Application of licochalcone as drug sensitizer and antitumor drug composition

Abstract

The invention discloses application of licochalcone as a drug sensitizer and an anti-tumor drug composition. The application comprises an application of the acanthochalcone in preparation of an anti-tumor drug sensitizer and an application of the acanthochalcone in improvement of the anti-tumor efficacy of platinum chemotherapeutic drugs, and the invention further provides an anti-tumor composition capable of efficiently inhibiting platinum drug-resistant gastric cancer cells based on the application. Through a gastric cancer organ model and an in-vitro cytology experiment, it is found that the acanthochalcone has an inhibiting effect on proliferation of gastric cancer cells, and the sensitivity of the gastric cancer cells to platinum drugs can be remarkably enhanced. The invention also finds that IC50 of gastric cancer cells to cis-platinum and oxaliplatin, especially platinum acquired drug-resistant gastric cancer cells, can be significantly reduced by combined use of the acanthochalcone and platinum drugs. Therefore, the invention provides a new medication basis and choice for overcoming acquired platinum drug resistance of gastric cancer by utilizing the acanthochalcone.

Description

Technical Field

[0001] This invention relates to an antitumor drug, and more particularly to the application of glycyrrhizin chalcone as a drug sensitizer and an antitumor drug composition. Background Technology

[0002] In systemic drug therapy for gastric cancer, platinum-based drugs play a central role. They directly damage the DNA double-strand structure by forming irreparable cross-links with cancer cell DNA, inhibiting DNA replication and transcription, and ultimately inducing apoptosis in cancer cells. This powerful DNA damage mechanism gives them a broad-spectrum and effective role in gastric cancer treatment. Platinum-based drugs, including cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin, are widely used in chemotherapy regimens. For surgically resectable locally advanced gastric cancer, platinum-based drugs are commonly used in postoperative adjuvant chemotherapy and preoperative neoadjuvant chemotherapy to eliminate micrometastases and shrink the primary tumor, thereby improving the success rate of radical surgery and long-term patient survival. Furthermore, platinum-based drugs combined with fluorouracil constitute the standard first-line chemotherapy regimen for advanced metastatic gastric cancer.

[0003] Despite their significant therapeutic effects, platinum-based drugs (especially acquired resistance) have become a bottleneck limiting their efficacy and severely restricting their clinical benefits. The mechanisms of platinum-based drug resistance are complex and diverse, involving enhanced DNA repair capabilities in cancer cells, abnormal drug transport, enhanced cellular detoxification, dysregulation of cell cycle and apoptosis pathways, and metabolic alterations. Therefore, developing novel drugs is of great significance for optimizing traditional treatment regimens and overcoming the challenges of tumor drug resistance faced by existing therapies.

[0004] In recent years, natural products have become a hot topic in anticancer drug development due to their clear anticancer efficacy and abundant candidate resources. For example, paclitaxel, etoposide, and irinotecan are all plant-derived anticancer compounds. Echinatin, a flavonoid extracted from the roots and rhizomes of licorice, possesses clear anti-inflammatory, antioxidant, and cardiovascular protective activities, and has shown antitumor activity in various tumor cells, such as esophageal and colorectal cancer. However, there is currently no literature reporting the role of echinatin as a platinum-based drug sensitizer in the treatment of gastric cancer. Summary of the Invention

[0005] Objectives of this invention: The first objective is to provide the application of glycyrrhizin chalcone in the preparation of antitumor drug sensitizers, thus solving the problem of how to prepare antitumor drug sensitizers. The second objective is to propose the application of glycyrrhizin chalcone in improving the antitumor efficacy of platinum-based chemotherapy drugs, thus solving the problem of how to improve the antitumor efficacy of platinum-based chemotherapy drugs. The third objective is to provide an antitumor composition capable of efficiently inhibiting platinum-resistant gastric cancer cells.

[0006] Technical solution: This invention discloses the application of chalcone from licorice in the preparation of antitumor drug sensitizers.

[0007] Preferably, the antitumor drug is a platinum-based chemotherapy drug.

[0008] In this invention, chalcone from glycyrrhiza is used as an antitumor drug sensitizer, which can effectively enhance the sensitivity of tumor cells to platinum-based drugs. In particular, when chalcone from glycyrrhiza is used in combination with cisplatin, it can significantly increase the sensitivity of platinum-resistant gastric cancer cell lines to cisplatin and substantially reduce the IC50 of cisplatin. 50 Value. Glycyrrhiza chalcone can enhance cisplatin-induced apoptosis in gastric cancer cells and downregulate the gastric cancer cell cycle.

[0009] The second aspect of this invention discloses the application of chalcone from licorice in enhancing the antitumor efficacy of platinum-based chemotherapy drugs.

[0010] Preferably, the tumor is platinum-resistant gastric cancer.

[0011] Preferably, the platinum-based chemotherapy drug includes at least one of cisplatin, carboplatin, oxaliplatin, lobaplatin, and nedaplatin.

[0012] A third aspect of the present invention discloses an antitumor pharmaceutical composition comprising a chemotherapy drug and licorice chalcone.

[0013] Preferably, the chemotherapy drug includes at least one of cisplatin, carboplatin, oxaliplatin, lobaplatin, and nedaplatin, and the molar ratio of the chemotherapy drug to chalcone is 1-20:10-100.

[0014] Preferably, the final concentration of the licorice chalcone is 10-100 μM.

[0015] Preferably, the chemotherapy drug is cisplatin at a final concentration of 5-100 μM.

[0016] The gastric cancer cells described in this invention are human gastric cancer cell lines HGC 27 or AGS, and the PDO is a gastric cancer organoid obtained from human gastric cancer tissue culture. The anti-gastric cancer effect described in this invention is the inhibition of gastric cancer cell proliferation or gastric cancer organoid growth. The effective concentration of the chalcone from glycyrrhizin is 10-100 μM. When the gastric cancer cells are HGC 27, the preferred concentration is 12.5-50 μM; when the gastric cancer cells are AGS, the preferred concentration is 25-100 μM; and the preferred concentration against PDO is 25-100 μM.

[0017] In some embodiments, compared to cisplatin or glycyrrhizin chalcone treatment alone, the combination of cisplatin and glycyrrhizin chalcone significantly inhibited the proliferation of gastric cancer cells or the growth of gastric cancer organoids, and the two drugs exhibited a very significant synergistic effect. The molar ratio of glycyrrhizin chalcone to cisplatin was 25:5 when the gastric cancer cells were HGC 27; the molar ratio was 25:5 when the gastric cancer cells were AGS; and the molar ratio was 25:5 when the target cells were PDO. The dosage can be adjusted appropriately according to the cell's sensitivity to the drug.

[0018] This invention, through analysis of the target proteins of glycyrrhizin chalcone, found that platinum-resistant proteins were significantly enriched within it. The target proteins were obtained from 20K human proteome microarray analysis, and the enrichment was based on KEGG signaling pathway enrichment analysis. This invention constructed a platinum-acquired resistance cell line, AGS-PR (AGS Platinum Resistance), using the gastric cancer cell line AGS. It was found that its sensitivity to glycyrrhizin chalcone was no different from that of the wild-type AGS cell line. However, the combined use of glycyrrhizin chalcone and cisplatin significantly reduced the IC50 of cisplatin in both wild-type AGS and the resistant AGS-PR cell line. 50 Furthermore, the combination of the two drugs significantly aggravated the apoptosis of the aforementioned two types of gastric cancer cells. The molar ratio of licorice chalcone and cisplatin was 25-50:1.5625-100;

[0019] Preferably, when the gastric cancer cells are wild-type AGS, the molar ratio of glycyrrhizin chalcone to cisplatin is 25-50:5-10; when the gastric cancer cells are the drug-resistant strain AGS-PR, the molar ratio of glycyrrhizin chalcone to cisplatin is 25-50:5-50.

[0020] Preferably, the antitumor drug composition further includes pharmaceutically acceptable excipients, and the dosage form of the antitumor drug composition includes at least one of capsules, aqueous injections, powder injections, tablets, gels, suspensions, and solutions.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0022] This invention, through gastric cancer organoid models and in vitro cell experiments, found that chalcone from glycyrrhiza uralensis inhibits the proliferation of gastric cancer cells and significantly enhances their sensitivity to cisplatin. This invention also found that the combined use of chalcone from glycyrrhiza uralensis and platinum-based drugs significantly reduces the IC50 of gastric cancer cells to cisplatin. 50 This is especially true for gastric cancer cells with acquired platinum-based resistance. Therefore, this invention provides a new basis and option for using glycyrrhizin chalcone to overcome acquired platinum-based resistance in gastric cancer. Attached Figure Description

[0023] Figure 1The results of CCK-8 assays on human gastric cancer cell lines HGC 27 and AGS after 48 hours of treatment with a specific concentration of glycyrrhizin chalcone combined with cisplatin, or the same concentration of glycyrrhizin chalcone combined with 5-fluorouracil.

[0024] Figure 2 Image of CCK-8 assay results and microscopic photograph of gastric cancer organoids (PDO) after 5 days of combined treatment with cisplatin at a specific concentration of glycyrrhizin chalcone;

[0025] Figure 3 KEGG pathway enrichment analysis of differentially expressed genes between the licorice chalcone combined with cisplatin treatment group and the cisplatin alone treatment group, showing the top 20 pathways that were upregulated and downregulated after combined treatment.

[0026] Figure 4 A KEGG pathway enrichment analysis diagram for the target protein of glycyrrhiza chalcone is shown, displaying the top 20 pathways. The target protein was obtained by 20K human proteome microarray detection and liquid chromatography / tandem mass spectrometry analysis.

[0027] Figure 5 IC50 of wild-type AGS and platinum-resistant cells AGS-PR against cisplatin and glycyrrhizin chalcone 50 Line graph;

[0028] Figure 6 The IC50 of wild-type AGS and platinum-resistant cells AGS-PR against cisplatin when cisplatin is used in combination with glycyrrhizin chalcone. 50 Line graph;

[0029] Figure 7 Figure 1 shows the results of flow cytometry detection of apoptosis in wild-type AGS and platinum-resistant AGS-PR cells after 48 hours of treatment with a specific concentration of licorice chalcone and cisplatin.

[0030] Figure 8 This is a KEGG pathway enrichment analysis diagram of differentially expressed genes between the glycyrrhiza chalcone combined with cisplatin treatment group and the control group, showing the top 20 pathways that were upregulated and downregulated after the combined treatment. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0032] Example 1: The experimental method for enhancing the sensitivity of gastric cancer cells to cisplatin using licorice chalcone is as follows:

[0033] CCK-8 gastric cancer cell line experiment: HGC 27 and AGS cells in good growth condition were harvested, digested, counted, and their density adjusted. Cell density was maintained at 5 x 10⁻⁶ cells per well for HGC 27 cells.3 Cells / 100μL, AGS: 1*10 4 Cells were seeded at 100 μL / 100 μL in 96-well plates. After overnight cell adhesion, six groups were established for each cell type: control group (DMSO), cisplatin group (5 μM), 5-fluorouracil group (5-FU 5 μM), echinatin group (25 μM), cisplatin + echinatin group (5 μM + 25 μM), and 5-fluorouracil + echinatin group (5-FU 5 μM + 25 μM), with five replicates per group. After 48 h, absorbance was measured using the CCK-8 assay and plotted.

[0034] Gastric cancer PDO CCK-8 assay: Well-grown PDO was digested and mixed with Matri gel at a 1:1 ratio, and then applied to each well at a density of 6 x 10⁻⁶ mm. 3 Cells were resuspended at 5 μL / well in a 96-well plate, and 100 μL of PDO-specific culture medium was added to each well. After culturing for 3-5 days, four groups were set up: control group (DMSO), cisplatin group (Cisplatin 5 μM), glycyrrhizin chalcone group (Echinatin 25 μM), and combination group (Cisplatin 5 μM + Echinatin 25 μM), with 5 replicates per group. The cells were further cultured at 37°C in a 5% CO2 incubator for 5 days. PDO activity was measured using the CCK-8 assay and plotted after photographing.

[0035] Transcriptome sequencing (RNA-seq) and bioinformatics analysis: AGS were processed according to 2*10 6Cells were cultured at 10 mL in 10 cm cell culture dishes (12 dishes in total). After overnight cell adhesion, four treatment groups were set up: control group (DMSO), cisplatin group (Cisplatin 5 μM), licorice chalcone group (Echinatin 50 μM), and combination group (Cisplatin 5 μM + Echinatin 50 μM), with 3 dishes in each group. After 48 h, RNA was extracted from each dish of cells using Trizol reagent according to the manufacturer's instructions. Subsequently, RNA sequencing was performed by Shanghai OE Biotech Co., Ltd. In bioinformatics analysis, differentially expressed genes were screened based on the fold change compared to the control group (screening threshold set as: fold change ≥ 1.5 and P value < 0.05, considered as significantly upregulated or downregulated). Subsequently, enrichment analysis was performed on differentially expressed genes that were upregulated and downregulated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http: / / www.genome.jp / kegg / ). Based on the corrected P-value, the 20 pathways with the most significant enrichment were selected and visualized using bubble charts.

[0036] The experimental results are as follows:

[0037] Figure 1 Figures A and B in Chinese and Figure 2 The results showed that the combination of cisplatin and glycyrrhizin significantly enhanced the inhibitory effect of cisplatin on gastric cancer, and the two drugs exhibited a strong synergistic effect. However, the same concentration of glycyrrhizin combined with 5-FU showed an antagonistic effect (e.g., Figure 1 (Figures C and D). Figure 1 and Figure 2 In the diagram, C+E represents the cisplatin and glycyrrhizin chalcone combination group, Ctrl represents the control group, F+E represents the 5-fluorouracil and glycyrrhizin chalcone combination group, Cisplatin5 represents the cisplatin group, Echinatin 25 represents the glycyrrhizin chalcone group, 5-FU 5 represents the 5-fluorouracil group, and ns represents no significant difference.

[0038] CDI stands for Coefficient of Drug Interaction, calculated as CDI = AB / (A*B). AB is the ratio of the absorbance of the combined drug group to the control group, while A or B is the ratio of the absorbance of either drug alone to the control group. A CDI < 1 indicates a synergistic effect.

[0039] CDI = 1: This indicates that there is an additive effect.

[0040] CDI>1: Indicates antagonistic effect

[0041] When CDI < 0.7, it indicates that the synergistic effect is very significant.

[0042] Figure 3 Transcriptome sequencing analysis further indicated that the combination therapy group promoted apoptosis more effectively than the cisplatin-only treatment group (e.g., ...). Figure 3 As shown in Figure A), inhibiting the cell cycle (e.g. Figure 3 As shown in Figure B, cisplatin chalcone increases the sensitivity of gastric cancer cells to cisplatin through this mechanism, thereby enhancing the antitumor efficacy of cisplatin.

[0043] Example 2: The experimental method for enhancing the sensitivity of platinum-acquired cisplatin-resistant gastric cancer cells with cisplatin using licorice chalcone and clarifying its mechanism of action is as follows:

[0044] Identification of target proteins of chalcone from Glycyrrhiza uralensis: Arrayit HuProt TM Human proteome microarrays were purchased from Dajite Pharmaceutical Technology, Shanghai. Free biotin or biotinylated glycyrrhizin chalcone (Ech) was incubated on the microarrays for 1 hour at room temperature. After three washes, Cy3-labeled streptavidin was added, and the proteome microarrays were scanned using a Genepix 4000B, with data analysis performed using GenePix Pro-v6.0. The signal-to-noise ratio (SNR) of each protein spot was determined using the formula (mean foreground signal intensity minus mean background intensity) (Cheng et al., 2017). Protein binding strength was expressed as the IMean value (the ratio of the median fluorescence signal at each site to the background intensity). Specific binding targets for glycyrrhizin chalcone were screened based on the IMean_Ratio value (Ech-biotinylated histone IMean value divided by the corresponding IMean value of the control group). Based on previously reported techniques, we identified potential intracellular interaction targets of glycyrrhizin chalcone using liquid chromatography / tandem mass spectrometry (LC-MS / MS) analysis (Cheng et al., 2021). Then, we performed enrichment analysis on direct-binding proteins of glycyrrhizin chalcone using the KEGG database (http: / / www.genome.jp / kegg / ), and selected the 20 most significantly enriched pathways based on corrected p-values, which were then visualized using a bubble chart.

[0045] Gastric cancer cells IC 50 Assay: Wild-type AGS (AGS-WT) and platinum-resistant AGS (AGS-PR) cells in good growth condition were collected, digested, counted, and their density adjusted to 1*102 cells per well. 4Cells were seeded at 100 μL per well in 96-well plates. After overnight cell adhesion, working solutions were prepared for cisplatin, glycyrrhizin chalcone, and a combination of both drugs. Specifically: Cisplatin: A 10 mM cisplatin stock solution was diluted to a 100 μM intermediate solution with 1640 medium, and then serially diluted in 2-fold increments to obtain 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 μM cisplatin working solutions. Glycyrrhizin chalcone: A 200 mM glycyrrhizin chalcone stock solution was diluted in the same manner to obtain 400, 200, 100, 50, 25, 12.5, 6.25, and 0 μM glycyrrhizin chalcone working solutions. Two-drug combination: First, three groups of cisplatin working solutions were prepared as described above, each at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 μM. Then, cisplatin chalcone was added to each group at concentrations of 50, 25, and 0 μM. The original culture medium was removed, and 100 μL of the above drug solutions at different concentrations was added to each well, with five replicates for each concentration. After 48 h, the absorbance was measured using the CCK-8 assay, and the IC50 values ​​of AGS-WT and AGS-PR for each drug group were calculated using GraphPadPrism. 50 And draw a diagram.

[0046] The experimental results are as follows:

[0047] To further clarify the mechanism of action of glycyrrhizin chalcone combined with cisplatin in inhibiting gastric cancer, this invention used a 20K human proteome microarray combined with liquid chromatography / tandem mass spectrometry to detect and analyze the intracellular specific target proteins of glycyrrhizin chalcone. The results are as follows: Figure 4 As shown, the target proteins of glycyrrhizin chalcone were significantly enriched in the platinum-resistant pathway, suggesting that glycyrrhizin chalcone may have an inhibitory effect on platinum resistance in gastric cancer. Subsequently, the IC50 values ​​of AGS-WT and AGS-PR against cisplatin and glycyrrhizin chalcone were determined. 50 and through IC 50 The concentration of chalcone from glycyrrhiza was selected for subsequent combination therapy, among which... Figure 5 Figure A shows the IC50 of the AGS-PR cell line to cisplatin. 50 The IC50 of AGS-WT cells was significantly higher than that of AGS-WT cells, indicating platinum-based resistance; AGS-WT cells showed significantly higher IC50 values ​​against chalcone. 50 The IC50 value of AGS-PR against glycyrrhizin chalcone was 52.4 μM. 50 48.01 μM (e.g.) Figure 5 As shown in Figure B, there was no significant difference between the two. Based on this, the concentration of glycyrrhizin chalcone for combination therapy with cisplatin was selected as 25 or 50 μM. Under these conditions, the IC50 of AGS-WT and AGS-PR against cisplatin was determined. 50 The results showed that, after combined use of 25 or 50 μM glycyrrhizin chalcone, the IC50 of AGS-WT against cisplatin was significantly reduced. 50The concentration decreased from 7.623 μM to 3.559 μM and 2.989 μM, respectively. Figure 6 (As shown in Figure A), and after combined use of 25, 50 μM glycyrrhizin chalcone, AGS-PR showed an IC50 of cisplatin. 50 The concentrations decreased from 24.23 μM to 8.257 μM and 3.626 μM, respectively. Figure 6 (As shown in Figure B) These results indicate that glycyrrhizin chalcone can effectively increase the sensitivity of platinum-resistant gastric cancer cells to cisplatin.

[0048] Example 3: The experimental method for enhancing cisplatin-induced apoptosis using licorice chalcone is as follows:

[0049] Flow cytometry detection of apoptosis: AGS-WT and AGS-PR cells in good growth condition were harvested, digested, counted, and their density adjusted to 5*10⁶ cells per well. 5 Cells were seeded at 2 mL / well in 6-well plates. After overnight cell adhesion, each cell type was divided into four groups: control group (DMSO), cisplatin group (Cisplatin 5 μM), licorice chalcone group (Echinatin 25 μM), and combination group (Cisplatin 5 μM + Echinatin 25 μM). After 48 h, the supernatant and cells were collected, centrifuged, and the supernatant was discarded. The cells were resuspended once with pre-cooled PBS, then resuspended in 300 μL of binding buffer, and Annexin V-FITC and PI dye were added. A negative control group and a single-stain compensation group were also set up. After staining in the dark for 15 min, the cells were immediately analyzed. The data were analyzed and displayed using FlowJo software.

[0050] The experimental results are as follows:

[0051] Figure 7 Flow cytometry results showed that the combined use of glycyrrhizin chalcone and cisplatin significantly promoted apoptosis in AGS-WT and AGS-PR cells, and enhanced the apoptosis-inducing effect of cisplatin, indicating that glycyrrhizin chalcone can enhance the sensitivity of cells to cisplatin by promoting apoptosis.

Claims

1. Application of licorice chalcone in the preparation of antitumor drug sensitizers.

2. The application according to claim 1, characterized in that, The antitumor drug is a platinum-based chemotherapy drug.

3. Application of licorice chalcone in enhancing the antitumor efficacy of platinum-based chemotherapy drugs.

4. The application according to claim 3, characterized in that, The tumor is platinum-resistant gastric cancer.

5. The application according to claim 2 or 3, characterized in that, The platinum-based chemotherapy drugs include at least one of cisplatin, carboplatin, oxaliplatin, lobaplatin, and nedaplatin.

6. An antitumor drug composition, characterized in that, This includes chemotherapy drugs and licorice chalcone.

7. The antitumor pharmaceutical composition according to claim 6, characterized in that, The chemotherapy drug includes at least one of cisplatin, carboplatin, oxaliplatin, lobaplatin, and nedaplatin, and the molar ratio of the chemotherapy drug to chalcone is 1-20:10-100.

8. The antitumor pharmaceutical composition according to claim 6, characterized in that, The final concentration of the chalcone from the licorice root is 10-100 μM.

9. The antitumor pharmaceutical composition according to claim 6, characterized in that, The chemotherapy drug is cisplatin at a final concentration of 5-100 μM.

10. The antitumor pharmaceutical composition according to claim 6, characterized in that, It also includes pharmaceutically acceptable excipients, and the dosage form of the antitumor drug composition includes at least one of capsules, aqueous injections, powder injections, tablets, gels, suspensions, and solutions.

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