Application of ferulic acid derivative FA-30 in preparation of antitumor drugs

Abstract

The invention relates to the technical field of biological medicines, in particular to a ferulic acid derivative FA-30 with an anti-tumor function. The ferulic acid derivative FA-30 disclosed by the invention has good anti-cervical cancer activity and relatively good safety. The ferulic acid derivative FA-30 has the potential of being developed into anti-cervical cancer drugs. Structural formula of ferulic acid derivative FA-30 is as follows: # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of a ferulic acid derivative FA-30 in the preparation of anti-tumor drugs. Background Art

[0002] Cancer is a malignant disease that afflicts humanity, among which cervical cancer is the fourth most common cancer worldwide and a major global health challenge. Although organized screening and HPV (human papillomavirus, HPV) vaccination programs have made remarkable progress in reducing mortality, treatment options are still very limited for women with metastatic or recurrent disease. After the discovery of lymph node metastases during radical hysterectomy with pelvic lymphadenectomy for early-stage cervical cancer, gynecologists may adopt one of two treatment strategies: abandon surgery and then perform primary (chemo)radiotherapy (PRT), or complete radical hysterectomy followed by adjuvant (chemo)radiotherapy (RHRT). Current guidelines recommend PRT rather than RHRT because combined therapy may be associated with increased morbidity. The research and development of new anti-cervical cancer drugs is very urgent and valuable.

[0003] The active ingredients of traditional Chinese medicine are an important source of bioactive molecules and play an important role in the research and development of innovative drugs. Ferula, which belongs to the Apiaceae family, is mainly distributed in Xinjiang Uygur Autonomous Region, China. Its resin has long been used as a folk medicine to treat rheumatoid arthritis and gastric disease, and its anti-tumor biological activity has attracted the attention of scientists. Today, scientists have found that Ferula species also show anti-cervical cancer effects. As a representative ingredient, ferulic acid (FA), 4-hydroxy-3-methoxycinnamic acid, is a polyphenolic compound with an acrylic acid group on the benzene ring. Modern pharmacological studies have shown that ferulic acid has antioxidant, antithrombotic, and anti-tumor effects. It is reported that ferulic acid can also treat cervical cancer by downregulating the phosphatidylinositol 3-kinase (PI3K) / Akt signaling pathway, inducing cell cycle arrest and autophagy, and increasing ROS (reactive oxygen species, ROS) levels. In order to make the compound more lipophilic and increase its ability to pass through the cell membrane, we synthesized a series of derivatives, and after screening, the compound named FA-30 was the most effective among them. In order to promote the future application of this compound, it is of great significance to comprehensively study the mechanism of action of FA-30 in cervical cancer. Summary of the invention

[0004] The purpose of the present invention is to provide a ferulic acid derivative FA-30 with anti-tumor function. Experiments of the present invention show that the ferulic acid derivative FA-30 has good anti-cervical cancer activity and good safety.

[0005] The first object of the present invention is to provide a ferulic acid derivative FA-30 having anti-tumor function, wherein the ferulic acid derivative FA-30 has the following structural formula:

[0006]

[0007] The second purpose of the present invention is to use the ferulic acid derivative FA-30 in the preparation of anti-tumor drugs.

[0008] The third purpose of the present invention is to use the ferulic acid derivative FA-30 in the preparation of anti-cervical cancer drugs.

[0009] Preferably, the anti-tumor drug is an anti-cervical cancer drug.

[0010] The dosage form of the drug includes oral preparations and injections, and can be prepared into tablets, capsules or injections by conventional methods; especially including injections.

[0011] The fourth object of the present invention is to provide a pharmaceutical composition, comprising a therapeutic amount of the ferulic acid derivative FA-30 having anti-tumor function and other pharmaceutically acceptable excipients.

[0012] Furthermore, the above-mentioned pharmaceutical composition comprises a therapeutic amount of the ferulic acid derivative FA-30 having anti-tumor function, a therapeutic amount of a commercially available anti-tumor drug, and other pharmaceutically acceptable excipients.

[0013] The beneficial effects of the present invention are as follows: FA-30 is an active derivative of ferulic acid, and the purity of FA-30 used in this study is above 98%. FA-30 shows an inhibitory effect on the proliferation of Hela cells, a human cervical cancer cell line, and the cell CCK-8 test shows that the IC 50 The value was about 36.99 μM, treated for 48 hours. In addition, FA-30 showed the effect of inducing apoptosis of Hela cells. Gefitinib was used as a positive control, and the apoptosis rate induced by gefitinib reached 60%. Compared with cells treated with the same amount of dimethyl sulfoxide (appropriately 3%), the apoptosis rate induced by FA-30 was significantly higher (18.50 μM, 6%; 36.99 μM, 12%; 55.50 μM, 23%). As the concentration of FA-30 increased, the apoptosis rate also gradually increased. And the ferulic acid derivative FA-30 has the potential to be developed into an anti-cervical cancer drug; specifically, FA-30 showed better anti-tumor activity than ferulic acid and other derivatives with similar structures. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The effect of different concentrations of FA-30 on Hela cell viability after 48 hours of treatment in Example 1 of the present invention;

[0015] Figure 2 This is the apoptotic effect of Hela cells treated with different concentrations of FA-30 in Example 2 of the present invention; DETAILED DESCRIPTION

[0016] The present invention is further described in detail below with reference to the embodiments, but is not limited thereto.

[0017] Chemicals and reagents

[0018] Gefitinib (catalog number: HY-50895) was purchased from MedChemExpress. 4-Chloro-DL-phenylalanine (catalog number: S4586) was purchased from Sellect. Fetal bovine serum (catalog number: 10099141C) and Dulbecco's modified Eagle medium (catalog number: 11965092) were purchased from Gibco (ThermoFisher). Cell counting kit-8 (CCK-8, catalog number: C0039), Annexin V-FITC apoptosis detection kit (catalog number: C1062M) and BCA protein detection kit (catalog number: P0011) were all from Biotech Company. Chromatographic grade reagents included menthol (catalog number: 34851), acetonitrile (catalog number: 34860), dimethyl sulfoxide (catalog number: D2650) and H 2 O (Catalog No.: 270733) were purchased from Merck. We synthesized the FA-30 compound ourselves, and the related mass spectrum and NMR spectra are shown in Figure S1 in the supplementary information file.

[0019] Example 1: Cell proliferation analysis

[0020] HeLa cells were cultured in Dulbecco's modified eagle medium with 10% fetal bovine serum at 37°C and 5% CO 2 . Hela cells were seeded in multiple 96-well plates to 70% confluence. In the cell proliferation assay, cells were treated with a series of concentrations of compound FA-30 from 0 to 500 μM for 48 hours (0, 3.09, 18.52, 55.56, 166.67, 500 μM). The culture medium was discarded, and after washing twice with phosphate buffer, the cells were treated with 100 μL CCK-8 solution and incubated for another 4 hours at 37°C. The absorbance value of the plate was then read at 450 nm. The IC50 value was calculated using GraphPad Prism software. The relevant values ​​are as follows:

[0021] Test samples <![CDATA[IC 50 ]]> FA-30 36.99 FA 362.44

[0022] Example 2: Cell apoptosis analysis

[0023] Hela cells were seeded in multiple 6-well plates to 80% confluence. Hela cells were treated with 36.99 μM FA-30 or with 10 μM gefitinib for 48 hours. After discarding the culture medium, the cells were washed twice with phosphate buffer and harvested with trypsin. After resuspending with culture medium, the cells were stained according to the protocol of Annexin V-FITC apoptosis detection kit and detected by flow cytometry.

[0024] Example 3: Effect of FA-30 on Cell Metabolomics

[0025] Sample preparation

[0026] Hela cells were seeded in 6-well plates to 80% confluence and treated with 36.99 μM FA for 48 h. The medium was discarded and the cells were harvested with methanol after washing twice with phosphate buffer. Metabolites were extracted after resuspension and sonication. The samples were centrifuged at 12,000 g for 10 min, the supernatant was saved and the precipitate was discarded. After lyophilization, the samples were redissolved in 50% menthol. For the control group and FA-30 group, n=6.

[0027] Chromatography and mass spectrometry

[0028] Agilent 1100 HPLC was used for sample analysis. To obtain a comprehensive metabolic analysis of FA-30, samples were analyzed in hydrophilic interaction liquid chromatography (HILIC) and reverse phase mode. A Waters XBridgeTM BEHHILIC column ( 2.5 μm, 2.1 mm × 100 mm, catalog number: 186006079) for hydrophilicity detection, using Waters XSelectTM Premier HSS T3 column ( 2.5μm, 2.1mm×100mm, catalog number: 186009831) for reverse phase detection. The mobile phase was H2O (0.1% formic acid, A) and acetonitrile (0.1% formic acid, B). The gradient conditions were 0-2 minutes, 5-5% B; 2-13 minutes, 5-95% B; 13-15 minutes, 95-95% B, and the flow rate was set to 0.4ml / min. The mass spectrometer used the ESI source positive and negative modes for data acquisition, and the parameters were as follows. The capillary voltage was positive 4kV and negative 3.5kV. The dryer flow rate was 11L / min. The gas temperature was 350°C. The nebulizer pressure was 45psi. The fragmentation voltage was 120V. The skimmer voltage was 60V. The m / z range was 50-1500. The reference ions were 121.0509, 922.0098 in positive mode and 112.98558700, 1033.98810900 in negative mode.

[0029] Data analysis

[0030] The data were analyzed by Mass Hunter B.07.00 (Agilent). The parameters were set as follows. The UPLC-QTOF / MS raw data were converted to (.mzdata) format files, isotope peaks were removed, and the absolute peak intensity threshold of the MS peak was set to 300. The XCMS online tool (https: / / metlin.scripps.edu / landing_page.php?pgcontent=mainPage) package was used to identify, calibrate, and integrate peaks to generate a .csv data matrix. Internal standards were further used for normalization. SIMCA-14.1 (Umetrics, Umea, Sweden) was used for centralization, proportionality (Pareto), and multivariate statistical analysis. First, the principal component analysis (PCA) method was used to separate the quality control from the sample, describe the stability of the system during the detection process, and use score plots to describe the results. A supervised OPLS-DA model was used to screen different metabolites, and the results were characterized by score plots and side plots. VIP>1 and P value <0.05 were selected as the principle. The Human Metabolome Database (http: / / www.hmdb.ca / ) was used to identify relevant metabolites.

[0031] Example 4: Effect of FA-30 on Cellular Proteomics

[0032] Sample preparation

[0033] Hela cells were seeded in a 10 cm dish to 80% confluence and treated with 36.99 μM FA for 48 h. The medium was discarded, and after washing twice with phosphate buffer, the cells were harvested with trypsin. After resuspending with lysis buffer, the cells were sonicated to extract cellular proteins. The conditions were set as follows: power 80 W, sonication 1 s, off 1 s, on ice for a total of 3 min. The samples were centrifuged at 12,000 g for 10 min, the supernatant was saved, and the precipitate was discarded. The protein concentration was determined by the BCA method according to the instructions. 200 μg of cell protein lysate was measured, 4 μL 1M DTT was added, and incubated at 55 ° C for 1 h. After adding 10 μL 1M IAA and incubating for another 1 h at room temperature in the dark, the sample was transferred to an ultrafiltration tube. After centrifugation at 14,000 g for 15 min, the supernatant was discarded and washed three times with 120 μL guanidine hydrochloride buffer. To hydrolyze the protein into peptides, 4 μg of trypsin was added to the ultrafiltration tube and placed at 37°C overnight. The tube was centrifuged at 2000 g and the solution was collected. For the control group and FA-30 group, n=4.

[0034] iTRAQ labeling

[0035] According to the instructions, iTRAQ labeling was performed using the iTRAQ8-plex kit (ABSciex, USA). Each bottle of iTRAQ reagent was allowed to stand at room temperature, and then the iTRAQ reagent was centrifuged to the bottom of the tube. Isopropanol was added to each reagent bottle. 100 μg of peptide sample was transferred to a new test tube. The samples of the control group were labeled with isotopes 113, 114, 115, and 116, while the samples of the FA-30 treatment group were labeled with 117, 118, 119, and 121. iTRAQ reagent was added and incubated at room temperature for 2 hours. Afterwards, water was added to stop the labeling reaction. The samples were dried in a vacuum freeze dryer.

[0036] Reverse phase separation by HPLC

[0037] The labeled samples were combined and lyophilized, and the chromatography was performed using an Agilent 1100 HPLC and an Agilent Zorbax ExtendTM C18 column ( 5μm, 2.1×150mm, catalog number: 773700-902) for sample segmentation. The mobile phase was 20mM ammonium formate (pH=10, A) and 20mM ammonium formate 80% acetonitrile (pH=10, B). The detection wavelength was set to 214nm. The gradient conditions were 0-5 minutes, 5-5% B; 5-30 minutes, 5-15% B; 30-45 minutes, 15-38% B; 45-46 minutes, 38-90% B; 46-54.5 minutes, 90-90% B; 54.5-55 minutes, 90-5% B; 55-65 minutes, 5% B. The flow rate was set to 0.8ml / min. After 5 minutes, the eluate was collected into a centrifuge tube every minute, and 48 eluates were combined into 12 eluates.

[0038] Chromatography and mass spectrometry

[0039] Sample from acapillary C 18 The trapping column (3 cm × 100 μm) was loaded and then analyzed on an Eksigent Nano LC-1Dplus system (AB SCIEX) using C 18 The sample was separated by a column (15 cm × 75 μm). The flow rate was 5 μL / min and the linear gradient was 90 min. The sample analysis was performed on a Triple TOF 5600 system (ABSCIEX) equipped with a Nanospray III source. The sample was further separated by a trapping column (ChromeXP C18_CL-3 μm, 350μm×0.5μm) and C18 reverse phase column (0.075×150mm, 3μm, ) for analysis. The mobile phase was acetonitrile:H2O:formic acid (2:98:0.1, v / v, A) and acetonitrile:H2O:formic acid (98:2:0.1, v / v, B). The gradient conditions were 0-1 min, 5-7% B; 1-55 min, 7-28% B; 55-70 min, 28-55% B; 70-75 min, 55-80% B; 75-81 min, 80-80% B; 81-82 min, 80-5% B; 82-90 min, 5-5% B. The flow rate was set to 5 μl / min. The mass spectrometer was collected in ESI source positive ion IDA mode with the following parameters. The ESI ion source temperature was 150°C; the nebulizer gas (ion source gas 1, GS1) pressure was 15 psi; the curtain gas (curtain gas, CUR) pressure was 35 psi; the ion spray voltage (IonSpray Voltage, ISVF) was 2300 V; the cumulative scan time in IDA mode was 250 ms; and the number of charged fragment ions was set to 2-5.

[0040] Data analysis and protein quantification

[0041] The data were analyzed using Protein Pilot 4.5 (ABSCIEX) with the following parameters: Sample type: iTRAQ-8-plex (peptide-labeled); Digestion: Trypsin; Cysteine ​​alkylation: Iodoacetyl methoxide; Instrument: TripleTOF 5600+; Species: Homo sapiens; Specified treatment: Quantification-bias correlation; Database: human_uniprot.fasta; Search job: By ID; Result quality: Threshold 0.05%; FDR (false discovery rate analysis): Threshold 0.05%.

[0042] GO and KEGG enrichment analysis

[0043] GO and KEGG functional enrichment analysis of metabolites and proteins was performed using the DAVID online tool (DAVID version 6.8; http: / / david.ncifcrf.gov / ). GO terms were divided into three categories: biological process (BP); cellular component (CC); and molecular function (MF). P values ​​< 0.05 were considered to indicate statistically significant differences.

[0044] Statistical analysis

[0045] Data are shown as mean ± SE for each group. Statistical significance was determined by Student's t-test and a P value of < 0.05 was used as the standard.

[0046] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A ferulic acid derivative FA-30 with anti-tumor function, characterized in that The structural formula of the ferulic acid derivative FA-30 is as follows:

2. Use of the ferulic acid derivative FA-30 according to claim 1 in the preparation of anti-tumor drugs.

3. Use of the ferulic acid derivative FA-30 according to claim 1 in the preparation of anti-cervical cancer drugs.

4. A pharmaceutical composition, characterized in that The invention comprises a therapeutic amount of the ferulic acid derivative FA-30 with anti-tumor function as claimed in claim 1 and other pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition comprises a therapeutic amount of the ferulic acid derivative FA-30 with anti-tumor function as claimed in claim 1, a therapeutic amount of a commercially available anti-tumor drug and other pharmaceutically acceptable excipients.

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