CN109999182B

CN109999182B - Application of blood coagulation factor in preparing antitumor drug

Info

Publication number: CN109999182B
Application number: CN201910252121.8A
Authority: CN
Inventors: 黄文�; 李幼平; 余睽; 张俊华; 辛光; 钮海; 张伯礼
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2019-03-29
Filing date: 2019-03-29
Publication date: 2023-03-07
Anticipated expiration: 2039-03-29
Also published as: CN109999182A

Abstract

The invention provides application of coagulation factor VIII and coagulation factor IX in preparation of antitumor drugs, wherein tumors comprise melanoma, breast cancer, colon cancer, cervical cancer and pancreatic cancer. Experiments prove that the blood coagulation factor VIII and the blood coagulation factor IX have obvious inhibition effect on tumor cells, the anti-tumor effect and mechanism of the blood coagulation factor VIII and the blood coagulation factor IX are clearly researched and verified, the new understanding on the paracrine regulation effect of endothelial cells is increased, a new thought is provided for the diagnosis, prognosis and treatment of tumors, and a reliable experimental basis is also provided for the development of new anti-tumor drugs.

Description

Application of blood coagulation factor in preparing antitumor drug

Technical Field

The invention relates to an application of a blood coagulation factor, in particular to an application of blood coagulation factor VIII and blood coagulation factor IX in preparation of antitumor drugs.

Background

Coagulation Factor VIII (FVIII) is an important coagulation factor synthesized by hepatocytes and endothelial cells, and consists of two peptide chains, wherein a heavy chain consists of A1-A2-B or A1-A2 and has a molecular weight of 90-200 kDa, a light chain consists of A3-C1-C2 and has a molecular weight of 80kDa, and Ca is arranged between the two peptide chains ²⁺ And (4) connecting. FVIII is in Ca during coagulation ²⁺ Under the participation condition, FIXa is assisted to activate FX to FXa. Normal persons contain about 80IU of FVIII per 400CC of new plasma, however patients with hemophilia a lack FVIII, which manifests as coagulation dysfunction, and therefore FVIII is commonly used clinically for the treatment of hemophilia.

At present, the research on FVIII at home and abroad mainly stays in the blood coagulation activity of FVIII, and the anti-tumor activity of FVIII is not reported yet. Meanwhile, the anti-tumor activity of the coagulation Factor IX (FIX) is not reported.

Disclosure of Invention

The invention aims to prove that the blood coagulation factors VIII and IX have anti-tumor activity, can be applied to preparation of anti-tumor drugs, and provides a new research and development idea for development of the anti-tumor drugs.

Experiments prove that the coagulation factor VIII and the coagulation factor IX have obvious inhibition effects on tumor cells and tumor tissues, can induce mitochondrial damage of the tumor cells, activate autophagy-induced autophagy-related protein expression, and further induce vWF-mediated apoptosis.

In particular, in vitro experiments prove that the blood coagulation factor VIII and the blood coagulation factor IX have obvious inhibition effects on the in vitro growth of mouse melanoma cells B16F10, human melanoma cells A375, mouse colon cancer cells C26 and human breast cancer cells MCF-7, the blood coagulation factor VIII and the blood coagulation factor IX can obviously damage mitochondrial functions and can activate autophagy-induced autophagy-related protein expression, so that vWF-mediated apoptosis is induced; the coagulation factors VIII and IX can inhibit the adhesion and migration ability of tumor cells. The invention proves that the blood coagulation factor VIII has obvious inhibiting effect on a Balb-c mouse tumor model inoculated with mouse melanoma cells B16F10 subcutaneously through in vivo experiments, and the blood coagulation factor VIII has obvious inhibiting effect on a tumor lung metastasis model of a Balb-c mouse inoculated with mouse melanoma cells B16F 10.

Based on the experimental results, the invention provides the application of the blood coagulation factor VIII and the blood coagulation factor IX in preparing antitumor drugs.

In the technical scheme of the application of the blood coagulation factor VIII and the blood coagulation factor IX in preparing the antitumor drugs, the tumors comprise melanoma, breast cancer, colon cancer, cervical cancer and pancreatic cancer.

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

experiments prove that the blood coagulation factor VIII and the blood coagulation factor IX have obvious inhibition effects on tumor cells and tumor tissues, the anti-tumor effects and mechanisms of the blood coagulation factor VIII and the blood coagulation factor IX are clearly researched and verified, new knowledge on the paracrine regulation effect of endothelial cells is increased, new ideas are provided for diagnosis, prognosis and treatment of tumors, and reliable experimental basis is also provided for development of new anti-tumor medicaments.

Drawings

Fig. 1 is a graph of results of FVIII and FIX inhibition of cell viability of tumor cells.

FIG. 2 is a graph showing the results of FVIII involvement in endothelial cell paracrine regulation of tumors.

FIG. 3 is a graph showing the results of VWF-mediated apoptosis of FVIII induced by stimulation of autophagy, wherein panel A shows the results of apoptotic protein expression after FVIII treatment and panel B shows the results of expression of autophagy protein and vWF after FVIII treatment.

FIG. 4 is a result of mitochondrial damage after FVIII treatment, wherein Panel A shows the results of mitochondrial ROS changes in cells after FVIII treatment, curves (a) and (B) show the results of mitochondrial ROS changes in cells after FVIII treatment with 0U/mL and 4U/mL, respectively, and Panel B shows the results of membrane potential detection of mitochondria with JC-1 after FVIII treatment.

Figure 5 is the results of cell streaking experiments after FVIII and FIX treatments.

FIG. 6 is a graph showing the results of inhibition of tumor growth and migration by FVIII in vivo, wherein (A) is a photograph showing the morphology of tumors after FVIII treatment, (B) is a graph showing the results of quantification of tumor weight, and (C) is a graph showing the results of lung metastatic nodules.

Detailed Description

The following examples further illustrate the use of the blood coagulation factors of the present invention in the preparation of anti-tumor drugs. The following examples are intended to be illustrative only and should not be construed as limiting the scope of the invention, which is intended to be covered by the appended claims.

Example 1

In this example, FVIII and FIX were evaluated for efficacy in vitro.

1. After incubation with different concentrations of FVIII or FIX (specifically 0, 0.25, 0.5, 1, 2, 4U/mL) for 24h with mouse melanoma cells B16F10, human melanoma cells A375, mouse colon cancer cells C26 and human breast cancer cells MCF-7, MTT examined the effect of FVIII and FIX on the viability of the different cells.

The results of FVIII and FIX inhibiting the cell viability of tumor cells A375, B16, C26 and MCF-7 are shown in FIG. 1. It can be seen from FIG. 1 that FVIII and FIX significantly inhibit the cell viability of tumor cells A375, B16, C26 and MCF-7.

2. The method comprises the steps of adopting endothelial cell line HUVEC culture medium supernatant, treating the endothelial cell culture medium alone or with FVIII antibody, detecting the expression condition of a proliferation marker Ki67 in tumor cells through immunoblotting, and analyzing the condition that FVIII participates in paracrine regulation.

The results of the participation of FVIII in the endothelial paracrine regulatory tumor are shown in FIG. 2, and it can be seen from FIG. 2 that antibodies against FVIII abolish the expression of the cell proliferation antigen Ki67 that is inhibited by the epithelial cell culture medium.

3. After incubating human melanoma cells A375 with FVIII of different concentrations (specifically 0, 1, 2, 4U/mL) for 24h, total cell protein was extracted, and expression of apoptosis protein, autophagy-related protein and vWF was detected by immunoblotting.

FIG. 3 is a graph showing the results of VWF-mediated apoptosis of FVIII induced by challenge autophagy, wherein panel A shows the results of apoptotic protein expression after FVIII treatment, and panel B shows the results of autophagy protein and vWF expression after FVIII treatment.

4. FVIII with different concentrations (specifically 0 and 4U/mL) is incubated with human melanoma cells A375 for 24h, and then a mitochondrial membrane potential detection probe JC-1 and a mitochondrial ROS probe are used for detecting mitochondrial function changes in the cells, and the specific changes are observed by a fluorescence microscope.

FIG. 4 is the result of mitochondrial damage after FVIII treatment, where Panel A is the result of mitochondrial ROS changes in cells after FVIII treatment and Panel B is the result of membrane potential measurements of mitochondria with JC-1 after FVIII treatment.

As can be seen from fig. 3 and 4, FVIII can induce mitochondrial damage in tumor cells, activate autophagy and induce autophagy-related protein expression, thereby inducing vWF-mediated apoptosis.

5. The change in the migratory capacity of human melanoma cells a375 after incubation of FVIII and FIX with human melanoma cells a375 for 24h was assessed using a scratch assay.

The results of the cell scratch test after FVIII treatment are shown in Panel A of FIG. 5, the results of the cell scratch test after FIX treatment are shown in Panel B of FIG. 5, and the control group (Ctr) in Panel A and Panel B refers to the case where FVIII and FIX are not used. As can be seen in FIG. 5, FVIII and FIX inhibit tumor cell adhesion and migration.

Example 2

FVIII was evaluated for efficacy in vivo.

Culturing mouse melanoma cell B16F10 in vitro, dividing mouse melanoma cell B16F10 into A, B two groups, culturing group A under normal condition, incubating group B with 4U/mL FVIII, inoculating two groups of cells to Balb-c mouse by subcutaneous and caudal vein injection respectively at right abdomen, inoculating two groups of cells with equal inoculum size, and inoculating 1 × 10 cells subcutaneously ⁶ Establishing subcutaneous model of mouse tumor, and inoculating tail vein with 3 × 10 ⁵ Establishing lung metastasis model, observing melanoma formation and lung metastasis, and recording subcutaneous tumor weight and lung metastasis. The group A cells were inoculated as a control group, the group B cells were inoculated as a FVIII group, and a normal mouse group was set without any cells.

FIG. 6 is a graph showing the results of inhibition of tumor growth and migration by FVIII in vivo, wherein (A) is a photograph showing the morphology of tumors after FVIII treatment, (B) is a graph showing the results of quantification of tumor weight, and (C) is a graph showing the results of lung metastatic nodules. As is clear from the graphs (A) and (B) in FIG. 6, FVIII showed significant inhibition of the Balb-C mouse tumor model inoculated with the mouse melanoma cell B16F10 subcutaneously, and as is clear from the graph (C) in FIG. 6, FVIII showed significant inhibition of the tumor lung metastasis model of the Balb-C mouse inoculated with the mouse melanoma cell B16F 10.

Claims

1. The coagulation factor VIII is used as an active ingredient only for preparing the antitumor drug, the tumor is melanoma, and when the coagulation factor VIII is used, the coagulation factor VIII induces mitochondrial damage of tumor cells, activates autophagy-induced autophagy-related protein expression and induces vWF-mediated tumor cell apoptosis.