Use of cinobufotalin combined with gemcitabine

Abstract

The present invention discloses the use of bufotalin combined with gemcitabine. The combination of bufotalin and gemcitabine is used as a drug for treating pancreatic cancer, wherein the molar concentration ratio of bufotalin to gemcitabine is 0.1 - 0.2:5 - 80 or 0.8 - 1.6:5 - 40 or 0.8:80. The present invention proves through in vitro experiments that the combined treatment of bufotalin and gemcitabine on pancreatic cancer cells can inhibit their survival, and through in vivo experiments, it proves that the combined treatment of bufotalin and gemcitabine can inhibit the growth of pancreatic cancer cells, promote DNA damage and cell death. Thus, it is demonstrated that the combined use of bufotalin and gemcitabine can promote the treatment of pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the field of pharmacy, and particularly to the use of bufotalin in combination with gemcitabine. Background Art

[0002] Pancreatic cancer (PC) is a digestive system tumor with extremely high malignancy and extremely poor prognosis, and is called the "king of cancers". Globally, the incidence and mortality of pancreatic cancer show a gradually increasing trend. According to the global cancer statistics in 2022, the incidence of pancreatic cancer ranks 12th (about 500,000 cases), and at the same time its mortality ranks 6th among cancer-related mortalities (about 470,000 cases). The incidence and mortality of pancreatic cancer are higher in men than in women. The number of new male cases is 256,000, and that of women is 240,000. According to the results of epidemiological investigations, the incidence and mortality of pancreatic cancer vary considerably among countries in the world, and the incidence and mortality of pancreatic cancer in high-income countries are significantly higher than those in middle- and low-income countries. The diagnosis rate of PDAC in young individuals is on the rise, and the most significant increase is observed in young women. The increase in obesity, diabetes, and alcohol use is considered a possible factor contributing to the increase in young-onset PDAC. Clinically, the main methods for treating tumors include radiotherapy, chemotherapy, and surgical resection. Chemotherapy plays a major role in the treatment process. However, in the current medical context, chemotherapy also faces intractable problems - chemotherapy drug tolerance, and its formation mechanism is closely related to the activation of the DNA damage response (DDR) pathway. Therefore, to solve this key problem, understanding its complex chemotherapy resistance mechanism and exploring small molecule drugs targeting the DDR pathway have become the main tasks at the present stage.

[0003] Gemcitabine is a deoxycytidine analogue, and its main mechanism is to inhibit DNA synthesis. It can inhibit tumor growth by causing tumor DNA damage, and the DNA damage caused by external adverse stimuli to cells [3] will activate the DNA damage response signaling pathway. In the DNA damage signal pathway, the DNA damage repair mechanism is an important reason for the generation of chemotherapy resistance in tumor cells.

[0004] Bufo bufo gargarizans or Bufo melanostictus parotid gland and skin gland secretion of white slurry after processing and drying, which has anti-tumor, analgesic, anesthetic, anti-infective and other effects, has good curative effect on a variety of cancers such as liver cancer, esophageal cancer, lung cancer, skin cancer, etc. A variety of active ingredients in bufalin have anti-cancer effects, and bufotalin has the best effect among these ingredients. Research shows that in hepatocellular carcinoma cell line HepG2, bufotalin inhibits the AKT-mediated signaling pathway, induces up-regulation of p53 gene expression, makes the cells arrest in the G2 / M phase, thereby inhibiting the proliferation and migration of hepatocellular carcinoma cell line HepG2; in HEP3B cells, bufotalin can activate caspase-3 and caspase-9 by activating caspase-8, increase mitochondrial tBid and cause mitochondrial dysfunction, disrupt mitochondrial membrane potential and cause the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, thereby leading to cell apoptosis and further inhibiting the growth of HEP3B cells; in non-small cell lung cancer A549 cells, promoting the degradation of GPX4 and the accumulation of Fe2+ trigger ferroptosis and inhibit the growth of tumor cells; in esophageal squamous cell carcinoma ESCC and ECA-109 cells, bufotalin can promote the activation of caspase-3, caspase-8 and caspase-9, promote the expression and phosphorylation of P53 and inhibit the expression of Ki-67, thereby promoting cell apoptosis. From the above research, it can be seen that bufotalin can inhibit the proliferation of tumor cells through multiple pathways such as cell cycle arrest, apoptosis and ferroptosis, but there is less research on the relationship with DNA damage, and it has not been reported whether it can promote tumor cell death through the DNA damage pathway. Therefore, the research of this drug in DNA damage has the opportunity to become a breakthrough to overcome tumor drug resistance and promote the research of chemotherapy drug resistance mechanism. Summary of the Invention

[0005] The object of the present invention is to provide a use of bufotalin combined with gemcitabine.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A use of bufotalin combined with gemcitabine, bufotalin combined with gemcitabine is used as a drug for treating pancreatic cancer, wherein the molar concentration ratio of bufotalin to gemcitabine is 0.1-0.2:5-80 or 0.8-1.6:5-40 or 0.8:80.

[0008] Further improvement, the combination of bufotalin and gemcitabine is used as a drug for inhibiting the growth of pancreatic cancer cells.

[0009] Further improvement, the combination of bufotalin and gemcitabine is used as a drug for inhibiting the survival of pancreatic cancer cells.

[0010] Further improvement: bufotalin combined with gemcitabine is used as a drug for promoting DNA damage in pancreatic cancer cells.

[0011] Further improvement: bufotalin combined with gemcitabine is used as a drug for promoting the death of pancreatic cancer cells.

[0012] Further improvement: the molar concentration ratio of bufotalin to gemcitabine is 0.1:5.

[0013] The advantages of the present invention are as follows:

[0014] The present invention has confirmed in vitro experiments that the combined treatment of bufotalin and gemcitabine on pancreatic cancer cells can inhibit their survival, and has confirmed in in vivo experiments that the combined treatment of bufotalin and gemcitabine inhibits the growth of pancreatic cancer cells, promotes DNA damage and cell death. Thus, it is proved that the combined use of bufotalin and gemcitabine can promote the treatment of pancreatic cancer. Description of the Drawings

[0015] Figure 1A It is a diagram showing that the combined use of bufotalin and gemcitabine significantly inhibits the growth of pancreatic cancer cells.

[0016] Figure 1B It is a diagram showing whether the combined treatment of bufotalin and gemcitabine on pancreatic cancer has a synergistic effect, an antagonistic effect or an additive effect.

[0017] Figure 2 It is a diagram showing that the combined use of bufotalin and gemcitabine inhibits the survival of pancreatic cancer cells. Among them, A is the colony formation diagram of pancreatic cancer cells Patu-8988T treated with the combined drug of bufotalin and gemcitabine, B is the quantification diagram of A, C is the colony formation diagram of pancreatic cancer cells Patu-8988S treated with the combined drug of bufotalin and gemcitabine, and D is the quantification diagram of C.

[0018] Figure 3 It is a diagram showing that the combined use of bufotalin and gemcitabine promotes DNA damage in pancreatic cancer cells. Among them, A is the Tunel labeling diagram of pancreatic cancer cells Patu-8988T treated with the combined drug of bufotalin and gemcitabine, and B is the quantification diagram of A.

[0019] Figure 4 It is a diagram showing that the combined use of bufotalin and gemcitabine promotes the death of pancreatic cancer cells. Among them, A is the apoptosis rate diagram of pancreatic cancer cells Patu-8988T treated with the combined drug of bufotalin and gemcitabine, and B is the quantification diagram of A.

[0020] Figure 5It is a graph showing the inhibition of tumor growth by bufotalin combined with gemcitabine. Among them, A is the fluorescence imaging graph of the subcutaneous tumor of mice treated with bufotalin combined with gemcitabine, B is the graph showing the relationship between the volume of the subcutaneous tumor of mice treated with bufotalin combined with gemcitabine and the administration time, and C is the graph of the solid tumor of the bufotalin combined with gemcitabine drug group and each single-drug group. Detailed implementation manners

[0021] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of the present invention. The terms used in the specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0023] The experimental materials used in the following examples are as follows: Cell lines: The human pancreatic cancer cell lines Patu-8988T and Patu-8988S were provided by Zhejiang Meisen Cell Technology Co., Ltd.; DMEM medium was purchased from Sangon Biotech (Shanghai) Co., Ltd. (Catalog number: E600003-0500); Fetal bovine serum was purchased from Sangon Biotech (Shanghai) Co., Ltd. (Catalog number: E600001-0500); CCK-8 kit was purchased from Selleckchem.com (Catalog number: B34304); DMSO was purchased from coolaber (Catalog number: CD4731C-100mL); Bufotalin was purchased from Chengdu Pufa Technology Development Co., Ltd. (Catalog number: 471-95-4); Gemcitabine was purchased from aladdin (Catalog number: G127944-1g); Annexin V-FITC / PI Apoptosis Detection Kit was purchased from Vazyme (Catalog number: A211-01); TUNEL FITC Apoptosis Detection Kit was purchased from Vazyme (Catalog number: A111-01); 4% PFA tissue cell fixative was purchased from Solarbio (Catalog number: P1110); Crystal violet staining solution was purchased from BBI (Catalog number: E607309-0100); Trypsin 0.25% (1x) Solution was purchased from cytiva (Catalog number: SH30042.01); Penicllin-Streptomycin Solution was purchased from cytiva (Catalog number: SH40003.01); Fetal Bovine Serum, Standard was purchased from BBI (Catalog number: E600001-0500); Bufotalinin (CS-6) was purchased from Chengdu Pufa Co., Ltd., with HPLC purity greater than 98%, and stored sealed at -20°C.

[0024] The experimental instruments used in the following examples are as follows:

[0025] Experimental instruments: Biological safety cabinet (Qingdao Haier model HR1780-IIA2), CO2 incubator (Eppendorf AG model GALAXY), Microplate reader (Thermo Varioskan Flash Rev.1.2), Flow cytometer (Suzhou Saijing model DxFLEX); Fluorescence microscope (Nanjing Nikon model ECLIPSE Ci-S). Multifunctional in vivo animal imaging system (BIO COVER model Ancel).

[0026] Example 1

[0027] Bufotalin and gemcitabine have a significant synergistic effect in the treatment of pancreatic cancer cells:

[0028] Test article: Add 1.1247 mL of DMSO to a vial containing 5 mg of bufotalin to dissolve it and prepare a 10 mM solution; weigh 5 mg of gemcitabine and add 3.7994 mL of DMSO to dissolve it and prepare a 5 mM solution.

[0029] Experimental protocol: (A) Detect the growth inhibitory effect of the combined use of bufotalin and gemcitabine on human pancreatic cancer cells through a colony formation assay: Digest human pancreatic cancer cells (Patu-8988T) with trypsin, count them, and mix them with a complete medium (1% double antibody + 10% fetal bovine serum + 89% DMEM medium) to prepare a cell suspension. Add 2 mL of the cell suspension (1×10 3 cells per well) to each well of a 6-well plate. Set the drug to be diluted to the required concentration gradient with the complete medium (the concentration gradient of bufotalin is 0 μM, 0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, 1.6 μM; the concentration gradient of gemcitabine is 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM; after cross-matching, there are a total of 36 concentration gradients). Add 2 mL of the corresponding drug-containing medium to each well, and then place the 6-well plate in an incubator at 37°C with 5% CO2 for culture. Count the number of cell colonies with more than 100 cells under a microscope, aspirate the drug-containing medium in the wells, wash 3 times with 1×PBS, add 500 μL of 4% PFA tissue cell fixative to each well for fixation, gently shake on a shaker for 15 min, wash 2 times with 1×PBS, add 500 μL of crystal violet staining solution to each well for staining, gently shake on a shaker for 15 min, rinse with distilled water, and air dry. (B) Use the CCK-8 method to calculate the inhibition rate of the combined use of bufotalin and gemcitabine on human pancreatic cancer cells, and calculate the CI index at different concentrations through CompuSyn software: Digest human pancreatic cancer cells (Patu-8988T) with trypsin, count them, and mix them with a complete medium (1% double antibody + 10% fetal bovine serum + 89% DMEM medium) to prepare a cell suspension. Add 100 μL of the cell suspension (5×10 3Cells), and then cultured overnight in an incubator at 37°C with 5% CO2; aspirate the medium in the wells, dilute the drug to the required concentration gradient with complete medium (the concentration gradients of bufotalin are 0 μM, 0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, 1.6 μM; the concentration gradients of gemcitabine are 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM; after cross-matching, there are a total of 36 concentration gradients, add 100 μL of the corresponding drug-containing medium to each well, and then place the 96-well plate in an incubator at 37°C with 5% CO2 and culture for 48 hours. Aspirate the drug-containing medium in the wells, add 100 μL of CCK-8 dilution (CCK-8 reagent: DMEM medium = 1:9) to each well, continue to culture for 1 hour, measure the absorbance of each well, i.e., the OD value, with an enzyme-linked immunosorbent assay at a wavelength of λ = 450 nm. Subtract the average value of the cell-free medium from the average value of each replicate well as the OD value of the cells in this group, compare with the control, calculate the cell inhibition rate after drug treatment, and calculate the CI index of different concentrations through CompuSyn software.

[0030] Experimental results: The experimental results of (A) were determined by colony formation assay. The combined use of bufotalin and gemcitabine significantly inhibited the growth of pancreatic cancer cells as Figure 1A shown. For the results of (B): To evaluate whether bufotalin has a synergistic effect with GEM, we calculated the combination index (CI) values for each dose combination. CI < 1 indicates a synergistic effect between the two drugs; when the CI index is 1, it indicates an additive effect between the two drugs; CI >= 1.2 indicates an antagonistic effect between the two drugs. Among the 25 combined treatments of GEM and bufotalin, we observed a significant synergistic effect of the combination of bufotalin and GEM at specific ratios as Figure 1B shown.

[0031] Among the 36 treatments, there is one pure control group without adding any agents, five experimental groups with only BT added, five experimental groups with only Gem added, and 25 groups with BT and Gem added in combination. Among them, the first four groups with a BT concentration of 0.4 μm showed obvious antagonistic effects. The fifth group with a BT concentration of 0.4 μm and 1.6 μm and the first group with a BT concentration of 0.2 μm showed slightly antagonistic effects. The second and fourth groups with a BT concentration of 0.2 μm showed synergistic effects, but were closer to additive effects. The remaining sixteen groups showed obvious synergistic effects. (From Figure 1A and Figure 1B it can be seen that when the BT concentration is 0.1 and the Gem concentration is 5, there is a good synergistic effect, and there is no toxicity to normal cells in this concentration range. Therefore, this concentration ratio is used in subsequent experiments.)

[0032] Example 2

[0033] Bufotalin combined with gemcitabine in the treatment of pancreatic cancer can inhibit cell survival and promote DNA damage:

[0034] Test articles: The bufotalin solution and gemcitabine solution were prepared by the method described in Experiment 1.

[0035] Experimental method: The inhibitory effect of the combined use of bufotalin and gemcitabine on the growth of human pancreatic cancer cells was detected by colony formation assay.

[0036] Human pancreatic cancer cells (Patu-8988T, Patu-8988S) were digested with trypsin, counted, and mixed with complete medium (1% double antibody + 10% fetal bovine serum + 89% DMEM medium) to prepare cell suspensions. 2 mL of the corresponding drug-containing medium (1×10 3 cells per well) was added to each well of a 6-well plate. The drugs were diluted to the required concentration gradients with complete medium (gemcitabine 5 μM, bufotalin 0.1 μM; 2 mL of complete medium was added to the control group tube, 2 mL of complete medium and 2 μL of 5 mM gemcitabine solution were added to the gemcitabine single-drug group tube, 2 mL of complete medium and 0.2 μL of 1 mM bufotalin solution were added to the bufotalin single-drug group tube, and 2 mL of complete medium, 0.2 μL of 1 mM bufotalin solution and 2 μL of 5 mM gemcitabine solution were added to the combined drug group tube), and then cultured in an incubator at 37 °C with 5% CO2 until colonies of 50 - 100 cells were formed; the medium in the wells was aspirated, washed 3 times with 1×PBS, 500 μL of 4% tissue cell fixative was added to each well for fixation, gently shaken on a shaker for 15 min, washed 2 times with 1×PBS, 500 μL of crystal violet staining solution was added to each well for staining, gently shaken on a shaker for 15 min, rinsed with distilled water, and air-dried. The results are as Figure 2 shown (the same experiment was performed on two pancreatic cancer cell lines, namely Patu-8988T and Patu-8988S).

[0037] Experimental method: The effect of the combined treatment of bufotalin and gemcitabine on DNA damage in human pancreatic cancer was detected by Tunel assay:

[0038] Human pancreatic cancer cells (Patu-8988T) were digested with trypsin, counted, and mixed with complete medium (1% double antibody + 10% fetal bovine serum + 89% DMEM medium) to prepare cell suspensions. Cell slides were placed in each well of a 6-well plate, and 2 mL of cell suspension was added to each well (the number of cells in four wells was 12×10 4) Then, it was placed in an incubator at 37°C with 5% CO2 and cultured overnight. The culture medium in the wells was aspirated, and the drug was diluted to the required concentration with complete medium (gemcitabine 5 μM, bufotalin 0.1 μM; 2 mL of complete medium was added to the control group tube, 2 mL of complete medium and 2 μL of 5 mM gemcitabine solution were added to the gemcitabine monotherapy group tube, 2 mL of complete medium and 0.2 μL of 1 mM bufotalin solution were added to the bufotalin monotherapy group tube, and 2 mL of complete medium, 0.2 μL of 1 mM bufotalin solution and 2 μL of 5 mM gemcitabine solution were added to the combination therapy group tube). 2 mL of the corresponding drug-containing culture medium was added to each well, and then the 6-well plate was placed in an incubator at 37°C with 5% CO2 and cultured for 48 hours. The drug-containing culture medium in the wells was aspirated, washed 3 times with 1×PBS, 500 μL of 4% tissue cell fixative was added and fixed at 4°C for 25 min, washed twice with 1xPBS, and gently shaken on a shaker for 5 min each time. 500 μL of 0.2% Triton X-100 solution prepared with 1xPBS was added, incubated at room temperature for 5 min, and rinsed 2 - 3 times with 1xPBS solution. 5x Equilibration Buffer was diluted to 1x Equilibration Buffer with ddH2O, 100 μL of 1x Equilibration Buffer was added dropwise to completely cover the coverslip, and equilibrated at room temperature for 30 min. Then, 50 μL of TdT incubation buffer (34 μL of ddH20 μL, 10 μL of 5x Equilibration Buffer, 5 μL of FITC-12-dUTP Labeling Mix, 1 μL of Recombinant TdT Enzyme per well) was added to the sample, and a coverslip of the same size as the coverslip was gently covered on the coverslip to ensure uniform distribution of the reagent, and incubated at 37°C for 60 min. Washed 2 times with 1xPBS solution for 5 min each time at room temperature, washed 3 times with 1xPBS containing 0.1% Triton X-100 and 5 mg / mL BSA for 5 min each time, 150 μL of DAPI staining solution was added to each well, counterstained at room temperature for 5 min, washed 3 times with 1xPBS for 5 min each time at room temperature, 50 μL of 20% glycerol prepared with 1xPBS was added dropwise on the glass slide for mounting, and the sample was analyzed under a fluorescence microscope.

[0039] Experimental results: TUNEL staining assay can be used to detect DNA damage. The results of TUNEL assay showed that after the combined treatment of bufotalin and gemcitabine on pancreatic cancer cells Patu-8988T, the DNA damage in the combination therapy group was significantly increased compared with the gemcitabine monotherapy group and the bufotalin monotherapy group, as shown specifically in Figure 3 the following. These results indicate that bufotalin promotes DNA damage induced by chemotherapeutic drugs.

[0040] Example 4

[0041] Bufotalin combined with gemcitabine in the treatment of pancreatic cancer promotes cell death:

[0042] Test samples: The bufotalin solution and gemcitabine solution were prepared by the methods described in Experiment 1 and Experiment 2. Weigh 5 mg of camptothecin and dissolve it in mL of DMSO (as a positive control) to prepare a 5 mM solution.

[0043] Experimental method: Detect the effect of the combination of bufotalin and gemcitabine on the apoptosis of human pancreatic cancer cells by Anxin V / PI assay: Digest human pancreatic cancer cells (Patu-8988T) with trypsin, count them, and mix them with complete medium (1% double antibody + 10% fetal bovine serum + 89% DMEM medium) to make a cell suspension. Add 2 mL of the cell suspension to each well of a 6-well plate (2.4×10 5 cells per well), then place it in an incubator at 37°C with 5% CO2 overnight. Aspirate the medium in the wells, dilute the drugs to the required concentrations with complete medium (5 μM gemcitabine, 0.1 μM bufotalin, 16 μM camptothecin (positive); add 2 mL of complete medium to the negative control tube, add 2 mL of complete medium and 6.4 μL of 5 mM camptothecin to the positive control tube, add 2 mL of complete medium and 2 μL of 5 mM gemcitabine solution to the gemcitabine single-drug group tube, add 2 mL of complete medium and 0.2 μL of 1 mM bufotalin solution to the bufotalin single-drug group tube, add 2 mL of complete medium, 0.2 μL of 1 mM bufotalin solution and 2 μL of 5 mM gemcitabine solution to the combination drug group tube), add 2 mL of the corresponding drug-containing medium to each well, and then place the 6-well plate in an incubator at 37°C with 5% CO2 and culture for 48 hours. Aspirate the drug-containing medium in the wells, wash 3 times with 1×PBS, add 500 μL of 1×PBS and 200 μL of 0.25% trypsin to each well for digestion, add 100 μL of complete medium to terminate digestion, centrifuge at 1800 rpm for 5 min, discard the supernatant, wash twice with 1 mL of 1×PBS, centrifuge at 1800 rpm for 5 min, count the cells, take 1×10 5 ~5×10 5 cells into new EP tubes. Take two tubes for the negative control group (blank and double staining), take two tubes for the positive control group (Anxin V single staining and PI single staining), and take one tube for each of the other groups (both double staining), centrifuge at 1800 rpm for 5 min, discard the supernatant, resuspend the cells with 100 μL of Binding Buffer, add 5 μL of the required dye to each tube, incubate in the dark at 25°C for 10 min, and add 400 μL of Binding Buffer for on-machine detection.

[0044] Experimental results: After detecting the combined treatment of bufotalin and gemcitabine on pancreatic cancer cells Patu-8988T by Annexin V-FITC / PI staining method, the results showed that the apoptosis ratio of the combined treatment group was significantly increased compared with that of the single-drug gemcitabine group and the bufotalin single-drug treatment group( Figure 4 ).

[0045] Example 5

[0046] Bufotalin combined with gemcitabine inhibits the growth of subcutaneously transplanted pancreatic cancer cells Patu-8988T in nude mice:

[0047] Experimental animals: BALB / c nude mice, male, 4 weeks old, weighing about 17 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., production license number: SYXK(Beijing)2022-0052, animal certificate number: 110011241103906223. Breeding environment: SPF level.

[0048] All experimental animals were raised in independent ventilated boxes with constant temperature and humidity. The temperature of the breeding room was 20-26 °C, the humidity was 40-70%, the air change was 10-20 times / hour, and the day-night light-dark cycle was 12 h / 12 h; cobalt-60 irradiated sterilized rat complete pellet feed was continuously supplied, and the animals could freely ingest it without limit. Tap water (used after high-pressure steam sterilization) was provided, and the water bottles were continuously supplied with water for free ingestion. The breeding mouse boxes were polysulfone mouse boxes, used after high-pressure sterilization, with a specification of 325 mm × 210 mm × 180 mm; the bedding was high-pressure sterilized corn cob, with 5 animals in each box; the experimental animals were marked with ear tags.

[0049] Drug preparation: Weigh powdered bufotalin and gemcitabine and dissolve them in a solvent solution (10% DMSO + 30% PEG300 + 60% PBS) respectively.

[0050] Experimental method: Human pancreatic cancer Patu-8988T cells were cultured in DMEM medium containing 10% fetal bovine serum. Collect Patu-8988T cells in the exponential growth phase, resuspend them with PBS to a suitable concentration (1×10 7 / ml) cell suspension for subcutaneous tumor inoculation in nude mice. 200 μL of cell suspension (containing 1×10 7 Patu-8988T cells) was inoculated subcutaneously under the right armpit of nude mice. When the tumors grew to an average volume of about 70 mm 3 , they were randomly grouped according to the tumor size, and drugs were administered on the day of grouping, and the day of drug administration was defined as day 0. The nude mice were administered by oral gavage, and the administration volume was 200 μL / mouse. The specific administration scheme is shown in Table 1. The major axis a and minor axis b of the tumors were measured with vernier calipers every other day. V = (length × width 2) / 2. After 4 weeks of observation, the mice were euthanized by inhaling carbon dioxide, and the transplanted tumors were isolated for analysis.

[0051] Table 1. Instructions for the dosing regimen

[0052]

[0053] Experimental results

[0054] We conducted in vivo xenograft experiments to investigate whether bufotalin and gemcitabine have a synergistic inhibitory effect on PDAC tumors when used in combination. As expected, we observed a statistically significant reduction in tumor volume in mice that received combination therapy for one week. Notably, the effect of the combination of bufotalin and gemcitabine was significantly stronger than that of GEM or bufotalin alone, as shown in Figure 5 the figure.

[0055] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A use of bufaline combined with gemcitabine, characterized in that: The bufotoxin combined with gemcitabine is used to prepare a drug for treating pancreatic cancer, wherein the molar concentration of the bufotoxin is 0.1 μM, and the molar concentration of gemcitabine is 5-80 μM.

2. The use of bufaline combined with gemcitabine according to claim 1, characterized in that: The bufaline combined with gemcitabine is used for preparing a medicine for inhibiting the growth of pancreatic cancer cells.

3. The use of bufaline combined with gemcitabine according to claim 1, characterized in that: The bufotolin combined with gemcitabine is used for preparing a medicine for inhibiting the survival of pancreatic cancer cells.

4. The use of bufaline combined with gemcitabine according to claim 1, characterized in that: The bufotolin combined with gemcitabine is used for preparing a drug for promoting DNA damage of pancreatic cancer cells.

5. The use of bufaline combined with gemcitabine according to claim 1, characterized in that: The bufotolin combined with gemcitabine is used for preparing a drug for promoting the death of pancreatic cancer cells.

6. The use of bufaline combined with gemcitabine according to claim 1, characterized in that: The molar concentration of bufaline is 0.1 μM, and the molar concentration of gemcitabine is 5 μM.

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