Use of episuberinol in preparing anti-lung cancer substances and anti-lung cancer compositions

Abstract

The present invention belongs to the technical field of pharmaceutical applications, specifically relating to the use of epifriedelanol in the preparation of anti-lung cancer substances and anti-lung cancer compositions. Western blot experiments demonstrate that epifriedelanol inhibits PAICS protein expression in the lung cancer cell line A549 / PC9, with a dose-dependent relationship with epifriedelanol concentration. CCK8 experiments also reveal that the inhibitory effects of epifriedelanol and CDDP alone on A549 / PC9 cells are significantly weaker than those of epifriedelanol combined with cisplatin (CDDP).

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical applications, and particularly relates to an application of episuberinol in preparing an anti-lung cancer substance and an anti-lung cancer composition. Background Art

[0002] Lung cancer, also known as primary bronchogenic carcinoma, is the most common lung malignancy originating from the trachea, bronchial mucosa, or glands. Lung cancer can be divided into non-small cell carcinoma (including adenocarcinoma and squamous cell carcinoma) and small cell carcinoma based on its histopathological characteristics. Lung adenocarcinoma, a common type of non-small cell carcinoma, is more common in women and smokers. Early clinical symptoms are often asymptomatic, making it easily overlooked, and most patients present with advanced disease by the time they present. Currently, the preferred treatment is surgical resection of the cancerous tissue, supplemented with postoperative chemoradiotherapy. While 50%-60% of lung adenocarcinoma patients harbor EGFR gene mutations and can be treated with targeted therapy with the drugs osimertinib and ametinib, chemotherapy is unsatisfactory in other patients. Therefore, further research into genes associated with lung adenocarcinoma and the discovery of new targeted drugs hold significant clinical value.

[0003] Phosphoribosylaminoimidazole succinamide synthetase (PAICS) is a bifunctional enzyme that catalyzes the de novo synthesis of purines. It possesses both 5-aminoimidazole nucleotide carboxylase and 4-(N-succinyl)-5-aminoimidazole nucleotide synthetase (SAICARs) activities. Studies have shown that PAICS is highly expressed in various cancers, including breast, gastric, and prostate cancers, and is a marker of poor prognosis. PAICS has been identified as a novel therapeutic target for EGFR wild-type non-small cell lung cancer (NSCLC). Therefore, the search for novel small molecule drugs targeting PAICS represents a new avenue for the treatment of lung adenocarcinoma.

[0004] Epifriedelanol is a triterpenoid compound isolated from the root bark of Ulmus pumila and Amaranthus chinensis, which has anti-inflammatory, analgesic and antibacterial effects.

[0005] Characterization and Analysis of Chemical Components of Aster tataricus and Study on the Inhibitory Effect of Epicorinol on Human Liver Microsomal CYP450 Enzymes, Yang Haotian, Master's thesis of Hebei Medical University, published on March 1, 2016, disclosed the inhibitory effect of epicorinol on human liver microsomal CYP3A4, 2C9 and 2C19 enzymes, and the protective effect of epicorinol on UV-induced photoaging of human dermal fibroblasts, Quan Hexiu et al., Shizhen Traditional Chinese Medicine, Vol. 28, No. 8, 2017, disclosed that epicorinol inhibits UVB-induced skin aging by regulating the expression of p53. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a use of epicorbyl alcohol in the preparation of anti-tumor substances and an anti-tumor composition, so as to improve the anti-lung cancer effect of the anti-lung cancer substances.

[0007] An embodiment of the present invention provides a use of episuberinol in preparing an anti-lung cancer substance.

[0008] Preferably, the episuberinol is used to prepare a sensitizer for anti-lung cancer drugs.

[0009] Preferably, the lung cancer is non-small cell lung cancer.

[0010] Preferably, the anti-lung cancer drug is cisplatin.

[0011] An embodiment of the present invention provides an anti-lung cancer composition, comprising an anti-lung cancer drug and epicorkol.

[0012] Preferably, it is composed of anti-lung cancer drugs and epicorkol.

[0013] Preferably, the anti-lung cancer drug is cisplatin.

[0014] Preferably, the molar concentration ratio of cisplatin to episuberol is 5-20:30-100.

[0015] Preferably, the molar concentration ratio of cisplatin to episuberol is 20:80-100.

[0016] Preferably, the dosage of cisplatin is 5-20 μM, and the dosage of episuberinol is 30-100 μM.

[0017] The present invention has the beneficial effect of demonstrating strong binding between epifriedelanol and PAICS, based on the molecular structure of phosphoribosylaminoimidazole succinamide synthetase (PAICS). Using AutoDock Vina, the present invention demonstrated that epifriedelanol and PAICS have a strong binding affinity. Different concentrations of epifriedelanol were used to treat the lung cancer cell line A549 / PC9. Western blot experiments demonstrated that epifriedelanol inhibited PAICS protein expression in the A549 / PC9 lung cancer cell line, with a dose-dependent relationship with epifriedelanol concentration. CCK8 experiments revealed that the inhibitory effects of epifriedelanol and CDDP alone on A549 / PC9 cells were significantly weaker than the effects of epifriedelanol combined with cisplatin (CDDP).

[0018] The present invention also found through CCK8 experiments that the inhibitory effect of CDDP alone on HN30 cells is equivalent to that of Epifriedelanol combined with cisplatin on cells, and Epifriedelanol alone has no obvious inhibitory effect on HN30 cells, indicating that the inhibitory effect of epifriedelanol on lung cancer cells is significantly better than that on other cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Detailed schematic diagram of the binding model and binding site of PAICS and Epifriedelanol molecular docking in the embodiment of the present invention; Figure 1 A is the binding model, Figure 1 B is a detailed schematic diagram of the binding site of PAICS and Epifriedelanol.

[0020] Figure 2 For the present invention, molecular dynamics simulation experiments were performed on the binding of PAICS and Epifriedelanol using Gromacs, and the root mean square deviation (RMSD) was calculated based on the respective simulation trajectories.

[0021] Figure 2 A is the PAICS root mean square deviation graph, Figure 2 B is the root mean square deviation graph of the binding of PAICS and Epifriedelanol.

[0022] Figure 3 In the present invention, the lung cancer cell line A549 / PC99 was treated with different concentration gradients of Epifriedelanol for 6 h, and then the expression level of PAICS protein was detected by Western Blot experiment;

[0023] Figure 4 In the present invention, different concentrations of Epifriedelanol and different concentrations of cisplatin (CDDP) were used to treat the lung cancer cell line A549 / PC9. Cell viability was detected by CCK8, and the synergistic combination index was calculated using CompuSyn software, reflecting that Epifriedelanol and CDDP have a synergistic effect.

[0024] Figure 5 In the present embodiment, the CCK8 assay was used to detect the cell viability of the lung cancer cell line A549 / PC9 at different time gradients after treatment with Epifriedelanol alone, CDDP alone, or a combination of Epifriedelanol and CDDP.

[0025] Figure 6This is the layout of the 96-well plate in Example 6.

[0026] Figure 7 In Example 6, the cell viability of human oral squamous carcinoma cell line HN30 cells at different time gradients after treatment with Epifriedelanol alone, CDDP alone, or Epifriedelanol and CDDP in combination was detected by CCK8 assay. DETAILED DESCRIPTION

[0027] Example 1

[0028] Based on the molecular structure of PAICS, epifriedelanol was molecularly docked with PAICS using AutoDock Vina, and the molecular docking score was -19.7, indicating that the two had a strong binding force.

[0029] Pymol was used to visualize the molecular docking results of PAICS-Epifriedelanol, and the combined model was as follows Figure 1 As shown in A. The PAICS-Epifriedelanol molecular docking results were processed using online tools to form a detailed model diagram of the binding site between PAICS and Epifriedelanol, as shown in Figure 1 As shown in B.

[0030] Example 2

[0031] To further investigate the interaction between PAICS and epifriedelanol, molecular dynamics simulations were performed using Gromacs to predict the stability of the PAICS-epifriedelanol binding. Molecular dynamics simulations were performed for 90 ns, and the root mean square deviation (RMSD) was calculated from each simulation trajectory.

[0032] like Figure 2 As shown in A, during the molecular simulation process, when PAICS was docked with Epifriedelanol, the fluctuation from 0 to 90 ns was within 0.2 nm, which was within an acceptable range.

[0033] like Figure 2 As shown in Figure B, the molecular conformation of the epifriedelanol ligand remained stable within 0.05 nm from 0 to 90 ns during the simulation. Molecular dynamics simulations confirmed the stability of the binding of PAICS to epifriedelanol.

[0034] Example 3

[0035] Western Blot Experiment

[0036] PC9 lung cancer cells were seeded in 1640 medium supplemented with 10% calf serum solution, and A549 lung cancer cells were seeded in F12 medium supplemented with 10% calf serum solution and cultured for 24 hours at 37°C and 5% CO2. A549 / PC9 lung cancer cells were treated with different doses of Epifriedelanol (0 μM, 20 μM, 40 μM, 80 μM, 100 μM, and 120 μM) for 96 hours.

[0037] Western Blot Experimental Steps:

[0038] ① Protein sample extraction and preparation: The treated cells were washed twice in phosphate-buffered saline (PBS), digested with trypsin, lysed on ice for 30 min using lysis buffer supplemented with protease inhibitors, centrifuged at 15,000 g for 15 min at 4°C, and the protein concentration was determined by the BCA assay. IP lysis buffer and loading buffer were added according to the calculated results, and the cells were denatured in a 99°C metal bath for 5 min.

[0039] ② Electrophoresis: 25 μg of protein lysate was added to the electrophoresis channel and electrophoresed at 90 V for 20 min on 10% SDS-PAGE. After the marker was separated, the electrophoresis was increased to 130 V for 60 min for separation.

[0040] ③ Transfer and staining: The separated gel was transferred to a polyvinylidene fluoride (PVDF) membrane and transferred at 260 mA for 90 minutes. The membrane was then blocked in TBST (TBS containing 0.1% Tween-20) containing 10% skim milk powder at 37°C for 2 hours. PAICS and ACTIN antibodies were added and incubated overnight on a shaker at 4°C. The next day, the membrane was washed three times with TBST and incubated with the corresponding goat secondary antibody for 1 hour at room temperature. The membrane was then washed three more times with TBST and developed.

[0041] The results are as follows Figure 3 As shown in the figure, whether in A549 lung cancer cells or PC9 lung cancer cells, as the dose of Epifriedelanol increases, the protein expression level of PAICS in A549 / PC9 cells decreases, indicating that Epifriedelanol has a dose-dependent inhibitory effect on the expression of PAICS protein in A549 / PC9 cells.

[0042] Example 4

[0043] A549 lung cancer cells and PC9 lung cancer cells were treated with different concentrations of Epifriedelanol (30, 60, 70, 75, 80, 85, 90, 95, 100 μM) combined with different concentrations of CDDP (5, 10, 20 μM) for 72 hours. Cell viability was detected by CCK8 assay, and the drug combination index was calculated based on cell viability using Compusyn software.

[0044] The results are as follows Figure 4 As shown, the combination index of Epifriedelanol and CDDP in A549 / PC9 lung cancer cells was less than 1, indicating that the combined use of Epifriedelanol and CDDP had a synergistic inhibitory effect on lung cancer cells.

[0045] Example 5

[0046] The CCK8 assay was used to evaluate the cell viability of A549 / PC9 lung cancer cells at different time points (1, 2, 3, 4, and 5 days) after the combined use of epifriedelanol and CDDP. PC9 lung cancer cells were seeded in 1640 medium supplemented with 10% calf serum, while A549 lung cancer cells were seeded in F12 medium supplemented with 10% calf serum. The cells were cultured at 37°C and 5% CO2 for 24 hours. The cultured A549 / PC9 cells were seeded at a density of 2000 cells / 100 μL onto 96-well plates and randomly divided into three groups after 24 hours of culture: ① 10 μM CDDP treatment group; ② 100 μM epifriedelanol treatment group; and ③ 10 μM CDDP + 100 μM epifriedelanol treatment group.

[0047] The experimental results are as follows Figure 5 As shown in the results, compared with CDDP or Epifriedelanol alone, the combination of CDDP and Epifriedelanol can more significantly reduce the cell viability of A549 / PC9 lung cancer cells. This indicates that Epifriedelanol can be used as a new sensitizer in combination with CDDP to exert a stronger inhibitory effect on the cell viability of lung cancer cells.

[0048] Example 6

[0049] Cell viability of HN30 cells (human oral squamous carcinoma cells) at different time points (24h, 48h, 72h, 96h, 120h) under the combined treatment of episuberin and cisplatin.

[0050] The experimental steps are:

[0051] 1. Observe the cells growing to a confluent state under a microscope;

[0052] 2. Trypsinize the cells in an incubator for 8 minutes to terminate the digestion. Collect the cell pellet by centrifugation and resuspend in serum-containing medium to prepare a cell suspension.

[0053] 3. Count the cells using a cell counting plate, dilute the cell suspension according to a certain ratio, and adjust the cell concentration to 2000 cells / 100 μl;

[0054] 4. Take a 96-well plate and add a circle of PBS around it to reduce evaporation. Use a pipette to draw up the diluted cell suspension (100μl, 2000 cells per well), and randomly divide it into three groups, with 5 replicates in each group: ①CDDP group; ②Epifriedelanol group; ③Combination drug group. At the same time, set up blank wells and control wells (also set up 5 replicates), repeat the plate layout 4 times, culture in the incubator for 24 hours, and then add drugs; CDDP is freshly prepared using DMF (the mother liquor concentration is 30mM), and the final concentration is 10μM (check the literature for the IC50 of CDDP in HN30 cells), filtered and sterilized before use, and the Epifriedelanol concentration is 100μM. The 96-well plate is arranged as follows Figure 6 shown.

[0055] 5. After adding the drug, culture in the incubator for 24 h, 48 h, 72 h, 96 h, and 120 h (change the medium after 72 h of culture for 96 h and 120 h), then add CCK8 reagent (mix 10 μl CCK8 reagent with culture medium in a ratio of 1:10 and add 100 μl), incubate in the incubator for 4 h, and use a microplate reader to detect the absorbance value at a wavelength of 450 nm.

[0056] Result processing

[0057] 1. The OD values ​​obtained from 5 replicate wells in each group were averaged after removing outliers, and the statistical differences between the experimental and control groups were compared.

[0058] 2. Calculation of cell viability: Cell viability (%) = [A(experimental) - A(blank)] / [A(control) - A(blank)] × 100;

[0059] A (experimental): OD values ​​of wells with cells, CCK8 solution, and drug solvent,

[0060] A (control): OD value of the well with cells and CCK8 solution but no drug solvent,

[0061] A (blank): OD value of the well with culture medium, CCK8 solution but no cells.

[0062] The experimental results are as follows Figure 7 As shown, from Figure 7It can be seen that compared with CDDP alone or Epifriedelanol alone, the combination of CDDP and Epifriedelanol did not significantly reduce the cell viability of HN30 cells. This indicates that Epifriedelanol, when used in combination with CDDP, does not enhance the inhibitory effect of CDDP on the activity of human oral squamous cell carcinoma cells.

[0063] Those 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 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.

[0064] The one or more embodiments of this application are intended to encompass all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of this application should be included in the scope of protection of this application.

Claims

1. Use of a composition of epicorbyl alcohol and cisplatin in the preparation of an anti-non-small cell lung cancer drug.

2. The use according to claim 1, characterized in that The molar concentration ratio of cisplatin to episuberyl alcohol is 5-20:30-100.

3. The use according to claim 2, characterized in that: The molar concentration ratio of cisplatin to episuberin is 20:80-100.

4. The use according to claim 1, characterized in that The dosage of cisplatin is 5-20 μM, and the dosage of episuberinol is 30-100 μM.

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