阅读说明：本技术 漆黄素在联合吉西他滨治疗胰腺癌中的应用 (Application of fisetin in combination with gemcitabine in treatment of pancreatic cancer ) 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：本发明提供漆黄素的一种新的用途,是在联合吉西他滨来制备用于治疗胰腺癌的制品中的应用；所述的制品,为癌症化疗用制品。本发明发现漆黄素能增加胰腺癌吉西他滨化疗疗效,可用于抗胰腺癌吉西他滨化疗耐药制品的制备。本发明所提供的技术方案在胰腺癌临床治疗及更新化疗方案的研发等方面具有重要意义,也具有极好的临床转化和应用前景。(The invention provides a new application of fisetin, namely the application of fisetin in combination with gemcitabine in preparing a preparation for treating pancreatic cancer; the product is a product for cancer chemotherapy. The invention finds that fisetin can increase the curative effect of chemotherapy of gemcitabine for pancreatic cancer, and can be used for preparing a drug-resistant product for chemotherapy of gemcitabine for pancreatic cancer. The technical scheme provided by the invention has important significance in the aspects of clinical treatment of pancreatic cancer, research and development of updating chemotherapy schemes and the like, and also has excellent clinical transformation and application prospects.)

1. Use of fisetin in the preparation of a preparation for the treatment of pancreatic cancer, characterized in that fisetin is used in combination with gemcitabine.

2. The use according to claim 1, wherein the product is a cancer chemotherapeutic product.

3. A preparation for chemotherapy treatment of pancreatic cancer, wherein the preparation comprises gemcitabine and fisetin in pharmacologically effective concentrations.

4. The article of claim 3, wherein the article is a chemotherapeutic injection.

5. The preparation of claim 4, wherein the concentration of fisetin in the injection solution is 50-100 μ M and the concentration of gemcitabine is 20 μ M.

Technical Field

The invention belongs to the technical field of preparation of cancer treatment medicines, and particularly relates to application of a traditional Chinese medicine small-molecular compound Fisetin (Fisetin) in combination with Gemcitabine (Gemcitabine, Gem) in treatment of pancreatic cancer.

Background

Pancreatic cancer (PDAC) is a common malignant tumor of a digestive system in clinic, and due to the special anatomical position and the biological behavior of the tumor, the pancreatic cancer is extremely high in malignancy degree and poor in prognosis, and the 5-year survival rate is only about 10%.

Gemcitabine, which is the first choice of chemotherapeutic drugs and radiotherapy sensitizers for advanced pancreatic cancer, can improve the survival time and the quality of life of patients to a certain extent, but some pancreatic cancer patients have tumor progression caused by gemcitabine acquired resistance, so that chemotherapy fails. But the therapeutic effect is very limited. Therefore, new therapeutic approaches are urgently needed.

Disclosure of Invention

The invention aims to provide application of fisetin in combination with gemcitabine treatment of pancreatic cancer, namely application of fisetin to increase curative effect of gemcitabine chemotherapy of pancreatic cancer, so as to make up for the deficiency of drug resistance of existing pancreatic cancer chemotherapy.

The invention firstly provides a new application of fisetin, namely the application of fisetin in combination with gemcitabine in preparation of a preparation for treating pancreatic cancer;

the product is a product for cancer chemotherapy;

the invention also provides a preparation for pancreatic cancer chemotherapy treatment, which comprises gemcitabine and fisetin in pharmacologically effective concentrations.

As a specific description of the examples, the fisetin concentration is 50-100. mu.M, and the gemcitabine concentration is 20. mu.M.

The invention finds that fisetin can increase the curative effect of chemotherapy of gemcitabine for pancreatic cancer, and can be used for preparing a drug-resistant product for chemotherapy of gemcitabine for pancreatic cancer. The technical scheme provided by the invention has important significance in the aspects of clinical treatment of pancreatic cancer, research and development of updating chemotherapy schemes and the like, and also has excellent clinical transformation and application prospects.

Drawings

FIG. 1: graph a is a graph of the effect of fisetin in combination with gemcitabine on pancreatic cancer cells, wherein graph a shows the effect of different concentrations and times of fisetin in combination with gemcitabine on PANC-1 cell killing, and absorbance is measured at 450 nm. Data are presented as mean ± SD, and panel B shows the effect of the combination of fisetin and gemcitabine.

FIG. 2: graph of the effect of fisetin in combination with gemcitabine on subcutaneous transplants in mice, wherein Con is the control group; gem is gemcitabine.

FIG. 3: graph of the effect of fisetin and gemcitabine on pancreatic cancer cell dryness, where Con is the control group; gem is gemcitabine.

Detailed Description

The applicant finds in research that fisetin can increase the killing effect of gemcitabine on pancreatic cancer cells, can increase the inhibition effect of gemcitabine on subcutaneous tumor-bearing mouse tumors, and can also inhibit the dryness of pancreatic cancer cells, thereby contributing to the invention.

The invention is described in detail below with reference to examples and figures:

example 1: fisetin can enhance the killing effect of gemcitabine on pancreatic cancer cells

1. CCK8 experiment

PANC-1 cells were seeded in 96-well cell culture plates, the number of plated cells was selected as appropriate, 100. mu.L of cell suspension per well, 5 replicate wells, cultured in 37 ℃ incubator, and after the cells were attached, treated with fisetin (50. mu.M and 100. mu.M), gemcitabine (20. mu.M) and both (fisetin 50. mu.M, 100. mu.M in combination with gemcitabine 20. mu.M, respectively) for 24 and 48 hours, serum-free medium was added in the form of 10% CCK8 medium, and the medium was added as a replacement solution, incubated in 37 ℃ incubator for 2 hours, and absorbance was measured at a wavelength of 450 nm.

The results are shown in fig. 1, the combination of fisetin and gemcitabine can obviously inhibit the activity of PANC-1 cells, and fisetin can obviously enhance the effect of gemcitabine.

Example 2: fisetin can enhance the inhibition effect of gemcitabine on subcutaneous transplantable tumors in mice

1. Xenograft mouse model and live imaging of small animals

Animal experiments were performed according to institutional guidelines for care and use of laboratory animals. 10 female BALB/c nude mice of 4 weeks old were obtained from Shanghai laboratory animals of Chinese academy of sciencesA center. 1X 10 injections in the shoulder were performed by incision under 3% isoflurane anesthesia through the right subcostal incision⁶PANC-1-luciferase cells, a subcutaneous xenograft mouse model was established in 4 to 6 week old mice. The mice were divided into two groups of five mice each. The control group was treated with 0.1mL of DMSO by intraperitoneal injection, the mice in the fisetin-treated group were treated with 0.1mL of fisetin (0.559mmol/kg body weight) every other day, the mice in the gemcitabine-treated group were treated with 0.1mL of gemcitabine (0.19mmol/kg body weight) every other day, and the mice in the gemcitabine-treated group were treated with fisetin (0.559mmol/kg body weight) and gemcitabine (0.19mmol/kg body weight) together at 0.1mL every other day. Mice were anesthetized with 3% isoflurane. The mice were then injected intraperitoneally with 200. mu.L (15mg/mL) of D-fluorescein. After 15 minutes, tumors were imaged using IVIS lumine Series III (Perkin Elmer, usa). The same ROI was applied to all bioluminescent tumors. 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, where mice treated with fisetin and gemcitabine alone were significantly reduced in size compared to the control group, and mice treated with fisetin and gemcitabine in combination were significantly reduced in tumor size compared to the two treatment groups alone.

Example 3: fisetin can enhance the inhibition effect of gemcitabine on the dryness of pancreatic cancer cells

1. Balling experiment

Well-grown cells (PANC-1 and HPC-Y5) were digested and centrifuged to remove serum-containing medium, washed 2 times with PBS, resuspended cells using stem cell medium, counted, and selected ultra-low adsorbed cell culture 24-well plates, approximately 5000 per well, supplemented with 4ml of medium, control group treated with DMSO, fisetin-treated group treated with fisetin (100. mu.M), gemcitabine-treated group treated with gemcitabine (20. mu.M), and combination treated with fisetin (100. mu.M) and gemcitabine (20. mu.M) to observe spheronization.

2、Western blot

The cell culture was performed according to the culture conditions of the cell line, the cells were harvested by a spatula, centrifuged at 6000rpm for 3 minutes, and the supernatant was 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.

The results of the experiments are shown in figure 3, where fisetin inhibits the balling-up of both cell lines compared to the DMSO-treated group, whereas gemcitabine does not, in combination, inhibit the balling-up of pancreatic cancer compared to the gemcitabine-treated group. Compared with a control group, the dryness indexes of CD44, CD24, Nanog, Sox2 and the like in the fisetin-treated cells are all reduced, which indicates that fisetin can inhibit the dryness of tumor cells.

In the technical idea of the present invention, those skilled in the art can determine other usage amount relationships of fisetin and gemcitabine through routine experiments.