阅读说明：本技术 漆黄素在抑制胰腺癌细胞和小鼠胰腺癌肿瘤增殖中的应用 (Application of fisetin in inhibition of pancreatic cancer cell and mouse pancreatic cancer tumor proliferation ) 是由 曹利平 贾盛楠 赵冰冰 徐晓冬 丁国平 于 2021-09-26 设计创作，主要内容包括：本发明首先提供了漆黄素的一种新的用途,即在制备用于抑制胰腺癌细胞或小鼠胰腺癌肿瘤增殖的制品中的应用。本发明再一个方面还提供一种用于抑制胰腺癌细胞或小鼠胰腺癌肿瘤增殖的制品,所述的制品中包含有药理有效浓度的漆黄素。本发明发现漆黄素能抑制胰腺癌细胞和小鼠胰腺癌肿瘤的增殖,可将漆黄素用于抗胰腺癌治疗新药物的制备。(The invention firstly provides a new application of fisetin, namely an application in preparing a product for inhibiting pancreatic cancer cells or mouse pancreatic cancer tumor proliferation. In still another aspect, the present invention provides a preparation for inhibiting pancreatic cancer cell or mouse pancreatic cancer tumor proliferation, wherein the preparation comprises fisetin in a pharmacologically effective concentration. The invention discovers that fisetin can inhibit proliferation of pancreatic cancer cells and mouse pancreatic cancer tumors, and fisetin can be used for preparing a new medicine for treating pancreatic cancer.)

1. The application of fisetin is application in preparing a product for inhibiting pancreatic cancer cells or mouse pancreatic cancer tumor proliferation.

2. A preparation for inhibiting pancreatic cancer cell or mouse pancreatic cancer tumor proliferation, wherein the preparation comprises fisetin at a pharmacologically effective concentration.

3. The article of claim 2, wherein the concentration of fisetin in the article is not less than 50 μ M.

4. The article of claim 2, wherein the concentration of fisetin in the article is from 80 to 400 μ M.

5. The article of any one of claims 2-4, wherein the article is a liquid article.

6. The preparation according to claim 5, wherein the liquid preparation is a culture medium for the culture of pancreatic cancer cells supplemented with fisetin.

7. The preparation of claim 6, wherein said fisetin is added at a concentration of 80-400 μ M.

8. The preparation of claim 5, wherein the liquid preparation is a DMSO solution to which fisetin is added.

9. The preparation of claim 8, wherein said fisetin is added at a concentration of 0.559 to 1.048 mmol/kg.

Technical Field

The invention belongs to the technical field of preparation of pancreatic cancer treatment medicines, and particularly relates to application of a traditional Chinese medicine small-molecule compound Fisetin (Fisetin) in inhibition of proliferation of pancreatic cancer cells and pancreatic cancer tumors of mice.

Background

Pancreatic cancer (PDAC), one of the most aggressive tumors in the digestive system that is common in the clinic, has a very poor prognosis due to its specific anatomical location and tumor biological behavior, and has a five-year survival rate that is penultimate in the persistence of all tumors, much lower than that of other tumors. Nowadays, the application of auxiliary chemotherapy, radiotherapy, immunotherapy, targeted therapy and other means in pancreatic cancer patients is increasingly common, and although the overall survival time of the patients is prolonged to a certain extent, the treatment effect is very limited. Therefore, new therapeutic approaches are urgently needed.

In recent years, with the continuous and deep research of traditional Chinese medicine treatment in the tumor field, the modern new traditional Chinese medicine preparation makes better progress in the clinical treatment process of tumor patients. The traditional Chinese medicine is improved into the Chinese patent medicine, so that the situations of complicated treatment application and the like in the traditional treatment process can be well avoided. Therefore, the screening of the traditional Chinese medicine small molecular compound for effectively inhibiting the pancreatic cancer proliferation and the deep research of the molecular mechanism of the effect of the compound have important significance for the clinical treatment of pancreatic cancer and the research and development of new chemotherapy schemes, and also have excellent clinical transformation and application prospects.

Disclosure of Invention

The invention aims to provide application of fisetin in inhibition of proliferation of pancreatic cancer cells and mouse pancreatic cancer tumors, namely fisetin is used for inhibiting proliferation of pancreatic cancer cells and mouse pancreatic cancer tumors, so that the defects of the existing pancreatic cancer treatment method are overcome.

The invention firstly provides a new application of fisetin, namely an application in preparing a product for inhibiting pancreatic cancer cells or mouse pancreatic cancer tumor proliferation.

In yet another aspect, the present invention provides a preparation for inhibiting pancreatic cancer cell or mouse pancreatic cancer tumor proliferation, wherein the preparation comprises fisetin in a pharmacologically effective concentration;

the pharmacological effective concentration is not lower than 50 mu M;

furthermore, the pharmacological effective concentration is 80-400 mu M;

the product is a liquid product;

as a specific liquid preparation, fisetin is added to a medium for culturing pancreatic cancer cells;

wherein the added concentration of fisetin in the culture medium is 80-400. mu.M;

another preparation is DMSO solution added with fisetin, wherein the addition concentration of fisetin is 0.559-1.048 mmol/kg.

The invention discovers that fisetin can inhibit proliferation of pancreatic cancer cells and mouse pancreatic cancer tumors, and fisetin can be used for preparing a new medicine for treating pancreatic cancer.

Drawings

FIG. 1: graph of the effect of fisetin on pancreatic cancer cell (PANC-1) proliferation, wherein Panel A is the cell viability of PANC-1 cells measured by the CCK-8 assay. Cells were treated with fisetin (0, 25, 50, 100, 200 and 400 μ M) for 24 and 48 hours, respectively. The absorbance was measured at 450 nm. Data are presented as mean ± SD; p is less than or equal to 0.05, # P is less than or equal to 0.05, panel B is real-time cell analysis (RTCA) of pancreatic cancer PANC-1 cells. Cells were tested for cell growth using an xCELLigence RTCA DP instrument for 72 hours after treatment with fisetin (0-400. mu.M). Every 15 minutes, each point represents the average from three replicates with SD.

FIG. 2: graph of the effect of fisetin on tumor proliferation in mouse pancreatic cancer.

FIG. 3: graph of the effect of fisetin on proliferation-associated proteins of pancreatic cancer tumor tissue in mice.

FIG. 4: graph of the effect of fisetin on proteins associated with the growth and proliferation of PANC-1 cells.

Detailed Description

Fisetin (Fisetin) is a natural flavonoid compound, has wide sources, low price and easy obtainment, and has wide pharmacological activities of resisting inflammation, resisting oxidation, resisting blood coagulation, resisting thrombus, treating diabetic kidney injury and the like. However, the applicant found in the research that the proliferation capacity of PANC-1 was significantly inhibited after treating pancreatic cancer cell line PANC-1 with fisetin. The proliferation ability of pancreatic cancer tumors was also significantly inhibited by treating xenograft pancreatic cancer mice with fisetin, which contributed to the present invention.

The present invention will be described in detail with reference to examples below:

example 1: fisetin can inhibit pancreatic cancer cell (PANC-1) proliferation

1. CCK8 experiment

The PANC-1 cells are inoculated in a 96-well cell culture plate, the proper number of plated cells is selected, 100 mu L of cell suspension is added into each well, 5 repeated wells are cultured in an incubator at 37 ℃, fisetin (0, 25, 50, 100, 200 and 400 mu M) with different concentrations is added for treatment for 24 and 48 hours after the cells adhere to the wall, a serum-free culture medium is added into a culture medium of 10 percent CCK8 in a liquid changing mode, the cells are cultured for 2 hours in the incubator at 37 ℃, and the absorbance is detected at the wavelength of 450 nm.

As shown in FIG. 1A, the activity of PANC-1 cells was not significantly inhibited by fisetin at concentrations of 25. mu.M and 50. mu.M, but proliferation of PANC-1 cells was effectively inhibited by fisetin at levels of 100. mu.M, 200. mu.M and 400. mu.M.

2. Real-time cell analysis (RTCA)

PANC-1 cells were seeded in 96-well cell culture plates and then treated with fisetin (0-400 μ M) for 72 hours, cell growth was monitored using an xcelligene RTCA DP instrument, the system recorded impedance every 15 minutes, the x-axis of the image represents time after treatment, the y-axis reflects a logarithmic relationship consistent with cell number, and each point represents the average from three replicates with SD.

The results are shown in FIG. 1, where fisetin inhibits the proliferation of PANC-1 cells in a dose and time dependent manner.

Example 2: fisetin can inhibit tumor proliferation of pancreatic cancer in mice

1. Xenograft mouse model

Animal experiments were performed according to institutional guidelines for care and use of laboratory animals. 10 female BALB/c nude mice, 4 weeks old, were obtained from Shanghai laboratory animal center, Chinese academy of sciences. By injecting 1X 10 under 3% isoflurane anesthesia in mouse axilla⁶Luciferase-labeled PANC-1 cells (Luc-PANC-1) were used to construct a subcutaneous xenograft mouse model. The mice were divided into two groups of five mice each. Treatment groups mice were treated with 0.1mL Fisetin (1.048mmol/kg body weight) by intraperitoneal injection every other day. The formula for the tumor volume is 0.52X length X width². Control group abdomen0.1mL of DMSO was injected intraperitoneally.

2. Bioluminescent imaging

Mice were anesthetized with 3% isoflurane and then injected intraperitoneally with 200 μ L (15mg/mL) of D-fluorescein. After 15 minutes, tumors were imaged using IVIS lumine Series III (Perkin Elmer, usa). Data are expressed as mean radiance (photons/sec/cm/steradian), which is a calibrated measurement of photon emission.

The results are shown in figure 2 and demonstrate that the tumor size in mice treated with fisetin is significantly reduced.

Example 3: fisetin can inhibit proliferation-related protein of mouse pancreatic cancer tissue

1. Tissue fixation

Fixing tissue with 4% paraformaldehyde solution, sequentially adding 70%, 80%, 90%, 95%, 100% ethanol for 30min, dehydrating and clearing xylene for 1h × 2 times, soaking in paraffin solution, embedding, slicing into 4 μm slices, placing on glass slide in water bath, and oven drying at 60 deg.C.

2. HE staining

Drying the paraffin sections overnight; dewaxing the xylene conventionally for 2 times and 5 min/time; eluting xylene with anhydrous ethanol for 2 times, 10 min/time; dewaxing in 95%, 90%, 85%, 80% and 75% ethanol for 5 min/time. Staining with hematoxylin for 5min, washing with running water for 2min, differentiating with 1% hydrochloric acid alcohol for several seconds, washing with running water, returning blue with dilute ammonia water for 30s, washing with running water for 1min, staining with eosin solution for 10min, and washing with running water for 30 s. Dehydrating with 75%, 80%, 95%, 100% alcohol for 1 min/time; the xylene is transparent for 2 times and 2 min/time; sealing the neutral resin, and baking in an oven at 60 ℃. The morphological changes of the cells were observed using optical microscope image acquisition.

3. Immunohistochemistry

Dewaxing in 100%, 95%, 90%, 80% and 70% ethanol for 10 min; rinsing with clear water, soaking in 3% H2O2 for 10min for hydration, adding 0.01M citrate buffer solution, heating in microwave oven for 3min, cooling to room temperature, and repeating twice. Washing twice with water, washing twice with PBS, wiping off surrounding liquid, adding 10% serum, sealing at 37 deg.C for 30 min. Wiping out surrounding serum, dripping PCNA primary antibody, standing at 4 ℃ overnight, and rewarming at room temperature for 1 h. PBS was washed three times, the surrounding PBS was wiped dry, secondary antibody was added dropwise, 30min at 37 ℃. PBS was washed three times, the surrounding PBS was wiped dry, 1% SABC was added dropwise, 30min at 37 ℃. PBS was washed three times, the surrounding PBS was wiped dry, and a color developing agent was added dropwise. Washing with clear water, and soaking in hematoxylin stain. Washing with clear water, and sequentially adding 70%, 80%, 90%, 95%, 100% ethanol and xylene for 2 min. The surrounding xylene was wiped dry, neutral resin was added dropwise, covered with a glass cover, and dried in a fume hood.

The experimental result is shown in figure 3, and the proliferation-related protein Proliferating Cell Nuclear Antigen (PCNA) in the tumors of the mice in the fisetin treatment group is remarkably reduced.

Example 4: protein related to inhibition of PANC-1 cell growth and proliferation by fisetin

1、Western blot

Pancreatic cancer PANC-1 cells were treated with fisetin and controls were treated with DMSO. Cells were harvested by spatula, centrifuged at 6000rpm for 3 minutes and the supernatant discarded. Adding lysis solution, performing ice lysis for 30min, performing centrifugation for 20min at 4 ℃ and 12000rpm, keeping supernatant, adjusting the concentration after the protein concentration is detected by the BCA kit, and adding 4 xSDS-PAGE loading buffer into the supernatant for mixing.

Preparing 10% SDS-PAGE separation gel according to the molecular size of the protein, pressing with deionized water, preparing 5% concentrated gel, inserting into a comb, coagulating, loading, and separating the protein by electrophoresis (constant pressure: concentrated gel 80V, separating gel 120V). The proteins were transferred onto a PVDF membrane activated with methanol by wet transfer (constant flow: 300mA) and blocked with a 5% skimmed milk solution for 1 h. Washing the membrane with TBST for 3 times, incubating the primary antibody diluent overnight at 4 deg.C, sucking out the primary antibody, washing the membrane with TBST for 3 times, incubating the secondary antibody diluent at room temperature for 1h, and developing with chemiluminescence apparatus.

Experimental results as shown in fig. 4, fisetin treatment reduced the expression of the cell growth and proliferation-related proteins PCNA, Ki67 and phosphorylated histone H3 (p-H3).