothelial growth factor is used for simulating the internal environment of a tumor to stimulate human umbilical vein endothelial cells to establish a cell abnormality model, and the anti-tumor effect and mechanism of heparin oligosaccharide are researched through experiments such as cell adhesion and qPCR (quantitative polymerase chain reaction), so that the result shows that the heparin oligosaccharide inhibits the adhesion of breast cancer cells to the endothelial cells, the mRNA and protein expression level of a cell surface receptor VEGFR2 is down regulated by the heparin oligosaccharide, the mRNA and protein expression level of cell surface adhesion molecules E-selectin and ICAM-1 is down regulated, and the VEGFR2 is one of important targets of the anti-tumor activity of the heparin oligosaccharide. The research can provide a new way for treating tumor growth and metastasis.)

1. The application of the heparin oligosaccharide in preparing the anti-tumor medicine is characterized in that the anti-tumor medicine is a medicine capable of inhibiting the growth and/or metastasis of tumor cells.

2. The use according to claim 1, wherein the heparin oligosaccharide is heparin dodecasaccharide.

3. Use according to claim 1, characterized in that the heparin oligosaccharide is heparin decadisaccharide with a molecular weight of 3200D.

4. The use of claim 1, wherein the heparin decadisaccharide is heparin decadisaccharide according to structural formula i:

5. the use of claim 1, wherein said antineoplastic agent exerts an antineoplastic effect through heparin oligosaccharides as VEGFR2 inhibitors.

6. The use according to claim 1, wherein the anti-tumor medicament exerts an anti-tumor effect by inhibiting the expression of vascular endothelial growth factor-induced endothelial cell adhesion molecules through heparin oligosaccharides.

7. An anti-tumor pharmaceutical composition is characterized in that heparin oligosaccharide is used as an active ingredient of the pharmaceutical composition, and a pharmaceutically acceptable carrier is used as an auxiliary ingredient of the pharmaceutical composition.

8. The antitumor pharmaceutical composition of claim 7, wherein said antitumor pharmaceutical composition is capable of inhibiting tumor cell growth and/or metastasis.

Technical Field

The invention belongs to the field of biological medicine, relates to heparin oligosaccharide with the molecular weight of 3200D, has the effect of inhibiting adhesion of breast cancer cells and endothelial cells, and provides application of the heparin oligosaccharide in preparing an anti-tumor medicine.

Background

Research finds that heparin and low molecular weight heparin can prolong the survival period of cancer patients and have obvious anti-tumor metastasis effect in animal bodies, and the anti-tumor activity of the heparin is independent of the anticoagulant activity of the heparin; the target organ in the body is vascular endothelium, and the tumor growth and metastasis are inhibited through the interaction of tumor-related factors such as selectin, heparinase, basic fibroblast growth factor (bFGF) and Vascular Endothelial Growth Factor (VEGF). The growth of tumor cells depends on the proliferation and migration of vascular endothelial cells, and the metastasis of tumor cells depends on the adhesion of tumor cells and vascular endothelial cells, which are related to the expression of vascular endothelial cell adhesion molecules. Thus, inhibition of vascular endothelial cell adhesion molecule expression may be a potential therapeutic strategy for inhibiting the growth and metastasis of malignant solid tumors.

However, heparin exerts antitumor activity and also brings about side effects such as bleeding and thrombocytopenia, so that a treatment mode for inhibiting tumor growth and metastasis and avoiding the side effects such as bleeding and thrombocytopenia is urgently needed, and a reference is provided for developing a blood vessel targeting heparin oligosaccharide antitumor drug.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to research the mechanism of inhibiting the adhesion of endothelial cells and tumor cells by heparin oligosaccharide and provide the application of the heparin oligosaccharide in preparing anti-tumor medicaments.

The purpose of the invention is realized by the following technical scheme:

the first purpose of the invention is to provide the application of the heparin oligosaccharide in preparing the anti-tumor medicine, wherein the anti-tumor medicine is a medicine capable of inhibiting the growth and/or metastasis of tumor cells.

Further, the heparin oligosaccharide is heparin dodecasaccharide, and the molecular weight of the heparin oligosaccharide is 3200D.

Further, the heparin decabiose is heparin decabiose shown in a structural formula I:

furthermore, the antitumor drug can play an antitumor role, namely, the role of inhibiting the growth and/or metastasis of tumor cells by using heparin oligosaccharide as a VEGFR2 inhibitor.

Furthermore, the antitumor drug can inhibit the expression of endothelial cell adhesion molecules induced by vascular endothelial growth factor through heparin oligosaccharide, thereby playing an antitumor role, namely inhibiting the growth and/or metastasis of tumor cells.

Furthermore, the heparin oligosaccharide is used as a VEGFR2 inhibitor, inhibits the interaction between Vascular Endothelial Growth Factor (VEGF) and VEGFR2, and further inhibits the expression of endothelial cell surface adhesion molecules, so that the antitumor drug has an antitumor effect.

The second purpose of the invention is to provide an anti-tumor pharmaceutical composition, which takes heparin oligosaccharide as an active ingredient and is assisted by a pharmaceutically acceptable carrier.

Furthermore, the anti-tumor drug is a drug capable of inhibiting the growth and/or metastasis of tumor cells.

Further, the heparin oligosaccharide is heparin dodecasaccharide, and the molecular weight of the heparin oligosaccharide is 3200D.

Further, the heparin decabiose is the heparin decabiose shown in the structural formula I.

Furthermore, the antitumor drug can play an antitumor role, namely, the role of inhibiting the growth and/or metastasis of tumor cells by using heparin oligosaccharide as a VEGFR2 inhibitor.

Further, the anti-tumor medicine composition can inhibit the growth and/or metastasis of tumor cells.

Furthermore, the anti-tumor medicine can be prepared into various preparations required clinically.

Compared with the prior art, the technical scheme of the invention has the beneficial effects that:

the invention researches a mechanism for inhibiting the adhesion of endothelial cells and tumor cells by heparin oligosaccharide, adopts heparin oligosaccharide with different concentrations to act on HUVEC of human umbilical vein endothelial cells, selects VEGF growth factors with the concentration of 10ng/mL to stimulate the endothelial cells to establish a cell abnormality model, and finds that the heparin oligosaccharide can inhibit the adhesion of the endothelial cells and the tumor cells in a dose-dependent manner, remarkably down-regulates the mRNA and protein expression level of a vascular endothelial growth factor receptor VEGFR2, and simultaneously down-regulates the mRNA and protein expression levels of cell surface adhesion molecules E-selectin and ICAM-1. The research can provide a new way for treating tumor growth and metastasis caused by blood vessel abnormality.

In the research, the heparin oligosaccharide can be obtained through a conventional channel, and Chinese patent No. CN201010139398.9 with the patent name: a heparin dodecasaccharide, its preparation method and its use for resisting vascular smooth muscle cell proliferation; the method for producing heparin oligosaccharide as described in (1).

The heparin dodecasaccharide with the molecular weight of about 3200D has an inhibition effect on the proliferation action of Vascular Endothelial Growth Factor (VEGF) promoting vascular endothelial cells, and can simultaneously reduce the expression levels of cell surface adhesion molecules E-selectin and ICAM-1, so that the anti-tumor effect of inhibiting tumor growth and metastasis is realized, VEGFR2 is one of important targets of anti-tumor activity of heparin oligosaccharide, and the heparin dodecasaccharide has the advantages of high bioavailability, lower bleeding tendency, less thrombocytopenia and the like, thereby being helpful for explaining the anti-tumor metastasis mechanism of the heparin oligosaccharide and providing a reference for developing anti-tumor drugs of heparin oligosaccharide targeting blood vessels.

Drawings

FIG. 1 is a data statistical analysis result chart of MTT experiment for determining the influence of heparin oligosaccharide on HUVEC cell proliferation

FIG. 2 is a cell diagram of the cell adhesion experiment for determining the effect of heparin oligosaccharide on the adhesion of HUVEC cells and tumor cells

FIG. 3 is a graph showing the result of statistical analysis of the data on the number of tumor cells adhered in each experimental group in the cell adhesion experiment

FIG. 4 is a data analysis chart of the Cell-based ELISA experiment for determining the effect of heparin oligosaccharide on VEGFR2 protein expression

FIG. 5 is a diagram of statistical analysis of data for determining the effect of heparin-oligosaccharide on the expression of E-selectin protein on the surface of HUVEC cells in Cell-based ELISA assay

FIG. 6 is a data statistical analysis chart of the effect of heparin oligosaccharide on HUVEC Cell surface ICAM-1 protein expression determined by Cell-based ELISA experiment

FIG. 7 is a graph of data statistical analysis of the qPCR assay to determine the effect of heparin oligosaccharides on VEGFR2 mRNA levels

FIG. 8 is a graph of data statistical analysis of the effect of qPCR experiments on E-selectin mRNA levels by heparin oligosaccharides

FIG. 9 is a graph of data statistical analysis for the qPCR experiment to determine the effect of heparin oligosaccharides on ICAM-1mRNA levels

Detailed Description

VEGFR2 described in the examples below refers to vascular endothelial growth factor receptor 2;

the E-Selectin in the following examples refers to E-Selectin;

ICAM-1 in the following examples refers to intercellular adhesion molecule 1;

in the following examples, HDO refers to heparin oligosaccharide, specifically heparin decadisaccharide (molecular weight of about 3200D) represented by structural formula i. Structural formula I:

in the following examples, LMWH refers to low molecular weight heparin, specifically, dalteparin sodium (molecular weight of about 6000D) represented by structural formula ii.

The following are examples in which Vorolanib refers to a tyrosine kinase inhibitor, specifically, as shown in structural formula iii:

example 1 preparation of heparin oligosaccharides

Step 1: weighing 1g of heparin sodium, dissolving the heparin sodium with distilled water to prepare a solution of 2% (w/v), adding 0.25g of sodium nitrite, adjusting the pH to 2.8 with hydrochloric acid to prepare a degradation solution, putting the degradation solution into a shaking table for reaction at 25 ℃, at 150rpm/min for 4h, measuring the pH at intervals during the reaction, and adjusting the pH to 2.8. Adjusting pH to 6-9 with sodium hydroxide after reaction to obtain heparin oligosaccharide crude product liquid, vacuum rotary steaming at 50 deg.C, and freeze drying.

Step 2: dissolving the above lyophilized powder with mobile phase, separating with Gel chromatography column (Bio-Gel P6, 1.0cm × 100cm) at flow rate of 0.3mL/min for 5min with mobile phase of 0.2mol/L NaCl.

And step 3: and (3) uniformly mixing 10 mu L of the collected liquid with 40% sucrose solution with the same volume, and carrying out electrophoresis by using bromophenol blue as an indicator. The concentration of the electrophoresis gel is 10% of separation gel and 5% of concentrated gel, and electrophoresis is carried out for 1 hour at 100V. After the electrophoresis was completed, the gel was stained with 0.25% (w/v) Ali-New blue staining solution for 30min, and eluted with 1% (v/v) acetic acid until the background was colorless. Observing the molecular weight distribution, selecting the components with the molecular weight of about 3200, and performing rotary evaporation and freeze drying to obtain the heparin dodecasaccharide shown in the structural formula I.

Before use, the prepared heparin decadisaccharide was dissolved in distilled water and the concentration was determined for use in the subsequent examples.

Example 2 MTT assay to examine the Effect of heparin oligosaccharides on HUVEC cell proliferation

CulturingHuman umbilical vein endothelial cells (HUVEC cells, source: ATCC) were fused to 90%, digested with 0.25% (w/v) pancreatin containing 0.02% (w/v) EDTA, mixed well and added to a 96-well plate at 100. mu.L per well of 5X 10⁴The cells were cultured at 37 ℃ for 24 hours in a cell suspension of one cell/ml. The cells were divided into a blank group (Control), a Model group (Model containing 10ng/mL VEGF), and HDO groups (0.01. mu.M, 0.1. mu.M, 1. mu.M, 10. mu.M, to which 10ng/mL VEGF was added, respectively) of different concentrations, each group was set with 6 duplicate wells, and the administration medium of each group was prepared with a DMEM medium containing 0.5% (v/v) FBS, and cultured at 37 ℃ for 24 hours. 20 mu L of MTT solution with the concentration of 5mg/mL is added into each well, the mixture is incubated at 37 ℃ for 4h, the culture medium is carefully sucked out, 150 mu L of DMSO is added into each well, crystals are dissolved by shaking a shaker for 10min, and the crystals are uniformly mixed and then the absorbance value is measured at 570 nm. The data obtained were statistically analyzed using Graph Pad Prism 5.0 software, and the results of quantitative analysis are shown in FIG. 1.

The results show that: HUVEC cells are added with 10ng/mL VEGF factor to stimulate, and then the proliferation of endothelial cells is remarkably promoted; and after the heparin oligosaccharide with different doses in the range of 0.01-10 mu M acts, the HUVEC cell proliferation induced by VEGF is inhibited in a dose-dependent mode.

Example 3 cell adhesion experiment to detect the Effect of heparin-oligosaccharide on the adhesion of HUVEC cells and tumor cells

Step 1: culturing human umbilical vein endothelial cells (HUVEC cells, source: ATCC) to 90% confluence, digesting with 0.25% (w/v) pancreatin containing 0.02% (w/v) EDTA, mixing well, adding 100. mu.L of 5X 10 per well of 96-well plate⁴Cell suspension at 37 ℃ overnight to 80% confluence. The cells were divided into a blank group (Control), a Model group (Model containing 10ng/mL VEGF), HDO groups of different concentrations (0.01. mu.M, 0.1. mu.M, 1. mu.M, to which 10ng/mL VEGF was added, respectively), LMWH group (0.1. mu.M LMWH +10ng/mL VEGF), and Vorolanib group (40nM Vorolanib +10ng/mL VEGF), each group was set with 6 duplicate wells, and the administration medium of each group was prepared with DMEM medium containing 0.5% (v/v) FBS, and cultured at 37 ℃ for 24 hours.

Step 2: after 23h administration, breast cancer (MDA-MB-231 cells, source: ATCC) was digested and centrifuged to obtain a cell pellet, 1ml of calcein AM was resuspended (in dark conditions), and incubated at 37 ℃ for 30 min. After incubation, the cells were resuspended in serum-free medium preheated at 37 ℃ after centrifugationCentrifugation and repetition twice, and finally adjustment of cell density to 1X 10 with serum-free medium preheated at 37 ℃⁴And obtaining the cell suspension of breast cancer MDA-MB-231.

And step 3: the 96-well plate was discarded from the old medium (i.e., DMEM medium containing 0.5% (v/v) FBS in step 1 to prepare each group of dosing medium), and 100. mu.L of the breast cancer MDA-MB-231 cell suspension treated in step 2 was added to each well and co-cultured at 37 ℃ for 1 hour. The breast cancer MDA-MB-231 cell suspension is discarded, PBS is reserved after being washed for 2 times, and the cell suspension is photographed in a dark place. After cell counting, the data obtained were analyzed using Graph Pad Prism 5.0 software and the results of quantitative analysis are shown in FIGS. 2 and 3. FIG. 2a is the graph of blank group tumor cell adhesion to endothelial cell, FIG. 2b is the graph of model group tumor cell adhesion to endothelial cell, FIG. 2c-e are the graph of HDO group tumor cell adhesion to endothelial cell at different concentrations, FIG. 2f is the graph of LMWH group tumor cell adhesion to endothelial cell, and FIG. 2g is the graph of Vorolanib group tumor cell adhesion to endothelial cell.

The results show that: the HUVEC cells are added with 10ng/mL VEGF factor to stimulate, so that the adhesion to the breast cancer cells is obviously increased, and the adhesion capability of the HUVEC cells is inhibited compared with that of a model group after the action of heparin oligosaccharide with different concentrations in the range of 0.01-1 mu M, and the HUVEC cells have dose dependence; after 0.1 μ M LMWH, the effect of inhibiting cell adhesion was present but not as good as that of the same concentration of heparin oligosaccharide after the action.

Example 4 Cell-based ELISA to examine the Effect of heparin oligosaccharides on the expression of VEGFR2, E-Selectin and ICAM-1 proteins in HUVEC

Step 1: culturing human umbilical vein endothelial cells (HUVEC cells, source: ATCC) to 90% confluence, digesting with 0.25% (w/v) pancreatin containing 0.02% (w/v) EDTA, mixing well, adding 100. mu.L of 5X 10 per well of 96-well plate⁴Each/mL cell suspension was cultured at 37 ℃ overnight to 80% confluence, and the cells were divided into a blank group (Control), a Model group (Model containing 10ng/mL VEGF), HDO groups (0.01. mu.M, 0.1. mu.M, 1. mu.M, to which 10ng/mL VEGF was added), an LMWH group (0.1. mu.M LMWH +10ng/mL VEGF), and a Vorolanib group (40nM Vorolanib +10ng/mL VEGF), each group was provided with 6 duplicate wells, and incubated for 24h by group administration.

Step 2: by usingWashing with PBS, drying, adding 4% (w/v) paraformaldehyde, and fixing at room temperature for 20-30 min. Washing with PBS-Triton X-100 for 5min × 3 times, adding 0.6% (v/v) H₂O₂Incubation with PBS-Triton X-100 for 20-30 min. Adding PBS-Triton X-100 for rinsing, 5min × 3 times, adding 100 μ L10% (w/v) BSA, and blocking at 37 deg.C for 100 min. The samples were divided into three groups, and 50 μ L of 5% (w/v) BSA diluted VEGFR2 primary antibody was added to the first 96-well plate and incubated overnight at 4 ℃. A second set of 96-well plates was incubated overnight at 4 ℃ with 50. mu.L of 5% (w/v) BSA diluted primary E-Selectin. A third set of 96-well plates was incubated overnight at 4 ℃ with 50. mu.L of ICAM-1 primary antibody diluted with 5% (w/v) BSA.

And 3, after the incubation of each group in the step 2 is finished, discarding the liquid, washing the solution by PBS-Triton X-100 for 5min multiplied by 3 times, washing the solution by PBS for 5min multiplied by 3 times, and finally adding 200 mu L of TMB color development liquid into each hole to develop the color for 30min in a dark place at 37 ℃. Add 50. mu.L of 1M H per well₂SO₄The reaction was terminated by the stop solution, and the absorbance at 450nm was immediately measured. The results of quantitative analysis using the data obtained with Graph Pad Prism 5.0 software are shown in FIGS. 4, 5 and 6.

The results show that: the HUVEC cells are added with 10ng/mL VEGF factor to stimulate to remarkably promote the expression of VEGFR2, E-Selectin and ICAM-1 protein, and the heparin oligosaccharide is in dose-dependent inhibition of the increase of VEGFR2, E-Selectin and ICAM-1 protein expression caused by VEGF stimulation.

Example 5 qPCR assay of the Effect of heparin oligosaccharides on the mRNA levels of VEGFR2, E-Selectin and ICAM-1 in HUVEC

Step 1: human umbilical vein endothelial cells (HUVEC cells, source: ATCC) were cultured to 90% confluence, digested with 0.25% (w/v) pancreatin containing 0.02% (w/v) EDTA, mixed well and 2mL of 5X 10 cells was added per well in 6-well plates⁴Each/mL cell suspension was cultured at 37 ℃ overnight to 80% confluence, and the cells were divided into a blank group (Control), a Model group (Model containing 10ng/mL VEGF), HDO groups with different concentrations (0.01. mu.M, 0.1. mu.M, 1. mu.M, to which 10ng/mL VEGF was added, LMWH group (0.1. mu.M LMWH +10ng/mL VEGF), and Vorolanib group (40nM Vorolanib +10ng/mL VEGF), and incubated for 24h by group administration.

Step 2: the cells of each group treated in the step 1 are subjected to total RNA extraction by TRIzol, concentration measurement and reverse transcription to cDNA. Real-time qPCR analysis was performed using SYBR Green fluorescence detection kit. The PCR reaction system was prepared in the eight tubes of the Real-time qPCR reaction (on ice) according to the following components. Amplification reactions of the target genes (VEGFR2, E-Selectin and ICAM-1) and the internal reference Gene (GAPDH) were performed simultaneously on each set of template cDNAs, 5 duplicate wells were set, and the experiment was repeated twice, with one positive control (calibration template) and one blank control (deionized water) set for each PCR plate.

A PCR reaction system is prepared by the following components:

the primer sequence is as follows:

the reaction solution was prepared on ice, as well as within 1 hour to prevent the decay of the fluorescent dye, and after brief centrifugation, the reaction tubes were placed in sequence on a qPCR apparatus for PCR. Setting reaction conditions of a PCR instrument, and performing pre-denaturation: at 95 ℃ for 10 min; denaturation: 95 ℃ for 15 s; annealing: 30s at 60 ℃; extension: amplification was carried out for 40 cycles at 72 ℃ for 30 s. Ct value of each sample was obtained by using 2^-△△CtAnd (4) calculating. GAPDH was used as internal reference. The data obtained were analyzed using Graph Pad Prism 5.0 software and the results of quantitative analysis are shown in FIGS. 7, 8 and 9.

The results show that: after HUVEC cells are added with 10ng/mL VEGF factor to stimulate, the expression of VEGFR2, E-Selectin and ICAM-1mRNA is remarkably promoted, and the dose-dependent inhibition of the increase of the expression of the E-Selectin and ICAM-1mRNA caused by VEGF stimulation is realized by heparin oligosaccharide; after heparin oligosaccharide action, VEGFR2 mRNA expression was inhibited to various degrees, but there was no dose dependence, with the heparin oligosaccharide inhibition effect at 0.1. mu.M concentration being most significant.

As can be seen from the above examples: the heparin oligosaccharide, particularly the heparin dodecasaccharide is used as an inhibitor of VEGFR2, has the functions of inhibiting the proliferation of Vascular Endothelial Growth Factor (VEGF) promoting vascular endothelial cells, inhibiting the interaction of the Vascular Endothelial Growth Factor (VEGF) and VEGFR2 and further inhibiting the expression of adhesion molecules on the surfaces of the endothelial cells, so that the tumor growth and metastasis are inhibited, and the antitumor drug plays an antitumor role; meanwhile, the molecular weight of the heparin oligosaccharide is smaller, the anticoagulation effect is reduced, and the side effect is reduced, so that the side effects of bleeding, thrombocytopenia and the like can be avoided, and a reference is provided for developing the blood vessel-targeted heparin oligosaccharide antitumor drug.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Application of heparin oligosaccharide in preparation of antitumor drugs

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