Composition containing cucurbitacine A and application of composition in preparation of pancreatic cancer resisting medicine

Abstract

The invention provides a composition which is composed of cucurbitacine A and gemcitabine. The invention further provides application of the composition in preparation of anti-pancreatic cancer drugs. The composition has the capacity of inhibiting clone formation of pancreatic cancer, inducing apoptosis of pancreatic cancer and / or inhibiting tumor progress. According to the invention, cucurbitacin A and gemcitabine are combined for use, gemcitabine can be synergistically sensitized to inhibit pancreatic cancer tumor activity, pancreatic tumor cells are inhibited, and toxicity of drugs to normal cells is reduced.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a composition comprising cucurbitacin A, and application of the composition in preparing an anti-pancreatic cancer drug. Background Art

[0002] Cucurbitacins are a class of tetracyclic triterpenoid compounds isolated and purified from various plants such as Cucurbitaceae and Cruciferae. They are often used in the treatment of chronic hepatitis and hepatitis caused by damp-heat toxicity. Studies have found that cucurbitacins have multiple biological activities such as anti-inflammatory, antioxidant, immunity enhancement, and gastrointestinal effects. Cucurbitacins A (CuA) is a type of cucurbitacins, but there are few studies on cucurbitacins A.

[0003] Gemcitabine (GEM) is a first-line drug for the treatment of pancreatic cancer. It is superior to the original first-line drug fluorouracil in terms of overall survival and pain control. However, pancreatic cancer is very easy to develop resistance to gemcitabine. Most patients begin to develop resistance a few weeks after taking the drug, which is an important reason for the short survival of patients. In clinical practice, in order to improve the sensitivity of pancreatic cancer to chemotherapy drugs and overcome drug resistance, a combination drug strategy is often used. Gemcitabine combined with cisplatin, gemcitabine combined with paclitaxel, and gemcitabine combined with fluorouracil are common clinical chemotherapy regimens. Although these combination chemotherapy regimens can play a certain role, the effects are generally poor. For example, gemcitabine combined with cisplatin can improve progression-free survival, but the patient's overall survival does not increase; gemcitabine combined with paclitaxel can increase the patient's overall survival and remission rate to a limited extent, but it has obvious side effects; gemcitabine combined with fluorouracil can increase the patient's overall survival but reduce the remission rate. Therefore, exploring new combinations of chemotherapy drugs for pancreatic cancer has always been one of the key issues that need to be urgently addressed in clinical and scientific research. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a composition containing cucurbitacin A, and the application of the composition in the preparation of anti-pancreatic cancer drugs, so as to overcome the above-mentioned defects in the prior art.

[0005] The present invention is achieved by adopting the following technical solutions:

[0006] The first aspect of the present invention is to provide a composition consisting of cucurbitacin A and gemcitabine.

[0007] The present invention finds that the combined use of cucurbitacin A and the chemotherapy drug gemcitabine has an inhibitory effect on pancreatic cancer, and the combined use of the two drugs can produce a significant synergistic effect.

[0008] In the composition of the present invention, the molar ratio of cucurbitacin A to gemcitabine is preferably 1:(0.5 - 100).

[0009] More preferably, the molar ratio of cucurbitacin A to gemcitabine is 1:(2 - 50). When the two components are in the above ratio, they have a significant synergistic effect on the inhibition of pancreatic cancer.

[0010] The second aspect of the present invention is to provide the application of the above composition in the preparation of anti - pancreatic cancer drugs.

[0011] In some embodiments, the cancer cells of pancreatic cancer are BXPC - 3 human - derived pancreatic duct cell line.

[0012] In some embodiments, the anti - pancreatic cancer includes inhibiting the colony - forming ability of pancreatic cancer, inducing apoptosis of pancreatic cancer and / or inhibiting the tumor process.

[0013] The third aspect of the present invention is to provide an anti - pancreatic cancer drug, which includes the above composition and pharmaceutically acceptable excipients.

[0014] The pharmaceutically acceptable excipients include at least one of solvents, diluents, binders, disintegrants, lubricants, flavoring agents, fillers, preservatives, suspending agents, coloring agents or emulsifiers.

[0015] In some embodiments, the pharmaceutically acceptable excipients at least include solvents. The solvents can be commonly used substances in the art, such as dimethyl sulfoxide, ethanol.

[0016] In some preferred embodiments, the solvent is dimethyl sulfoxide.

[0017] Specifically, the anti - pancreatic cancer drug contains dimethyl sulfoxide as the solvent, the concentration range of cucurbitacin A is 0.05 - 0.5 μmol / L, and the concentration range of gemcitabine is 0.25 - 5 μmol / L.

[0018] Preferably, the concentration range of cucurbitacin A is 0.05 - 0.5 μmol / L, and the concentration range of gemcitabine is 1 - 2.5 μmol / L.

[0019] Compared with the prior art, cucurbitacin A of the present invention sensitizes gemcitabine to inhibit the tumor activity of pancreatic cancer with a high - efficiency synergistic effect, rather than simply adding the effects of the two drugs. It can inhibit pancreatic tumor cells while reducing the toxicity of the drug to normal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the structural formula of cucurbitacin A.

[0021] Figure 2These are the results of a control experiment on the clonal inhibition of pancreatic cancer by different drugs. From left to right in the figure are: a - control group, b - cucurbitacin A, c - gemcitabine, d - photo after culturing with cucurbitacin A + gemcitabine, and the rightmost is the statistical chart of the number of clones formed.

[0022] Figure 3 These are the results of a control experiment on the apoptosis of pancreatic cancer cells. The apoptosis rate of cells was detected by flow cytometry. From left to right in the figure are: a - control group, b - cucurbitacin A, c - gemcitabine, d - detection result chart of cucurbitacin A + gemcitabine, and the rightmost is the statistical chart of the apoptosis rate.

[0023] Figure 4 These are the results of a control experiment on the inhibitory effect of different drugs on subcutaneous tumors in mice. Figure 4 A shows the tumor photos of each group. Figure 4 B shows the statistical chart of the tumor volume of each group. In the figure, group a - normal saline (control group), group b - gemcitabine (50 mg / kg), group c - cucurbitacin A (1 mg / kg), and group d - combination of gemcitabine (50 mg / kg) + cucurbitacin A (1 mg / kg).

[0024] Figure 5 These are the results of the experiment on the inhibition of the JAK2 / STAT3 signaling pathway by cucurbitacin A and gemcitabine. Specific proteins were detected by an enhanced chemiluminescence system, and the protein expression was analyzed using Image J software. Among them Figure 5 A, 5B correspond to the inhibition results of cucurbitacin A at different concentrations on the STAT3 signaling pathway. Figure 5 C, 5D correspond to the inhibition results of gemcitabine at different concentrations on the JAK2 signaling pathway. Figure 5 E, 5F correspond to the inhibition results of gemcitabine at different concentrations on the STAT3 signaling pathway.

[0025] In the figure, CuA represents cucurbitacin A, GEM represents gemcitabine, and μM is μmol / L. Detailed implementation manners

[0026] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments in the following description are only examples and do not limit the scope of the present invention. Those skilled in the art can think of other obvious variations. The basic principles defined in the following description can be applied to other implementation schemes, variant schemes, improvement schemes, equivalent schemes, and other technical schemes that do not deviate from the spirit and scope of the present invention.

[0027] Unless otherwise specified, technical terms or scientific terms that are not defined shall have the ordinary meaning in the field to which the present invention belongs.

[0028] For the instruments or raw materials in the present invention, if the manufacturer is not specified, they are all conventional commercial instruments or raw materials. For the detection indexes involved in the embodiments of the present invention, if not mentioned, the conventional detection methods in the art are used for detection.

[0029] Example 1

[0030] Prepare standard solutions. Dissolve cucurbitacin A in dimethyl sulfoxide to a concentration of 1 mmol / L; dissolve gemcitabine in dimethyl sulfoxide to a concentration of 500 μmol / L.

[0031] Detect the inhibitory effect of the combination of cucurbitacin A (CuA) and gemcitabine (GEM) on pancreatic cancer. Treat the pancreatic cancer cell line BXPC-3 with different concentrations of cucurbitacin A (0 μmol / L, 0.05 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.5 μmol / L) and different concentrations of gemcitabine (0 μmol / L, 0.25 μmol / L, 0.5 μmol / L, 1 μmol / L, 2.5 μmol / L, 5 μmol / L) in pairs. After 48 h, add CCK8 absorbance and calculate the cell viability (%). After converting to cell survival rate, use Compusyn software to calculate the combination index (CI). The experimental results (Table 1) show that the CI value is less than 1, that is, cucurbitacin A and gemcitabine have a synergistic effect.

[0032] Table 1 CI values of combined use of cucurbitacin and gemcitabine at various concentrations

[0033]

[0034] The experimental results show that all the CI values are less than 1 after the combination of cucurbitacin A and gemcitabine, so there is a general synergistic effect between the two drugs. Among them, when the concentration of cucurbitacin A is 0.1 μmol / L and the concentration of gemcitabine is 1 μmol / L, the synergistic effect value of their combination is 0.54, showing the most obvious synergistic effect.

[0035] Example 2

[0036] Drug inhibition experiment on pancreatic cancer colony formation.

[0037] Pancreatic cancer cells BXPC-3 were taken, and 750 cells per well were inoculated into 6-well plates and cultured for 3 days. Four working solutions were prepared using RPMI-1640 medium, namely normal medium (control group), cucurbitacin A at 0.1 μmol / L, gemcitabine at 10 μmol / L, and cucurbitacin A + gemcitabine (0.1 μmol / L + 1 μmol / L). Pancreatic cancer cells were cultured with the above four working solutions respectively. After 3 days of culture, the cells were replaced with complete medium without drugs and continued to be cultured for 7 days. The cells were washed twice with PBS buffer, fixed with 4% paraformaldehyde for 30 min, incubated with 0.1% crystal violet staining solution for 10 min, air-dried, photographed, and statistically analyzed. The experimental results are as Figure 2 shown.

[0038] The results showed that the number of colony formations in the control group was 79, in the cucurbitacin A group was 43, in the gemcitabine group was 31, and in the combination drug group was 13. The drug composition could significantly inhibit the colony formation of pancreatic cancer.

[0039] Example 3

[0040] Experiment on drug-induced apoptosis of pancreatic cancer.

[0041] 90,000 BXPC-3 pancreatic cancer cells were inoculated into 6-well plates. Four working solutions were prepared using RPMI-1640 medium, namely normal medium (control group), cucurbitacin A at 0.1 μmol / L, gemcitabine at 1 μmol / L, and cucurbitacin A + gemcitabine (0.1 μmol / L + 1 μmol / L). Pancreatic cancer cells were cultured with the above four working solutions respectively. After 72 h of culture, cell apoptosis was induced; the cells were digested and collected, stained with AnnexinV-PI, and detected and analyzed by flow cytometry. The experimental results are as Figure 3 shown.

[0042] The results showed that the apoptosis rate of the control group was 6.07%, in the cucurbitacin A group was 14.63%, in the gemcitabine group was 21.69%, and in the combination drug group was 35.7%. The drug composition could induce the apoptosis of pancreatic cancer.

[0043] Example 4

[0044] Experiment on the inhibition of pancreatic cancer progression by drugs in vivo.

[0045] Five-week-old immunodeficient nude mice (BALB / c-nu) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All animal research protocols were approved by the Experimental Animal Ethics Committee of Zhejiang Chinese Medical University (SYXK(Zhejiang)2018–0012). 2×10 BXPC-3 cells 6Cells were injected into the right abdomen of each nude mouse. After waiting for two weeks, 24 nude mice with the same tumor size were selected and randomly divided into 4 groups. Each group was injected with the following drugs every three days: group a - normal saline (control group), group b - gemcitabine (50 mg / kg), group c - cucurbitacin A (1 mg / kg), and group d - combination of gemcitabine (50 mg / kg) and cucurbitacin A (1 mg / kg). The volume of the tumor was measured using the formula: volume = length × width 2 × 0.5. After 21 days, the mice were sacrificed and the tumors were dissected. The experimental results are as Figure 4 shown.

[0046] The results showed that the tumor volume in the control group was always larger than that in the other three groups. The tumor volume in the cucurbitacin A group and the gemcitabine group was slightly smaller than that in the control group but larger than that in the combination treatment group, and the tumor volume in the combination treatment group was the smallest. It indicates that the pharmaceutical composition of the present invention can significantly inhibit the progression of pancreatic cancer.

[0047] Example 5

[0048] Inhibitory experiment of cucurbitacin A and gemcitabine on JAK2 / STAT3 signaling pathway.

[0049] For Western blot analysis, pancreatic cancer cells were washed with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitors. Proteins (30 μg) were loaded onto SDS-polyacrylamide gel electrophoresis (PAGE) gels. The cell lysates were subjected to polyacrylamide gel electrophoresis and then transferred to Millipore membranes. Nonspecific reactions were blocked by incubating the membranes in 4% bovine serum albumin (BSA) at room temperature for 1 hour. Then, diluted primary antibodies (p-STAT3, t-STAT3, p-JAK2, and t-JAK2) were added, followed by secondary antibody Goat anti-Rabbit IgG(H+L). Specific proteins were detected by enhanced chemiluminescence system. Analysis was performed using Image J software for protein expression. The experimental results are as Figure 5 shown.

[0050] The results found that( Figure 5 A, 5B) cucurbitacin A could inhibit the expression of phosphorylated STAT3, but did not affect the total protein expression of STAT3. It is suggested that cucurbitacin A may inhibit the proliferation of pancreatic cancer cells by inhibiting the STAT3 signaling pathway. Further detection of the effects of gemcitabine on the phosphorylated and total proteins of JAK2 and STAT3 in pancreatic cancer cells showed that( Figure 5 C, 5D, 5E, 5F) gemcitabine could inhibit the expression of phosphorylated JAK2 and phosphorylated STAT3, but did not affect the total protein expression of JAK2 and STAT3. It is suggested that cucurbitacin A can synergistically inhibit the expression of the STAT3 signaling pathway with gemcitabine.

[0051] Those skilled in the art should understand that the above embodiments are only examples and do not limit the present invention. The object of the present invention has been completely and effectively achieved. The functions and structural principles of the present invention have been shown and described in the embodiments. Without departing from the above principles, any deformation or modification of the embodiments of the present invention is possible.

Claims

1. A composition comprising cucurbitacin A and gemcitabine.

2. The composition according to claim 1, wherein The molar ratio of cucurbitacin A to gemcitabine is 1:(0.5 - 100).

3. The composition according to claim 1, wherein The molar ratio of cucurbitacin A to gemcitabine is 1:(2 - 50).

4. Use of the composition according to any one of claims 1 - 3 in the preparation of an anti - pancreatic cancer drug.

5. The application according to claim 4, wherein The anti - pancreatic cancer includes inhibiting the clonogenic ability of pancreatic cancer, inducing apoptosis of pancreatic cancer, and / or inhibiting the progression of the tumor.

6. The application according to claim 4, characterized in that The cancer cells of the pancreatic cancer are BXPC - 3 human - derived pancreatic duct cell line.

7. A drug for treating pancreatic cancer, characterized in that, It includes the composition according to any one of claims 1 - 3 and a pharmaceutically acceptable excipient.

8. The medicament according to claim 7, wherein, The excipient at least includes a solvent, and the solvent is dimethyl sulfoxide.

9. The drug according to claim 8, characterized in that, The concentration range of cucurbitacin A is 0.05 - 0.5 μmol / L, and the concentration range of gemcitabine is 0.25 - 5 μmol / L.

10. The drug according to claim 9, characterized in that, The concentration range of cucurbitacin A is 0.05 - 0.5 μmol / L, and the concentration range of gemcitabine is 1 - 2.5 μmol / L.

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