Application of bufalin combined with gemcitabine

Abstract

The invention discloses application of bufotalin combined with gemcitabine, the bufotalin combined with gemcitabine is used as a medicine for treating pancreatic cancer, and the molar concentration ratio of the bufotalin to the gemcitabine is (0.1-0.2): (5-80) or (0.8-1.6): (5-40) or (0.8: 80). In-vitro experiments prove that the survival of the pancreatic cancer cells can be inhibited by combined treatment of the bufotalin and the gemcitabine, and in-vivo experiments prove that the growth of the pancreatic cancer cells is inhibited by combined treatment of the bufotalin and the gemcitabine, and DNA damage and cell death are promoted. Therefore, the combined use of the bufotalin and the gemcitabine can promote the treatment of the pancreatic cancer.

Description

Technical Field

[0001] The invention relates to the field of pharmacy, and in particular to a use of bufalin combined with gemcitabine. Background Art

[0002] Pancreatic cancer (PC) is a highly malignant digestive system tumor with a very poor prognosis. It is known as the "king of cancers." Globally, the incidence and mortality of pancreatic cancer are gradually increasing. According to the 2022 global cancer statistics, the incidence of pancreatic cancer ranks 12th (about 500,000 cases), and its mortality rate ranks 6th among cancer-related mortality rates (about 470,000 cases). The incidence and mortality of pancreatic cancer in men are higher than those in women. The number of new cases in men is 256,000, and in women is 240,000. According to the results of epidemiological surveys, the incidence and mortality of pancreatic cancer vary greatly among countries in the world, among which the incidence and mortality of pancreatic cancer in high-income countries are significantly higher than those in the United States. Middle- and low-income countries. The diagnosis rate of PDAC in young individuals is increasing, with the most significant increase observed in young women. Increased obesity, diabetes, and alcohol use are considered possible factors leading 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 a difficult problem to solve - chemotherapy drug resistance, and its formation mechanism is closely related to the activation of the DNA damage response (DDR) pathway. Therefore, in order to solve this critical problem, understanding its complex chemotherapy resistance mechanism and exploring small molecule drugs targeting the DDR pathway have become the main tasks at this 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. DNA damage caused by adverse external stimuli in cells [3] It will activate the DNA damage response signaling pathway. In the DNA damage signaling pathway, the DNA damage repair mechanism is an important reason for tumor cells to develop chemotherapy resistance.

[0004] Toad venom is a white pulp secreted by the parotid glands and skin glands of the Chinese giant toad or the black-spotted toad, which is processed and dried. It has anti-tumor, analgesic, anesthetic, and anti-infective effects. It has good therapeutic effects on liver cancer, esophageal cancer, lung cancer, skin cancer and other cancers. The various active ingredients of toad venom have anti-cancer effects. Among these ingredients, toad venom has the best effect. Studies have shown that in liver cancer cells HepG2, toad venom inhibits the AKT-mediated signaling pathway, induces upregulation of p53 gene expression, and causes cells to stagnate in the G2 / M phase, thereby inhibiting the proliferation and migration of liver cancer cells HepG2; in HEP3B cells, toad venom can activate caspase-3 by activating caspase-8 It can promote the activation of caspase-3, caspase-8 and caspase-9, increase mitochondrial tBid and cause mitochondrial dysfunction, destroy mitochondrial membrane potential and cause apoptosis-inducing factor (AIF) to translocate from mitochondria to the nucleus, thus leading to cell apoptosis, and further inhibiting the growth of HEP3B cells; in non-small cell lung cancer A549 cells, it promotes GPX4 degradation and Fe2+ accumulation to induce ferroptosis, inhibiting the growth of tumor cells; in esophageal squamous cell carcinoma ESCC and ECA-109 cells, bufotolin 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. The above studies show that bufotolin can inhibit tumor cell proliferation through multiple pathways such as cell cycle arrest, apoptosis and ferroptosis, but there are few studies on its connection with DNA damage, and it has not been reported whether it can promote tumor cell death through the DNA damage pathway. Therefore, the research on this drug in DNA damage has the opportunity to become a breakthrough in overcoming tumor resistance and promote the research on the mechanism of chemotherapy resistance. Summary of the invention

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

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

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

[0008] In a further improvement, the bufotolin is combined with gemcitabine to be used as a drug for inhibiting the growth of pancreatic cancer cells.

[0009] As a further improvement, the bufotolin is combined with gemcitabine to be used as a drug for inhibiting the survival of pancreatic cancer cells.

[0010] In a further improvement, the bufotolin is combined with gemcitabine as a drug for promoting DNA damage in pancreatic cancer cells.

[0011] In a further improvement, the bufotolin is combined with gemcitabine to be used as a drug for promoting the death of pancreatic cancer cells.

[0012] In a further improvement, the molar concentration ratio of bufaline to gemcitabine is 0.1:5.

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

[0014] The present invention confirms from in vitro experiments that the combined treatment of bufaline and gemcitabine can inhibit the survival of pancreatic cancer cells, and from in vivo experiments confirms that the combined treatment of bufaline and gemcitabine inhibits the growth of pancreatic cancer cells and promotes DNA damage and cell death. Thus, it is proved that the combined use of bufaline and gemcitabine can promote the treatment of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A The figure shows that the use of bufotolin combined with gemcitabine significantly inhibited the growth of pancreatic cancer cells.

[0016] Figure 1B This is a graph showing whether the combined effect of bufotolin and gemcitabine in the treatment of pancreatic cancer is synergistic, antagonistic or additive.

[0017] Figure 2 The figures are as follows: A is the colony formation graph of pancreatic cancer cell Patu-8988T treated with bufotoxin combined with gemcitabine, B is the quantification graph of A, C is the colony formation graph of pancreatic cancer cell Patu-8988S treated with bufotoxin combined with gemcitabine, and D is the quantification graph of C.

[0018] Figure 3 This is a diagram of bufotoxin combined with gemcitabine promoting DNA damage in pancreatic cancer cells, where A is the Tunel labeling diagram of pancreatic cancer cells Patu-8988T treated with bufotoxin combined with gemcitabine, and B is the quantification diagram of A.

[0019] Figure 4 This is a graph showing that bufotolin combined with gemcitabine promotes the death of pancreatic cancer cells, wherein A is a graph showing the apoptosis rate of pancreatic cancer cells Patu-8988T treated with bufotolin combined with gemcitabine, and B is a quantification graph of A.

[0020] Figure 5Figure 3 is a graph showing the inhibition of tumor growth by tovastatin combined with gemcitabine, wherein A is a mouse fluorescence imaging graph of subcutaneous tumors in mice treated with tovastatin combined with gemcitabine, B is a graph showing the relationship between the volume of subcutaneous tumors in mice treated with tovastatin combined with gemcitabine and the administration time, and C is a graph showing solid tumors in the tovastatin combined with gemcitabine drug group and each single drug group. DETAILED DESCRIPTION

[0021] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are shown 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, the purpose of providing these embodiments is 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 those commonly understood by those skilled in the art of the present invention. The terms used in the specification are only for describing specific implementation purposes and are not intended to limit the present invention.

[0023] The experimental materials used in the following examples are as follows: cell lines: human pancreatic cancer cell lines Patu-8988T and Patu-8988S were provided by Zhejiang Meisen Cell Technology Co., Ltd.; DMEM culture medium was purchased from Sangon Biotechnology (Shanghai) Co., Ltd. (catalog number: E600003-0500); fetal bovine serum was purchased from Sangon Biotechnology (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); bufotolin was purchased from Chengdu Purifa 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 No.: A111-01); 4% PFA tissue cell fixative was purchased from Solarbio (Catalog No.: P1110); crystal violet stain was purchased from BBI (Catalog No.: E607309-0100); Trypsin 0.25% (1x) Solution was purchased from cytiva (Catalog No.: SH30042.01); Penicllin-Streptomycin Solution was purchased from cytiva (Catalog No.: SH40003.01); Fetal Bovine Serum, Standard was purchased from BBI (Catalog No.: E600001-0500); CS-6 was purchased from Chengdu Purifa Co., Ltd., HPLC purity greater than 98%, and stored in a sealed container 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 animal live imaging system (BIO COVER model Ancel).

[0026] Example 1

[0027] The combination of bufotolin and gemcitabine in the treatment of pancreatic cancer cells has a significant synergistic effect:

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

[0029] Experimental plan: (A) Colony formation assay to detect the inhibitory effect of bufotolin combined with gemcitabine on the growth of human pancreatic cancer cells: Human pancreatic cancer cells (Patu-8988T) were digested with trypsin, counted, and mixed with complete culture medium (1% double antibody + 10% fetal bovine serum + 89% DMEM culture medium) to prepare a cell suspension. 2 mL of cell suspension (1×10 cells per well) was added to each well of a 6-well plate. 3 cells), set the drug to the required concentration gradient with complete medium (the concentration gradient of bufotolin is 0μM, 0.1μM, 0.2μM, 0.4μM, 0.8μM, 1.6μM; the concentration gradient of gemcitabine is 0μM, 5μM, 10μM, 20μM, 40μM, 80μM; there are 36 concentration gradients after cross-matching, add 2mL of the corresponding drug-containing medium to each well, and then place the 6-well plate in a 37℃5%CO2 incubator for culture. Count the number of cell colonies greater than 100 under a microscope, aspirate the drug-containing medium in the well, wash 3 times with 1×PBS, and add 500μL to each well. Fix with 4% PFA tissue cell fixative, shake gently on a shaker for 15 minutes, wash twice with 1×PBS, add 500μL crystal violet stain to each well, shake gently on a shaker for 15 minutes, rinse with distilled water, and dry. (B) The inhibition rate of bufotolin combined with gemcitabine on human pancreatic cancer cells was calculated using the CCK-8 method, and the CI index of different concentrations was calculated using CompuSyn software: Human pancreatic cancer cells (Patu-8988T) were digested with trypsin, counted, and mixed with complete culture medium (1% double antibody + 10% fetal bovine serum + 89% DMEM culture medium) to prepare a cell suspension. Add 100μL of cell suspension (5×10 per well) to each well of a 96-well plate. 3cells), and then placed in a 37°C 5% CO2 incubator for overnight culture; aspirate the medium in the wells, and dilute the drug with complete medium to the required concentration gradient (the concentration gradient of bufotolin is 0μM, 0.1μM, 0.2μM, 0.4μM, 0.8μM, 1.6μM; the concentration gradient of gemcitabine is 0μM, 5μM, 10μM, 20μM, 40μM, 80μM; after cross-matching, there are 36 concentration gradients in total, 100μL of the corresponding drug-containing culture medium is added to each well, and then the 96-well plate is placed in a 37°C 5% CO2 incubator for 48 hours. Aspirate the drug-containing culture medium in the wells, and add 100μL of each well CCK-8 diluent (CCK-8 reagent: DMEM medium = 1:9) was added, and the culture was continued for 1 hour. The absorbance of each well, i.e., OD value, was detected by a microplate reader at a wavelength of λ = 450 nm. The OD value of the group of cells was taken as the average value of each duplicate well minus the average value of the cell-free medium. The cell inhibition rate after drug treatment was calculated by comparing with the control, and the CI index of different concentrations was calculated by CompuSyn software.

[0030] Experimental results: For the experimental results of (A), the colony formation assay showed that the combination of bufotolin and gemcitabine significantly inhibited the growth of pancreatic cancer cells. Figure 1A As shown. For the results of (B): In order to evaluate whether bufaline has a synergistic effect with GEM, we calculated the combination index (CI) value of each dose combination. CI < 1 indicates that there is a synergistic effect between the two drugs; when the CI index is 1, it means that the two drugs have an additive effect; CI> = 1.2 indicates that there is an antagonistic effect between the two drugs. In the 25 combined treatments of GEM and bufaline, we observed that the combination of bufaline and GEM at a specific ratio had a significant synergistic effect as shown in Figure 2. Figure 1B shown.

[0031] Among the 36 treatments, there was one pure control group without adding drugs, five experimental groups with BT alone and five experimental groups with Gem alone, and 25 groups with BT and Gem mixed. Among them, the first four groups with BT concentration of 0.4μm had obvious antagonistic effects. The fifth group with BT concentration of 0.4μm and 1.6μm and the first group with BT concentration of 0.2μm had a slight antagonistic effect. The second and fourth groups with BT concentration of 0.2μm had synergistic effects, but they were closer to additive effects. The remaining 16 groups had 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, so the subsequent experiments use this concentration ratio. )

[0032] Example 2

[0033] Combination therapy of bufotolin and gemcitabine in pancreatic cancer can inhibit cell survival and promote DNA damage:

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

[0035] Experimental method: The colony formation experiment was used to detect the growth inhibitory effect of bufotolin combined with gemcitabine on human pancreatic cancer cells.

[0036] Human pancreatic cancer cells (Patu-8988T, Patu-8988S) were digested with trypsin, counted, and mixed with complete culture medium (1% double antibody + 10% fetal bovine serum + 89% DMEM culture medium) to prepare a cell suspension. 2 mL of the corresponding drug-containing culture medium (1×10 cells / well) was added to each well of a 6-well plate. 3 cells), dilute the drug to the required concentration gradient (gemcitabine 5μM, bufotoxin 0.1μM; add 2mL of complete medium to the control group tube, add 2mL of complete medium and 2μL of 5mM gemcitabine solution to the gemcitabine monotherapy group tube, add 2mL of complete medium and 0.2μL of 1mM bufotoxin solution to the bufotoxin monotherapy group tube, add 2mL of complete medium, 0.2μL of 1mM bufotoxin solution and 2μL of 5mM gemcitabine solution to the combined drug group tube), then culture in a 37℃ 5% CO2 incubator until a colony of 50-100 cells is formed; aspirate the medium in the well, wash 3 times with 1×PBS, add 500μL of 4% tissue cell fixative to each well for fixation, shake gently on a shaker for 15min, wash 2 times with 1×PBS, add 500μL of crystal violet stain to each well for staining, shake gently on a shaker for 15min, rinse with distilled water, dry, and the results are as follows Figure 2 As shown (the same experiment was performed on two pancreatic cancer cell lines, Patu-8988T and Patu-8988S).

[0037] Experimental method: Tunel experiment was used to detect the effect of combined treatment of bufotoxin and gemcitabine on DNA damage in human pancreatic cancer:

[0038] Human pancreatic cancer cells (Patu-8988T) were digested with trypsin, counted, and mixed with complete culture medium (1% double antibody + 10% fetal bovine serum + 89% DMEM culture medium) to make a cell suspension. Place a cell slide in a 6-well plate and add 2 mL of cell suspension to each well (the number of cells in four wells is 12×10 4), then placed in a 37℃5%CO2 incubator for overnight culture, aspirate the medium in the wells, dilute the drugs to the required concentrations with complete medium (gemcitabine 5μM, bufotolin 0.1μM; add 2mL of complete medium to the control tube, add 2mL of complete medium and 2μL of 5mM gemcitabine solution to the gemcitabine monotherapy group tube, add 2mL of complete medium and 0.2μL of 1mM bufotolin solution to the bufotolin monotherapy group tube, add 2mL of complete medium, 0.2μL of 1mM bufotolin solution and 2μL of 5mM gemcitabine solution to the combined drug group tube), add 2mL of the corresponding drug-containing medium to each well, and then place the 6-well plate in a 37℃5%CO2 incubator for 48 hours. Aspirate the drug-containing medium in the wells, wash 3 times with 1×PBS, add 500μL of 4% tissue cell fixative and fix at 4℃ for 25min, wash twice with 1xPBS, shake gently on a shaker, and place for 5min each time. Add 500μL 0.2% TritonX-100 solution prepared with 1xPBS, incubate at room temperature for 5min, and rinse with 1xPBS solution 2-3 times. Dilute 5x Equilibration Buffer to 1xEquilibration Buffer with ddH2O, add 100μL 1x Equilibration Buffer to cover the slide completely, and equilibrate at room temperature for 30min. Then add 50μL TdT incubation buffer (34μL ddH20μL per well, 10μL 5xEquilibration Buffer, 5μL FITC-12-dUTPLabeling Mix, 1μL Recombinant TdT Enzyme) to the sample, cover the slide lightly with a sealing film of the same size as the slide to ensure uniform distribution of the reagents, and incubate at 37℃ for 60min. The slides were washed twice with 1xPBS solution, each time for 5 min at room temperature, washed three times with 1xPBS containing 0.1% Triton X-100 and 5 mg / mL BSA, each time for 5 min, 150 μL of DAPI staining solution was added to each well, counterstained for 5 min at room temperature, washed three times with 1xPBS, each time for 5 min at room temperature, 50 μL of 20% glycerol prepared in 1xPBS was added to the slides for sealing, and the samples were analyzed under a fluorescence microscope.

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

[0040] Example 4

[0041] Combination therapy of bufotolin and gemcitabine promotes cell death in pancreatic cancer:

[0042] Test sample: The bufaline solution and gemcitabine solution were prepared by the methods described in Experiment 1 and Experiment 2. 5 mg of camptothecin was weighed and added into mL DMSO to dissolve (as a positive control) to prepare a 5 mM solution.

[0043] Experimental method: The effect of bufotolin combined with gemcitabine on apoptosis of human pancreatic cancer cells was detected by Anxin V / PI experiment: human pancreatic cancer cells (Patu-8988T) were digested with trypsin, counted, and mixed with complete culture medium (1% double antibody + 10% fetal bovine serum + 89% DMEM culture medium) to prepare cell suspension. 2 mL of cell suspension (2.4×10 cells per well) was added to each well of a 6-well plate. 5 cells), and then placed in a 37°C 5% CO2 incubator for overnight culture, the culture medium in the wells was aspirated, and the drugs were diluted with complete culture medium to the required concentration (5 μM gemcitabine, 0.1 μM bufotoxin, 16 μM camptothecin (positive); 2 mL of complete culture medium was added to the negative control group tube, 2 mL of complete culture medium and 6.4 μL of 5mM camptothecin were added to the positive control group tube, 2 mL of complete culture medium and 2 μL of 5mM gemcitabine solution were added to the gemcitabine monotherapy group tube, 2 mL of complete culture medium and 0.2 μL of 1mM bufotoxin solution were added to the bufotoxin monotherapy group tube, and 2 mL of complete culture medium, 0.2 μL of 1mM bufotoxin solution and 2 μL of 5mM gemcitabine solution were added to the combination drug group tube), 2 mL of the corresponding drug-containing culture medium was added to each well, and the 6-well plate was placed in a 37°C 5% CO2 incubator for 48 hours. The drug-containing medium in the wells was aspirated, and the cells were washed three times with 1×PBS. 500 μL 1×PBS and 200 μL 0.25% trypsin were added to each well for digestion. 100 μL complete medium was added to terminate the digestion. The cells were centrifuged at 1800 rpm for 5 min, the supernatant was discarded, 1 mL 1×PBS was added to wash twice, and the cells were centrifuged at 1800 rpm for 5 min. The cells were counted and 1×10 5 ~5×10 5 Transfer the cells to 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 one tube for each of the other groups (all double staining). Centrifuge at 1800rpm for 5min, discard the supernatant, resuspend the cells in 100μL Binding Buffer, add 5μL of the required dye to each tube, incubate at 25℃ for 10min in dark, add 400μL Binding Buffer and detect on the machine.

[0044] Experimental results: Annexin V-FITC / PI staining was used to detect the combination of bufotoxin and gemcitabine in the treatment of pancreatic cancer cells Patu-8988T. The results showed that the apoptosis rate of the combined treatment group was significantly higher than that of the gemcitabine group and the bufotoxin monotherapy group ( Figure 4 ).

[0045] Example 5

[0046] Combination therapy of bufotolin and gemcitabine inhibits the growth of subcutaneously transplanted pancreatic cancer cell Patu-8988T tumors in nude mice:

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

[0048] The experimental animals were kept in independent ventilation boxes with constant temperature and humidity. The temperature of the breeding room was 20-26℃, the humidity was 40-70%, the ventilation was 10-20 times / hour, and the light and dark alternation time was 12h / 12h. The animals were continuously supplied with cobalt 60 radiation sterilized complete pellet feed for mice, and they could take it freely without limit. They also drank tap water (used after high-pressure steam sterilization) and the drinking water bottle was continuously supplied with water for free intake. The mouse box was a polysulfone mouse box, which was used after high-pressure sterilization and had a specification of 325mm×210mm×180mm. The bedding was high-pressure sterilized corn cobs, and there were 5 animals in each box. The experimental animals were marked with ear tags.

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

[0050] Experimental method: Human pancreatic cancer Patu-8988T cells were cultured in DMEM containing 10% fetal bovine serum. Patu-8988T cells in the exponential growth phase were collected and resuspended in PBS to an appropriate concentration (1×10 7 / ml) cell suspension was used for subcutaneous tumor inoculation in nude mice. 200 μL cell suspension (containing 1×10 7 Patu-8988T cells). When the tumor grows to an average volume of about 70 mm 3 At the time of the experiment, the mice were randomly divided into groups according to the tumor size and the drug was administered on the day of grouping, which was defined as day 0. The nude mice were given the drug by oral gavage, and the administration volume was 200 μL / nude mouse. The specific administration scheme is shown in Table 1. The long diameter a and short diameter b of the tumor were measured with a vernier caliper every other day. V = (length × width 2) / 2. After 4 weeks of observation, the mice were euthanized by carbon dioxide inhalation, and the transplanted tumors were isolated and analyzed.

[0051] Table 1. Dosage regimen instructions

[0052]

[0053] Experimental Results

[0054] We performed in vivo xenograft experiments to investigate whether bufotolin combined with gemcitabine has a synergistic inhibitory effect on PDAC tumors. As expected, we observed a statistically significant reduction in tumor volume in mice treated with the combination for one week. Notably, the effect of bufotolin combined with gemcitabine was significantly stronger than that of GEM or bufotolin alone, as shown in Figure 2. Figure 5 shown.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A use of bufaline combined with gemcitabine, characterized in that: The bufotolin combined with gemcitabine is used as a drug for treating pancreatic cancer, wherein the molar concentration ratio of bufotolin to gemcitabine is 0.1-0.2:5-80 or 0.8-1.6:5-40 or 0.8:

80.

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

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

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

6. The use of bufaline combined with gemcitabine according to any one of claims 1 to 5, characterized in that: The molar concentration ratio of bufaline to gemcitabine is 0.1:5.

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