阅读说明：本技术 Gl-v9在制备抑制胰腺癌药物中的应用 (Application of GL-V9 in preparation of pancreatic cancer inhibition drug ) 是由 卢娜 郭青龙 赵凯 赵越 曹汪佳 于 2020-12-07 设计创作，主要内容包括：为了研究GL-V9多环节、多途径的抗胰腺癌作用,在抑制胰腺癌细胞生长的基础上,本发明提供了GL-V9在制备抑制胰腺癌侵袭转移药物中的应用,所述GL-V9分子式为C-(24)H-(27)NO-(5),其化学结构式如式Ⅰ所示：本发明还提供一种抑制胰腺癌的药物组合物,所述药物组合物以GL-V9为活性成分,辅以药学上可接受的载体。本发明表明GL-V9能够有效抑制胰腺癌细胞增殖,迁移,侵袭,并抑制胰腺癌细胞转移相关蛋白表达。这些作用表明GL-V9可用于抑制胰腺癌细胞转移的治疗,具有开发药物的前景。(In order to research the pancreatic cancer resistance of GL-V9 through multiple links and pathways, the invention provides application of GL-V9 in preparation of a medicament for inhibiting pancreatic cancer invasion and metastasis on the basis of inhibiting pancreatic cancer cell growth, wherein the molecular formula of GL-V9 is C 24 H 27 NO 5 The chemical structural formula is shown as formula I: the invention also provides a pharmaceutical composition for inhibiting pancreatic cancer, which takes GL-V9 as an active ingredient and is assisted by a pharmaceutically acceptable carrier. The GL-V9 can effectively inhibit proliferation, migration and invasion of pancreatic cancer cells and inhibit the expression of pancreatic cancer cell metastasis related proteins. These effects indicate that GL-V9 can be used for inhibiting pancreatic cancer cell proliferationThe treatment of cell metastasis has the prospect of developing medicaments.)

The application of GL-V9 in preparing a pancreatic cancer inhibiting drug is characterized in that the GL-V9 structural formula is shown as a formula I:

2. the use of claim 1, wherein the medicament is a medicament capable of inhibiting pancreatic cancer cell proliferation.

3. The use of claim 1, wherein the medicament is a medicament capable of inhibiting pancreatic cancer cell migration.

4. The use of claim 1, wherein the medicament is a medicament capable of inhibiting pancreatic cancer cell invasion.

5. The use of claim 1, wherein the medicament is a medicament capable of inhibiting the expression of a protein associated with metastasis of pancreatic cancer cells.

6. The use of any one of claims 2 to 5, wherein the pancreatic cancer cells are human pancreatic cancer cells PANC-1.

7. The use of claim 6, wherein the related protein is one or more of MMP-9, MMP-14, Vimentin, CD 44.

8. A pharmaceutical composition for inhibiting pancreatic cancer, which comprises GL-V9 as an active ingredient, and a pharmaceutically acceptable carrier.

Technical Field

The invention discloses application of wogonin derivative GL-V9 in preparation of a medicine for inhibiting pancreatic cancer invasion and metastasis, and particularly relates to new application of GL-V9.

Background

Tumor metastasis is responsible for 90% of tumor-related deaths. In metastatic dissemination of tumors, tumor cells invade peripheral tissues from primary foci, enter capillaries of lymphatic and blood systems, metastasize through blood vessels to capillaries of distal tissues to survive, subsequently extravasate from the blood vessels, survive in the metastases and proliferate to form new tumor tissues. Pancreatic cancer is a highly malignant, difficult to diagnose and treat malignant neoplasm of the digestive tract worldwide, and about 90% is ductal adenocarcinoma of the glandular epithelium. Its morbidity and mortality has increased dramatically in recent years. Survival rate of less than 1% for 5 years is one of the worst prognosis tumors. In the current treatment of pancreatic cancer, the basic treatment principle is still mainly surgical operation treatment and combined with radiotherapy, chemotherapy and other comprehensive treatments, but the overall curative effect is poor; and the prognosis is very poor. The survival of a pancreatic cancer patient who is not treated is about 4 months, and a patient can generally survive 16 months after resection surgery. Meanwhile, pancreatic cancer is easy to metastasize and recur, so that clinical treatment of pancreatic cancer faces a serious challenge.

Therefore, aiming at the invasion and metastasis of pancreatic cancer, the search for safe and effective anti-metastasis drugs has important significance for treating pancreatic cancer and inhibiting pancreatic cancer metastasis.

Disclosure of Invention

In order to research the multi-link and multi-path anti-pancreatic cancer effect of wogonin derivative GL-V9, on the basis of inhibiting the growth of pancreatic cancer cells, the invention provides the application of GL-V9 in preparing a medicament for inhibiting pancreatic cancer invasion and metastasis, and provides a basis for developing a medicament for inhibiting pancreatic cancer metastasis.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the first purpose of the invention is to provide GL-V9 for preparing a medicine for inhibiting pancreasThe GL-V9 molecular formula is C in application of cancer drugs₂₄H₂₇NO₅The chemical structural formula is shown as formula I:

further, the drug is a drug capable of inhibiting pancreatic cancer cell proliferation.

Further, the drug is a drug capable of inhibiting pancreatic cancer cell migration.

Further, the medicament is capable of inhibiting pancreatic cancer cell invasion.

Furthermore, the medicine is capable of inhibiting the expression of the pancreatic cancer cell metastasis related protein.

Furthermore, the pancreatic cancer cell is human pancreatic cancer cell PANC-1.

Further, the related protein is one or more of MMP-9, MMP-14, Vimentin and CD 44.

The second purpose of the invention is to provide a pharmaceutical composition for inhibiting pancreatic cancer, which takes GL-V9 as an active ingredient and is supplemented with a pharmaceutically acceptable carrier.

When the pharmaceutical composition for inhibiting pancreatic cancer provided by the invention is used for inhibiting pancreatic cancer cells, the pharmaceutical composition can be used alone, can be matched with other medicines for simultaneous use, or can be prepared into a compound preparation together with other medicines for use, and the purpose of inhibiting pancreatic cancer can be achieved.

The pharmaceutically acceptable auxiliary materials refer to various conventional auxiliary materials required when different dosage forms are prepared, such as diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorbing materials and the like, and the pharmaceutically acceptable auxiliary materials are prepared into any one of common oral preparations by a conventional preparation method, such as granules, powder, tablets, capsules, pills, oral liquid, decoction, dropping pills and the like.

Compared with the prior art, the GL-V9 can effectively inhibit the proliferation, migration and invasion of pancreatic cancer cells and inhibit the expression of pancreatic cancer cell metastasis related proteins. These effects indicate that GL-V9 can be used for the treatment of inhibiting pancreatic cancer cell metastasis, and has the prospect of drug development.

Drawings

FIG. 1 is a graph showing the results of experiments in which GL-V9 inhibits proliferation of human pancreatic cancer cells PANC-1.

FIG. 2 is a graph showing the results of the experiment of GL-V9 inhibiting migration of PANC-1, a human pancreatic cancer cell.

FIG. 3 is a graph showing the results of experiments in which GL-V9 inhibits invasion of human pancreatic cancer cell PANC-1.

FIG. 4 is a graph showing the results of an experiment for GL-V9 inhibition of invasion metastasis associated protein expression.

FIG. 5 shows the structure of GL-V9.

Detailed Description

The invention is further elucidated with reference to the drawings and the specific examples.

Example 1

First, test materials

(1) Medicine

GL-V9(C₂₄H₂₇O₅N, molecular weight: 409.47) was supplied by university of Chinese pharmacy as a pale yellow powder with a purity of 98%, prepared as a stock solution in DMSO, and stored at-20 ℃. Diluting to required concentration before use.

(2) Cell line

The human pancreatic cancer cell strain PANC-1 is purchased from a cell bank of a biochemical and cytobiological research institute of Shanghai Life sciences institute of Chinese academy of sciences, and is cultured by DMEM culture solution containing 10% fetal bovine serum.

(3) Reagent

1) Culture solution: DMEM medium was purchased from GIBCO, USA, 10.4g of DMEM powder was dissolved in 1000mL of sterilized distilled water, and 3.7g of NaHCO was added₃Adjusting pH to 7.2-7.4, filtering with cylindrical filter, sterilizing, packaging, and storing in refrigerator at 4 deg.C. Before use, 10% fetal calf serum, 100U/mL penicillin and 100mg/L streptomycin are added.

2) Fetal bovine serum: product of GIBICO, USA. Inactivating in 56 deg.C water bath for 30min, subpackaging, and storing in-20 deg.C low temperature refrigerator.

3) Hematoxylin staining solution: purchased from Shanghai Biotechnology engineering, Inc.

4) Eosin dye liquor: purchased from Shanghai Biotechnology engineering, Inc.

5) Transwell cell (0.8 μm): purchased from Millipore corporation, usa.

6) Matrigel (phenol red containing): purchased from Corning, usa.

7) MTT solution: MTT powder was purchased from Sigma-Aldrich, USA. Weighing 500mg MTT powder, adding 100mL PBS solution, ultrasonic dissolving to mix uniformly to obtain 5mg/mL MTT solution, filtering with sterile filter membrane with pore diameter of 0.22 μm, sterilizing, packaging, and storing in refrigerator at 4 deg.C.

Second, experimental instrument

(1)3111 type water jacket CO₂An incubator: thermo Fisher Scientific, USA.

(2) Axiocam ICC5 upright fluorescence microscope: the company Carl Zeiss, Germany.

(3) Tanon 5200 full-automatic chemiluminescence image analysis system: shanghai Tianneng technologies, Inc.

(4) Power Pac Basic electrophoresis: BIO-RAD, USA.

(5) Mini-ProteaTetra System Total Wet type Membrane tank: BIO-RAD, USA.

(6) A Varioskan full-wavelength microplate reader; purchased from Thermo Fisher Scientific, usa.

(7) A blood counting chamber; purchased from Shanghai Biochemical instruments Ltd.

Third, test method and test result

(1) Tetramethyl azoazolate (MTT) method for verifying GL-V9 to inhibit pancreatic cancer cell PANC-1 proliferation

The MTT method, also known as MTT colorimetry, is a method for detecting cell viability. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce MTT into water-insoluble blue-purple crystal Formazan (Formazan), and dead cells do not have the function. When the formazan is solubilized with DMSO to form a purple solution, cell viability is directly proportional to the shade of the solution color. Therefore, the inhibition rate of the drug on cell proliferation can be calculated according to the OD value detected by the microplate reader.

Pancreatic cancer cells in logarithmic growth phase were centrifuged and resuspended in fresh medium, all at 1X 10⁴The density of individual cells/well was seeded in 96-well cell culture plates with a cell suspension volume of 100 μ L per well, with three sub-wells set. Setting a control group and an additive group, wherein the control group is added with 0 mu M GL-V9, the additive group is 10 groups, and 0.01 mu M,0.1 mu M,0.5 mu M,1 mu M,5 mu M,10 mu M,50 mu M,100 mu M,500 mu M and 1000 mu M GL-V9 are respectively added. Subsequently, 100. mu.L of GL-V9 at the corresponding concentration (0. mu.M, 0.01. mu.M, 0.1. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 50. mu.M, 100. mu.M, 500. mu.M, 1000. mu.M) was added to each well. After 24h, 20. mu.L of MTT solution at a concentration of 5mg/mL was added to each well and left to stand in a 37 ℃ thermostatted cell incubator for 4h in the dark. Subsequently, the 96-well cell culture plate was placed in a plate centrifuge and centrifuged at 4000rpm for 20 minutes, the supernatant was aspirated, 100. mu.L of DMSO was added to each well and placed on a micro shaker and shaken for 5 minutes to completely dissolve the crystals. Measuring absorbance with a full-wavelength microplate reader at a detection wavelength of 570nm and a reference wavelength of 630nm, and calculating the inhibition rate of the drug on cell proliferation (figure 1):

the inhibition rate is (1-drug-added group average cell absorbance/control group average cell absorbance) × 100%

The results show that: GL-V9 clearly inhibited cell proliferation activity of PANC-1 and was shown to be concentration dependent. Half maximal Inhibitory Concentration (IC) of GL-V9 when it acted on PANC-1 cells (50% inhibition Concentration)₅₀) It was 3.51. mu.M.

(FIG. 1)

(2) GL-V9 inhibits pancreatic cancer cell PANC-1 migration

PANC-1 cells were digested and seeded into 6-well plates and cultured to a density of 90% to prepare for scratching. A10. mu.l plastic tip was used to draw a line in the center of a well with a cell density of 90%, the line being of equal thickness. After scraping, the lifted cells were washed out with PBS. After adding culture medium containing GL-V9 (0. mu.M, 2. mu.M, 4. mu.M, 8. mu.M) at different concentrations to each well, the wells were incubated in an incubator at 37 ℃. Each dose group was scored at 24h for each photograph. Photographs were taken at the same time points to visually observe the change in migration distance of PANC-1 before and after treatment with GL-V9.

The results show that: compared with the non-drug administration, GL-V9 significantly reduced the migration distance of PANC-1 cells and the migration ability of PANC-1 cells in a dose-dependent manner after the administration (FIG. 2).

(3) GL-V9 inhibits pancreatic cancer cell PANC-1 invasion

Setting a control group and an administration group, wherein 0 mu M GL-V9 is added into the control group; the administration components were divided into 3 groups, and GL-V9 (2. mu.M, 4. mu.M, 8. mu.M) was administered to PANC-1 cells, respectively. The control group and the administration group are placed in a constant temperature incubator to be cultured for 24 h. Matrigel (matrigel: serum-free medium: 1:10) was diluted, 100. mu.L of diluted matrigel was added to each chamber, and the mixture was allowed to stand at 37 ℃ for 30min or more, followed by molding. The GL-V9-treated PANC-1 cells were digested, resuspended in serum-free medium, and diluted to 2X 10⁵Individual cells/mL. The transwell cell was removed and placed in a well of a 24-well plate. Each well had 600. mu.L of serum-containing medium, and 400. mu.L of cell suspension was added to the upper layer of the chamber. Putting into an incubator to be cultured for 24 h. The chamber was removed and the cells on the inside of the microporous membrane that did not pass through were wiped off with a cotton swab. Methanol was fixed for 5 minutes and washed with pure water. Staining with hematoxylin for 15 minutes, and washing with pure water; eosin staining for 20 seconds, washing with blown water, and wiping the cotton swab dry. Five visual fields in the left, right, upper and lower parts are taken under a microscope, photographed and averaged.

The results show that: after each of the PANC-1 cells was given GL-V9 (2. mu.M, 4. mu.M, 8. mu.M) for 24h, the same number of cells were seeded into a transwell chamber previously plated with Matrigel and incubated for 24 h. After 24h, it was seen that GL-V9 administration significantly reduced the number of PANC-1 cells that passed through the transwell chamber, inhibited PANC-1 cell invasion, and was dose-dependent (FIG. 3).

(4) Western blot experiment verifies that GL-V9 inhibits invasion and metastasis related protein expression

PANC-1 cells were treated with different concentrations of GL-V9 (0. mu.M, 2. mu.M, 4. mu.M, 8. mu.M) by: PANC-1 cell digestions were counted and counted at 30X 10⁴The cells/well were plated on 6-well plates at a density of 70-80%, and GL-V924h was administered. After the treatment is finished, the PANC-1 cells are digested by pancreatin and collected in a tube, cell lysate with proper volume is added, the cell lysate is cracked on ice, and the supernatant obtained after centrifugation is the extracted protein sample, wherein the protein sample comprises MMP-9, MMP-14, CD44 and Vimentin protein.Calculating the protein concentration and carrying out protein electrophoresis. Transferring the gel to a cellulose acetate membrane, and sequentially blocking, and incubating with primary antibodies and secondary antibodies (the primary antibodies are MMP-9, MMP-14, CD4 and Vimentin antibodies, and the secondary antibodies are HRP goat anti-rabbit secondary antibodies). Finally exposing protein bands, and detecting the concentrations of the target proteins MMP-9, MMP-14, CD44 and Vimentin respectively.

The results show that: the results of different concentrations of GL-V9 given by PANC-1 cells are shown in FIG. 4, and GL-V9 can effectively inhibit the expression levels of MMP-9, MMP-14, CD44 and Vimentin proteins of PANC-1 cells.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be within the scope of the invention.