CN106727629A - A kind of antitumor mechanism research of the derivatives of combretastatin A 4 of originating - Google Patents

Abstract

The present invention discloses that the present invention relates to the field of biomedicine, in particular to combretastatin A-4 derivative CPU-TX-008 inhibiting the Src-FAK signaling pathway to inhibit the migration of vascular endothelial cells induced by VEGF. CPU‑TX‑008 is a derivative of combretastatin A‑4 (CA‑4), by substituting the hydroxyl group on the structure of CA‑4 with a more soluble glucosamine group. In vitro cell experiments showed that it can inhibit the migration of human umbilical vein endothelial cells (HUVECs). By studying the effects of related migration proteins Src and FAK, it was found that CPU‑TX‑008 inhibited the phosphorylation of Src and FAK proteins in HUVECs. Inhibit cell migration, and ultimately achieve the purpose of inhibiting tumor angiogenesis. The CA-4 is prepared into a drug with anti-tumor angiogenesis by adding chemical groups, and its beneficial effect is to provide a new approach for clinical anti-tumor.

Description

Study on the anti-tumor mechanism of a derivative of combretastatin A-4

technical field

The invention relates to the field of biomedicine, in particular to the analysis of the migration mechanism of tumor vascular endothelial cells induced by VEGF by combretastatin A-4 derivative CPU-TX-008.

The invention relates to the field of biomedicine, in particular to the analysis of the migration mechanism of tumor vascular endothelial cells induced by VEGF by combretastatin A-4 derivative CPU-TX-008.

Background technique

Angiogenesis is a highly regulated process, and in mature mammalian organisms, physiological angiogenesis occurs only in the ovary, uterus and placenta, and in other tissues the turnover rate of vascular endothelial cells is quite low. For most tumors, due to their uncontrolled proliferation, the existing vascular system is difficult to meet the normal supply of nutrients and oxygen, and the metabolites continue to accumulate. Therefore, tumor cells can up-regulate some factors that promote the migration of vascular endothelial cells: vascular endothelial growth factor (VEGF ), Src kinase, focal adhesion kinase (FAK) and paxillin are activated to promote the migration of endothelial cells into tumor tissue to form blood vessels. People have studied the structure of CA-4 and found that the structure of CA-4 is similar to colchicine, with a basic structure of two benzene rings connected by a cis-vinyl bridge, and some methoxy groups are substituted on the ring. CA-4 has poor water solubility and is difficult to administer intravenously. CPU-XT-008 replaces the hydroxyl group on the CA-4 structure with a more water-soluble glucosamine group. The structure shows that it not only maintains the stronger CA-4 Biological activity and water solubility are also greatly improved. The present invention finds that CPU-TX-008 inhibits cell migration by inhibiting the phosphorylation of Src and FAK proteins in HUVECs, and finally achieves the purpose of inhibiting the growth of tumor blood vessels.

CPU-TX-008 is the hydroxyl group on the CA-4 structure replaced by a more water-soluble glucosamine group. The pharmacological structure shows that it not only maintains the strong biological activity of CA-4, but also greatly improves the water solubility. However, there is no report on the migration mechanism of CPU-TX-008 on human umbilical vein endothelial cells.

Contents of the invention

The purpose of the present invention is to provide CPU-TX-008 as the mechanism for inhibiting the migration of human umbilical vein endothelial cells as a drug for treating tumors.

The invention discloses a novel CA-4 derivative CPU-TX-008 to inhibit VEGF-induced migration signal pathway of human umbilical vein endothelial cells (HUVECs). The results of pharmacological experiments show that under the action of 10ng/ml VRGF, 0.1, 1, 10μmol/LCPU-XT-008 can significantly inhibit the migration of HUVECs. XT-008 can effectively inhibit the phosphorylation of Src and FAK proteins. It shows that the drug CPU-TX-008 can inhibit angiogenesis through the Src-FAK signaling pathway, so as to achieve the purpose of treating tumors.

Description of drawings

Figure 1 shows the results of cell scratch experiments in five groups of experiments.

Figure 2 is a statistical chart of the results of cell scratch experiments in five groups of experiments.

Figure 3 shows the Src protein results of Western blottingd in 5 experiments.

Figure 4 shows the p-Src protein results of Western blottingd in five experiments.

Figure 5 shows the p-FAK protein results of Western blottingd in five experiments.

detailed description

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited by the examples.

Example

The drug CPU-XT-008 was administered to human umbilical vein endothelial cells (HUVECs), and its anti-HUVECs proliferation activity was detected by MTT method, and the optimal concentration was obtained:

HUVECs were inoculated in 96-well culture plates, and after synchronization, the cells were divided into a blank group (DMEM medium without serum), a model group (DMEM medium with 10% neonatal bovine serum), a positive control group, and an administration group. The positive control group was 10 μmol/L CA-4, the administration group was 0.01, 0.1, 1, 10 μmol/L CPU-XT-008 respectively, the positive control group and the administration group both used 10% newborn calf serum DMEM medium, continued Cultivate for 24h. Add 20 μL of 5 mg/mL MTT solution to each well, and incubate at 37° C. for 4 h. After the culture solution was aspirated, 150 μL of DMSO was added to each well to dissolve the crystals, and the absorbance of each well was measured at 562 nm after shaking and mixing. Induction was carried out according to the concentration of factors that had been successfully explored in the laboratory before, namely VEGF 10ng/ml and PP2 10ng/ml.

Cell scratch experiment to study the effects of VEGF, CPU-XT-008, CA-4, PP2 on HUVECs cell migration:

When the HUVECs in the logarithmic growth phase were made into a cell suspension and inoculated on a 6-well plate and cultured to form a single layer of cells, use a sterile pipette tip to draw across the overgrown monolayer of cells in each well to make it A scratch with a width of 1 mm was formed. Rinse with PBS to remove the exfoliated cells, add DMEM culture fluid containing 0.5% serum, divide into blank group only 10% serum medium, model group 10% serum plus VEGF (10ng/ml) stimulating factor, experimental group 1 plus VEGF (10ng/ml) /ml) stimulating factor and positive drug CA-4 (10μmol/L), experimental group 2 plus VEGF (10ng/ml) and PP2 (10μmol/L), experimental group 3 VEGF (10ng/ml) and CPU-XT-008 ( 10μmol/L). The scratch width was measured at 4 pre-selected locations 24 h after drug addition. The average migration width was calculated for statistical analysis, see Figure 1.

Western-Blotting method to detect the changes in the protein levels of related migration proteins Src and FAK in HUVECs by CPU-XT-008, CA-4 and PP2 under the action of VEGF

Rinse the cultured human umbilical vein endothelial cells with cold 0.01mol/L PBS, add 150μl cell lysate, extract the total protein, measure the protein concentration and quantify it by BCA method, separate the protein by 10% polyacrylamide denaturing gel electrophoresis, Coomassie bright Blue staining was used to observe the protein electrophoresis, the wet electroporation method was used to transfer the protein to the PVDF membrane, 4% skimmed milk was blocked, and Src, p-Src, FAK, p-FAK antibodies were used to incubate overnight, horseradish enzyme-labeled goat anti-rabbit secondary antibody , developed by ECL chemiluminescence, and photographed by Bio-Rad imaging system. The Image J software system was used for analysis, and the protein expression was expressed by absorbance, and the result was expressed by the ratio of the target band to the internal reference band, as shown in Figure 3/4/5.

Claims (2)

1. Combretastatin A-4 derivative CPU-TX-008 inhibits VEGF-induced migration of vascular endothelial cells by inhibiting Src-FAK signaling pathway. 2. give medicine CPU-XT-008 to human umbilical vein endothelial cells (HUVECs) according to claim 1, MTT method detects its anti-HUVECs proliferative activity, and draw optimal action concentration, it is characterized in that: CPU- The administration concentration of XT-008 is 10 μmol/L.