Application of beta-amyrin in preparation of anti-tumor substance and anti-tumor composition

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of beta-amyrin in preparation of anti-tumor substances and an anti-tumor composition, and the beta-amyrin can improve the anti-tumor effect of anti-tumor drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the use of beta-amyrin in preparing anti-tumor substances and an anti-tumor composition. Background Art

[0002] Lung cancer (LC) is one of the most common cancers in the world. Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer. Lung adenocarcinoma (LUAD) is the most common subtype of NSCLC, accounting for half of NSCLC. Due to the high malignancy and rapid progression of lung adenocarcinoma, most patients are already in the advanced stage when diagnosed. Therefore, further research on genes related to the development of lung adenocarcinoma and exploring their potential as therapeutic targets are of great clinical significance.

[0003] The purine factor phosphoribosylaminoimidazole succinamide synthetase (PAICS) is a bifunctional enzyme that has both 5-aminoimidazole ribonucleotide carboxylase and 4-(N-succinylformamide)-5-aminoimidazole ribonucleotide synthetase activities and is a key enzyme in the de novo purine synthesis pathway. Previous studies have shown that cancer cells rely on a PAICS-dependent metabolic pathway to synthesize adenosine monophosphate and guanosine monophosphate, and inactivation of this new pathway can inhibit the proliferation of cancer cells in vitro and in vivo. PAICS has been shown to be highly expressed in a variety of cancers, including breast cancer, prostate cancer, bladder cancer, lung cancer, and acute lymphoblastic leukemia. In addition, overexpression of PAICS also predicts poor survival in tumor patients. Studies on the carcinogenic mechanism of PAICS have found that PAICS can induce epithelial-mesenchymal transition in bladder cancer by positively regulating SNAI1 and reducing the expression of E-cadherin.

[0004] Molecular docking is a structure-based computer simulation method widely used in drug discovery. Docking technology can identify novel compounds with therapeutic significance and predict ligand-target interactions at the molecular level. If a suitable target structure is available, molecular docking can be used to distinguish putative binders from non-binders in large compound databases, greatly reducing the number of compounds that need to be tested experimentally. AutoDock Vina is a new program for molecular docking and virtual screening that can verify the binding affinity between important active compounds and targets. Molecular dynamics can be used to complement AutoDock Vina to evaluate the stability of predicted complexes by exploring the conformational space of the studied target, ligand, and ligand-target complex. Studies have found that PAICS promotes the malignant phenotype of lung adenocarcinoma (LUAD). Therefore, in clinical practice, treating patients with lung adenocarcinoma (LUAD) with high expression of PAICS by using small molecule inhibitors may be an effective way to enhance the sensitivity of tumor cells to chemotherapeutic drugs and improve patient prognosis. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a use of beta-amyrin in preparing anti-tumor substances and an anti-tumor composition to improve the anti-tumor effect.

[0006] The embodiment of the present invention provides a use of β-amyrin in preparing an anti-tumor substance.

[0007] The structural formula of the β-amyrin is as follows: .

[0008] Optionally, the β-amyrin is used to prepare a sensitizer for anti-lung cancer drugs.

[0009] Optionally, the tumor is breast cancer, prostate cancer, bladder cancer, lung cancer or acute lymphoblastic leukemia.

[0010] Optionally, the tumor is lung cancer.

[0011] Optionally, the anti-lung cancer drug is cisplatin.

[0012] The embodiment of the present invention provides an anti-tumor composition, comprising an anti-tumor drug and β-amyrin.

[0013] Optionally, it consists of an anti-tumor drug and beta-amyrin.

[0014] Optionally, the anti-tumor drug is cisplatin.

[0015] Optionally, the molar concentration ratio of cisplatin to β-amyrin is 5-20:30-100, preferably 20:80-100.

[0016] The beneficial effect of the present invention is that the present invention uses AutoDock Vina to screen out the specific PAICS inhibitor β-amyrin (β-Amyrin) based on the molecular structure of PAICS, and through the inhibitor β-Amyrin combined with anti-lung cancer drugs to inhibit lung cancer cells, it is shown that it has the effect of a sensitizer. When the specific PAICS inhibitor is in a small dose, the inhibitory effect on lung cancer cells is small, but when used in combination with the anti-lung cancer drug cisplatin (CDDP), it can significantly increase the efficacy of the anti-lung cancer drug CDDP. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure is a docking binding model of the PAICS target site and the inhibitor β-Amyrin according to an embodiment of the present invention, and a detailed view of the binding site; wherein A is a diagram for visualizing the PAICS-β-Amyrin complex using Pymol, and B is a docking diagram of the compound β-Amyrin and the PAICS target site.

[0018] Figure 2 For the present embodiment, a molecular dynamics (MD) simulation of the binding of PAICS and β-Amyrin was performed using Gromacs, and the root mean square deviation (RMSD) was calculated according to the respective simulation trajectories; wherein A is the RMSD of the carbon skeleton of PAICS in the 100ns MD simulation, and B is the RMSD of the PAICS-β-Amyrin complex in the 100ns MD simulation.

[0019] Figure 3 Western Blot was used to detect the expression level of PAICS protein in human lung adenocarcinoma cell A549 / PC9 after being treated with β-Amyrin of different concentration gradients for 96 hours.

[0020] Figure 4 In the present invention, A549 / PC9 cells were treated with different concentrations of β-Amyrin and different concentrations of cisplatin (CDDP) and CCK8 was used to detect cell viability. The synergistic combination index was calculated using CompuSyn software, reflecting that β-Amyrin and CDDP had a synergistic effect.

[0021] Figure 5 The CCK8 experiment of the present invention was used to evaluate the cell viability of A549 / PC9 cells treated with β-Amyrin, CDDP or a combination of the two drugs over a time gradient.

[0022] Figure 6The figure shows the changes in A549 cell viability after treatment with different concentrations of β-Amyrin for 72 hours.

[0023] Figure 7 The graph shows the changes in PC9 cell viability after treatment with different concentrations of β-Amyrin for 72 hours. DETAILED DESCRIPTION

[0024] Example 1 The present invention takes PAICS as the starting point, and according to the molecular structure of PAICS, a large number of small molecule compounds are used to carry out molecular docking with the structure using the AutoDock Vina program, and the PAICS-β-Amyrin complex is visualized using Pymol ( Figure 1 A), docking diagram of compound β-Amyrin and PAICS target site Figure 1 B. Among them, the compound β-Amyrin can enter the action pocket of the PAICS target site and has multiple effects, such as hydrogen bond interactions. The molecular docking score is -19.1, which has a strong binding force and can be used as an inhibitor of PAICS.

[0025] Example 2 In order to analyze the interaction between PAICS and β-Amyrin, MD simulation was performed using Gromacs to predict the changes, stability and intermolecular interactions of the corresponding complexes of PAICS and β-Amyrin. The MD simulation execution time was 100 ns. In order to further verify the stability of the selected receptor-ligand complex, the root mean square deviation (RMSD) was calculated based on the respective simulation trajectories. When the carbon skeleton of PAICS was docked with the selected inhibitor (β-Amyrin), it showed an acceptable fluctuation of less than 0.2 during the simulation ( Figure 2 A), and remain in equilibrium until the end of the simulation. The small molecule ligand (β-Amyrin) did not diffuse from the initial site, and its conformation was stable at 0.1 nm ( Figure 2 B), confirming the reliability of the molecular dynamics simulation results.

[0026] Example 3 10% calf serum solution was added to 1640 medium, PC9 lung cancer cells were inoculated, 10% calf serum solution was added to F12 medium, A549 lung cancer cells were inoculated, and cultured at 37°C and 5% CO2. A549 / PC9 was then treated with different doses of β-Amyrin, with the doses being 0μM, 20μM, 40μM, 60μM, 80μM, and 100μM, and the treatment time was 96 hours. After treatment, the cells were washed twice in phosphate buffer (PBS), trypsinized, and then lysed on ice for 30 minutes using lysis buffer with protease inhibitors, centrifuged at 15,000 g for 15 minutes (4°C), and the protein concentration was determined by the BCA method. IP lysis buffer and loading buff were added according to the calculated results, and denatured in a 99°C metal bath for 5 minutes. Each 25 µg protein lysate was separated by 10% SDS-PAGE at 90V for 20 minutes and 130V for 1 minute, then transferred to a polyvinylidene fluoride (PVDF) membrane at 260mA for 90 minutes, and blocked at 37°C for 2 hours in TBST (TBS containing 0.1% Tween-20) containing 10% skim milk powder. After adding PAICS and ACTIN antibodies, the membrane was incubated overnight on a shaker at 4°C. The next day, the membrane was washed three times with TBST, and the corresponding goat secondary antibodies were added and incubated for 1 hour at room temperature, and then the membrane was washed three times with TBST again and developed to show the expression of PAICS protein. The results are shown in the figure. Figure 3 As shown, it was shown that β-Amyrin had a dose-dependent inhibitory effect on PAICS in A549 / PC9 cells.

[0027] Example 4 A549 and PC9 cells were treated with different concentrations of β-Amyrin (30, 60, 70, 75, 80, 85, 90, 95, 100 μM) and / or CDDP (5, 10, 20 μM) for 72 h. Cell viability was detected by CCK8, and then the drug combination index was calculated based on cell viability using Compusyn software. The results are shown in Figure 4 As shown in Table 1-6.

[0028] Table 1-3 shows the CI values ​​of A549 cells when different concentrations of β-Amyrin were used in combination with 5μM CDDP, 10μM CDDP, and 20μM CDDP, respectively.

[0029] Table 1 CI values ​​of different concentrations of β-Amyrin combined with 5 μM CDDP

[0030] Table 2 CI values ​​of different concentrations of β-Amyrin combined with 10 μM CDDP

[0031] Table 3 CI values ​​of different concentrations of β-Amyrin combined with 20 μM CDDP

[0032] Table 4-6 shows the CI values ​​of PC9 cells when different concentrations of β-Amyrin were used in combination with 5μM CDDP, 10μM CDDP, and 20μM CDDP, respectively.

[0033] Table 4 CI values ​​of different concentrations of β-Amyrin combined with 5 μM CDDP

[0034] Table 5 CI values ​​of different concentrations of β-Amyrin combined with 10 μM CDDP

[0035] Table 6 CI values ​​of different concentrations of β-Amyrin combined with 20 μM CDDP

[0036] The potency was calculated as the absorbance of drug-treated cells / the absorbance of drug-free cells.

[0037] The results showed that the combination index was less than 1, indicating that the combination of β-Amyrin and CDDP had a synergistic inhibitory effect on the proliferation of lung cancer cells.

[0038] Example 5 The effect of the combination of PAICS inhibitor β-Amyrin and CDDP at different time points was evaluated by CCK-8 experiment. 10% calf serum solution was added to 1640 medium, PC9 lung cancer cells were inoculated, and 10% calf serum solution was added to F12 medium, A549 lung cancer cells were inoculated, and cultured for one day at 37°C and 5% CO2. The cultured A549 / PC9 cells were inoculated on a 96-well plate at a density of 2000 cells / 100μL. After 24h of culture, they were randomly divided into three groups: chemotherapy drug CDDP group, inhibitor β-Amyrin group, and combination drug group (β-Amyrin+CDDP). The concentration of chemotherapy drug CDDP was 10μM, and the concentration of β-Amyrin was 80μM. The above concentrations of CDDP solution, inhibitor β-Amyrin solution, CDDP solution and inhibitor were added to the three groups respectively. The cell activities were recorded at 0h, 24h, 48h, 72h, 96h, and 120h. The results are shown in Figure 5This indicates that the anti-proliferation effect of the combined drug is significantly better than that of CDDP alone under various treatment conditions, and β-Amyrin can be used as a sensitizer in combination with CDDP to exert a more significant inhibitory effect on lung cancer cell proliferation.

[0039] Example 6 A549 and PC9 cells were treated with different concentrations of β-Amyrin to obtain the following results: Figure 6-7 The experimental results show that as the concentration of β-Amyrin increases, the activity of tumor cells decreases, indicating that it has the effect of inhibiting tumor cells.

[0040] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0041] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.

Claims

1. Use of β-amyrin in preparing anti-tumor substances.

2. The use according to claim 1, characterized in that: The beta-amyrin is used for preparing a sensitizer for anti-lung cancer drugs.

3. The use according to claim 1, characterized in that: The tumor is breast cancer, prostate cancer, bladder cancer, lung cancer or acute lymphocytic leukemia.

4. The use according to claim 3, characterized in that: The tumor is lung cancer.

5. The use according to claim 2, characterized in that: The anti-lung cancer drug is cisplatin.

6. An antitumor composition, characterized in that: Including anti-tumor drugs and beta-amyrin.

7. The antitumor composition according to claim 6, characterized in that: It is composed of anti-tumor drugs and β-amyrin.

8. The antitumor composition according to claim 6 or 7, characterized in that: The anti-tumor drug is cisplatin.

9. The antitumor composition according to claim 8, characterized in that: The molar concentration ratio of cisplatin to β-amyrin is 5-20:30-100.

10. The antitumor composition according to claim 9, characterized in that: The molar concentration ratio of cisplatin to β-amyrin is 20:80-100.

Images