Application of epifriedeltaol in preparation of anti-lung cancer substance and anti-lung cancer composition

Abstract

The invention belongs to the technical field of medicine application, and particularly relates to application of epifriedel in preparation of an anti-lung cancer substance and an anti-lung cancer compound.The Western Blot experiment proves that Epifriedel has an inhibition effect on PAICS protein expression in a lung cancer cell line A549 / PC9, and the Epifriedel and the concentration of Epifriedel have a dose-dependent relationship. A CCK8 experiment shows that the inhibition effect of the Epifriedel alone and the CDDP alone on the A549 / PC9 is obviously weaker than the effect of the Epifriedel in combination with the cis-platinum (CDDP) on the cells, and the effect of the Epifriedel alone and the CDDP alone on the cells is obviously better than that of the Epifriedel alone and the cis-platinum (CDDP).

Description

Technical Field

[0001] The invention belongs to the technical field of drug application, and in particular relates to an application of epicorkol in preparing an anti-lung cancer substance and an anti-lung cancer composition. Background Art

[0002] Lung cancer, also known as primary bronchial cancer, 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 according to its histopathological characteristics. Lung adenocarcinoma is a type of non-small cell carcinoma and a common pathological type. It is more common in women and smokers. There are generally no obvious clinical symptoms in the early stage and it is easy to be ignored. Most patients are in the middle and late stages when they seek medical treatment. At present, the preferred treatment method in clinical practice is surgical resection of cancerous tissue, supplemented by postoperative radiotherapy and chemotherapy. Except for 50%-60% of lung adenocarcinoma patients with EGFR gene mutations who can use targeted drugs osimertinib and ametinib for gene targeted therapy, the chemotherapy effect of other patients is not ideal. Therefore, further research on lung adenocarcinoma-related genes and the discovery of new targeted drugs have important clinical value.

[0003] Phosphoribosylaminoimidazole succinamide synthetase (PAICS) is a bifunctional enzyme that catalyzes the de novo synthesis of purines. It also has the activities of 5-aminoimidazole nucleotide carboxylase and 4-(N-succinamide)-5-aminoimidazole nucleotide synthetase (SAICARs). Studies have shown that PAICS is highly expressed in a variety of cancers, such as breast cancer, gastric cancer, and prostate cancer, and is an indicator of poor prognosis for patients. Currently, PAICS has been identified as a new therapeutic target for EGFR wild-type non-small cell lung cancer (NSCLC), so exploring new small molecule drugs targeting PAICS is a new direction 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 epicorbyl alcohol 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 epicorbyl alcohol on human liver microsomal CYP3A4, 2C9 and 2C19 enzymes, and the protective effect of epicorbyl alcohol on ultraviolet-induced photoaging of human dermal fibroblasts, Quan Hexiu et al., Shizhen Traditional Chinese Medicine, Vol. 28, No. 8, 2017, disclosed that epicorbyl alcohol inhibits skin aging caused by UVB irradiation 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 epicorkol 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] The embodiment of the present invention provides a use of epicorkol in preparing an anti-lung cancer substance.

[0008] Preferably, the epicorbyl alcohol 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] The 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 epicorkol is 5-20:30-100.

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

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

[0017] The beneficial effect of the present invention is that, based on the molecular structure of phosphoribosylaminoimidazole succinamide synthetase PAICS, the present invention finds that epifriedelanol and PAICS have a strong binding force by using AutoDock Vina. The lung cancer cell line A549 / PC9 was treated with different concentrations of Epifriedelanol, and the Western Blot experiment proved that Epifriedelanol had an inhibitory effect on the expression of PAICS protein in the lung cancer cell line A549 / PC9, and there was a dose-dependent relationship with the concentration of Epifriedelanol. The CCK8 experiment found that the inhibitory effect of Epifriedelanol alone and CDDP alone on A549 / PC9 was significantly weaker than the effect of Epifriedelanol combined with cisplatin (CDDP) on cells.

[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 of other cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The binding model and detailed schematic diagram of the 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 embodiment of the present invention, a molecular dynamics simulation experiment was 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; Figure 2 A is the PAICS root mean square deviation graph, Figure 2 B is the root mean square deviation diagram of the combination of PAICS and Epifriedelanol.

[0021] Figure 3 In the present invention, the lung cancer cell line A549 / PC99 was treated with Epifriedelanol at different concentration gradients for 6 hours, and then the expression level of PAICS protein was detected by Western Blot experiment; Figure 4 In the present invention, different concentrations of Epifriedelanol and different concentrations of cisplatin (CDDP) were used to treat lung cancer cell line A549 / PC9, CCK8 was used to detect cell viability, and the synergistic combination index was calculated using CompuSyn software, reflecting that Epifriedelanol and CDDP have a synergistic effect; Figure 5 In the present embodiment, the cell viability of lung cancer cell line A549 / PC9 at different time gradients was detected by CCK8 experiment after treatment with Epifriedelanol alone, CDDP alone or Epifriedelanol and CDDP in combination.

[0022] Figure 6 This is the layout of the 96-well plate in Example 6.

[0023] Figure 7In Example 6, the cell viability of human oral squamous carcinoma cell line HN30 cells at different time gradients after being treated with Epifriedelanol alone, CDDP alone, or Epifriedelanol and CDDP in combination was detected by CCK8 experiment. DETAILED DESCRIPTION

[0024] Example 1 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.

[0025] Pymol was used to visualize the molecular docking results of PAICS-Epifriedelanol, and the combined model was Figure 1 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.

[0026] Example 2 To further study the interaction between PAICS and Epifriedelanol, molecular dynamics simulation was performed using Gromacs to predict the stability of the binding between PAICS and Epifriedelanol. The molecular dynamics simulation was performed for 90 ns, and the root mean square deviation (RMSD) was calculated based on the respective simulation trajectories.

[0027] 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.

[0028] like Figure 2 As shown in Figure B, the molecular conformation of the ligand Epifriedelanol was stable within 0.05 nm from 0 to 90 ns during the simulation. Molecular dynamics simulation confirmed the stability of the binding of PAICS to Epifriedelanol.

[0029] Example 3 Western Blot Experiment PC9 lung cancer cells were inoculated in 1640 medium supplemented with 10% calf serum solution, and A549 lung cancer cells were inoculated in F12 medium supplemented with 10% calf serum solution at 37°C and 5% CO.2 The cells were cultured under the same conditions for 24 hours. A549 / PC9 lung cancer cells were treated with different doses of Epifriedelanol, with the concentrations being 0 μM, 20 μM, 40 μM, 80 μM, 100 μM, and 120 μM, respectively, and the treatment time was 96 hours.

[0030] Western Blot Experimental Steps: 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 method. IP lysis buffer and loading buffer were added according to the calculated results, and denatured in a 99°C metal bath for 5 min.

[0031] ② Electrophoresis: Take 25 µg of protein lysate and add it to the electrophoresis channel for 20 min at 10% SDS-PAGE at 90 V. After the marker is separated, increase the voltage to 130 V for 60 min for separation.

[0032] ③ Transfer and color development: The separated gel was transferred to a polyvinylidene fluoride (PVDF) membrane and transferred at 260 mA for 90 min. The membrane was then placed in TBST containing 10% skim milk powder (TBS containing 0.1% Tween-20) and blocked at 37°C for 2 h. After adding PAICS and ACTIN antibodies, the membrane was incubated on a shaker at 4°C overnight. The next day, the membrane was washed three times with TBST, and the corresponding goat secondary antibodies were added and incubated for 1 h at room temperature, and then the membrane was washed three times with TBST again and developed.

[0033] The results are as follows Figure 3 As shown, 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.

[0034] Example 4 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 h, and the cell viability was detected by CCK8 assay. Then, Compusyn software was used to calculate the drug combination index based on the cell viability.

[0035] The results are as follows Figure 4 As shown, the combined drug index of Epifriedelanol and CDDP in A549 / PC9 lung cancer cells was less than 1, proving that the combined use of Epifriedelanol and CDDP had a synergistic inhibitory effect on lung cancer cells.

[0036] Example 5 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 combination of Epifriedelanol and CDDP. PC9 lung cancer cells were inoculated in 1640 medium supplemented with 10% calf serum, and A549 lung cancer cells were inoculated in F12 medium supplemented with 10% calf serum. They were incubated at 37°C and 5% CO 2 After culturing for 24 h under the conditions, the cultured A549 / PC9 cells were seeded into 96-well plates at a density of 2000 cells / 100 μL and randomly divided into three groups after culturing for 24 h: ①10 μM CDDP treatment group; ②100 μM Epifriedelanol treatment group; ③10 μM CDDP+100 μM Epifriedelanol treatment group.

[0037] The experimental results are as follows Figure 5 As shown, compared with CDDP alone or Epifriedelanol alone, the combination of CDDP + Epifriedelanol can more significantly reduce the cell activity 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 activity of lung cancer cells.

[0038] Example 6 Cell activity of HN30 cells (human oral squamous carcinoma cells) at different time points (24h, 48h, 72h, 96h, 120h) under the combined action of epicortin and cisplatin.

[0039] The experimental steps are: 1. Observe the cells growing to a fusion state under a microscope; 2. Digest the cells with trypsin in an incubator for 8 minutes, terminate the digestion, collect the cell pellet by centrifugation, and resuspend it in serum-containing culture medium to make a cell suspension; 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; 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 replicate wells 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 replicate wells), repeat the layout of the plate 4 times, and add drugs after culturing in the incubator for 24 hours; 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), and it is used after filtration and sterilization. The concentration of Epifriedelanol is 100μM. The 96-well plate is arranged as follows: Figure 6 shown.

[0040] 5. After adding the drug, culture in the incubator for 24h, 48h, 72h, 96h, and 120h (change the medium after 72h of culture for 96h and 120h), then add CCK8 reagent (mix 10μl CCK8 reagent with culture medium at a ratio of 1:10 and add 100μl), incubate in the incubator for 4h, and use an enzyme reader to detect the absorbance value at a wavelength of 450nm.

[0041] Result processing 1. The OD values ​​obtained from 5 replicate wells in each group were averaged after removing outliers, and the statistical differences between the experimental group and the control group were compared.

[0042] 2. Calculation of viability value: Cell viability (%) = [A (experimental) - A (blank)] / [A (control) - A (blank)] × 100; A (experimental): OD values ​​of wells with cells, CCK8 solution and drug solvent, A (control): OD value of the well with cells, CCK8 solution but no drug solvent, A (blank): OD value of wells with culture medium, CCK8 solution but no cells.

[0043] The experimental results are as follows Figure 7 As shown, from Figure 7 It can be seen that compared with CDDP alone or Epifriedelanol alone, the combination of CDDP+Epifriedelanol cannot significantly reduce the cell activity of HN30 cells. This indicates that Epifriedelanol, when used in combination with CDDP, cannot enhance the inhibitory effect of CDDP on the activity of human oral squamous cell carcinoma cells.

[0044] 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.

[0045] 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. A use of epicorkol in preparing an anti-lung cancer substance.

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

3. The use according to claim 1 or 2, characterized in that: The lung cancer is non-small cell lung cancer.

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

5. An anti-lung cancer composition, characterized in that: Including anti-lung cancer drugs and epicorbyl alcohol.

6. The anti-lung cancer composition according to claim 5, characterized in that: It is composed of anti-lung cancer drugs and epicorbyl alcohol.

7. The anti-lung cancer composition according to claim 5 or 6, characterized in that: The anti-lung cancer drug is cisplatin.

8. The anti-lung cancer composition according to claim 7, characterized in that: The molar concentration ratio of cisplatin to epicorkol is 5-20:30-100.

9. The anti-lung cancer composition according to claim 8, characterized in that: The molar concentration ratio of cisplatin to epicorkol is 20:80-100.

10. The anti-lung cancer composition according to claim 8, characterized in that: The dosage of cisplatin is 5-20 μM, and the dosage of epicorbyl alcohol is 30-100 μM.

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