Use of isocryptomerin 7-o-glucuronide in preparation of anticancer drugs

Abstract

The application discloses application of isohomoerudifin 7-O-glucuronide in preparation of anticancer drugs, and the cancer includes nasopharyngeal carcinoma and the like. 50 The application proves by experiments that isohomoerudifin 7-O-glucuronide inhibits C666-1 cells in a time and concentration dependent manner, and the IC50 of 24, 48 and 72 hours is 152.0, 74.16 and 58.91 muM respectively; the isohomoerudifin 7-O-glucuronide can significantly inhibit cell migration, induce cell apoptosis in a concentration dependent manner, and arrest cell cycle in G0 / G1 phase; it is indicated that the isohomoerudifin 7-O-glucuronide can inhibit C666-1 cell proliferation and migration, induce cell apoptosis and arrest cell cycle in vitro, and the mechanism may be related to core target points such as ALB, MAPK8, HSPA8, CTSS and DHFR.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to the anti-tumor application of active ingredients in natural drugs, and particularly to the application of isocoryne 7-O-glucuronide in the preparation of anticancer drugs, especially anti-nasopharyngeal carcinoma drugs. This invention is applicable to the fields of anti-tumor drug development, research on the pharmacological mechanisms of natural products, and the preparation of drugs for the treatment of head and neck malignant tumors. Background Technology

[0002] Nasopharyngeal carcinoma is a common head and neck malignant tumor in southern my country, characterized by its high invasiveness, high recurrence and metastasis rates, and low early diagnosis rate, seriously threatening patients' lives and health. Currently, clinical treatment mainly involves radiotherapy, chemotherapy, and combination therapy, but these methods suffer from problems such as frequent drug resistance, significant side effects, poor patient compliance, and limited long-term efficacy. There is an urgent clinical need for novel, highly effective, low-toxicity, and clearly defined natural anti-tumor candidate drugs.

[0003] Isocoryne 7-O-glucuronide is a flavonoid glycoside compound isolated from medicinal plants such as safflower. Previous studies have shown that it possesses anti-inflammatory and antioxidant activities; however, its inhibitory effect on nasopharyngeal carcinoma cells, its target site, and its molecular mechanism have not been reported, and there are no patents or published literature related to its application in the preparation of anti-nasopharyngeal carcinoma drugs. This invention, through in vitro experiments combined with network pharmacology, molecular docking, and protein validation, clarifies the inhibitory effect and core regulatory target of isocoryne 7-O-glucuronide on human nasopharyngeal carcinoma cells C666-1, filling a technological gap and providing a new drug source and research direction for nasopharyngeal carcinoma treatment. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and address the problems of significant toxic side effects, easy drug resistance, and insufficient utilization of natural active ingredients in current nasopharyngeal carcinoma treatment drugs. This invention provides a novel application of isoscrolin 7-O-glucuronide in the preparation of anticancer drugs, especially anti-nasopharyngeal carcinoma drugs, clarifying its role in inhibiting proliferation, migration, inducing apoptosis, and arresting cell cycle, as well as its core target.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention discloses the application of isosorcinol 7-O-glucuronide in the preparation of anticancer drugs.

[0006] Specifically, the above-mentioned application, where the cancer includes but is not limited to nasopharyngeal carcinoma, discloses the application of isosaffron 7-O-glucuronide in the preparation of anti-nasopharyngeal carcinoma drugs.

[0007] Furthermore, in the above applications, the nasopharyngeal carcinoma includes, but is not limited to, human nasopharyngeal carcinoma C666-1 cells.

[0008] Furthermore, in the above applications, the isocoryne 7-O-glucuronide can significantly inhibit the migration of nasopharyngeal carcinoma cells, induce apoptosis, and arrest the cell cycle in the G0 / G1 phase.

[0009] Furthermore, in the above applications, the isocoryne 7-O-glucuronide can regulate the expression of ALB and MAPK8 / p-MAPK8 proteins.

[0010] This invention investigates the in vitro inhibitory effect and potential molecular mechanism of isocorycin 7-O-glucuronide on human nasopharyngeal carcinoma cell line C666-1. The CCK-8 assay was used to detect the inhibitory effect of isocorycin 7-O-glucuronide on the proliferation of C666-1 cells at 24, 48, and 72 h, and the IC50 was calculated. 50 The effects of the drug on the migration ability, apoptosis rate, and cell cycle distribution of C666-1 cells were detected by cell migration assay and flow cytometry, respectively. Network pharmacology was used to screen potential targets of isocorycin 7-O-glucuronide against nasopharyngeal carcinoma, and a protein-protein interaction (PPI) network was constructed to screen core targets. Molecular docking was used to verify the binding activity of the core targets and the drug. Western blot was used to verify the expression of the core target protein.

[0011] This invention has experimentally confirmed that isosorcinol 7-O-glucuronide has a time- and concentration-dependent inhibitory effect on C666-1 cells, with IC50 values ​​at 24, 48, and 72 h. 50 The concentrations were 152.0, 74.16, and 58.91 μM, respectively. It significantly inhibited cell migration, induced apoptosis in a concentration-dependent manner, and arrested the cell cycle at the G0 / G1 phase. Network pharmacology screening identified 113 potential targets of isocorycin 7-O-glucuronide, 3092 nasopharyngeal carcinoma-related targets, and 37 intersection targets. PPI network analysis identified ALB, MAPK8, HSPA8, CTSS, and DHFR as core targets. Molecular docking results showed that the binding energies of isocorycin 7-O-glucuronide to the five core targets were all below -5.0 kcal / mol, indicating good binding activity. Western blot successfully detected the protein expression of the core targets ALB, MAPK8, and the internal control GAPDH. This indicates that isocoryne 7-O-glucuronide can inhibit the proliferation and migration of C666-1 cells in vitro, induce apoptosis and arrest the cell cycle. Its mechanism of action may be related to the regulation of core targets such as ALB, MAPK8, HSPA8, CTSS and DHFR.

[0012] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1) This invention is the first to demonstrate that isocoryne 7-O-glucuronide has significant in vitro antitumor activity against human nasopharyngeal carcinoma cells C666-1, with a clear effect and significant efficacy; 2) This invention clearly demonstrates that it exerts its effects by inhibiting proliferation, migration, inducing apoptosis, and arresting the G0 / G1 phase; the mechanism is clear and the target can be verified. 3) This invention screens and validates core targets such as ALB and MAPK8, providing new pathways for the development of targeted drugs for nasopharyngeal carcinoma; 4) The isosorcinol 7-O-glucuronide described in this invention is derived from natural plants, has high safety and low toxicity, and is suitable for development as a novel low-toxicity anti-nasopharyngeal carcinoma candidate drug. Attached Figure Description

[0013] Figure 1 The effect of isochordin 7-O-glucuronide on the proliferation of C666-1 cells; changes in cell viability of C666-1 cells after treatment with isochordin 7-O-glucuronide for 24 h (left), 48 h (middle), and 72 h (right). n=3; *P<0.05, **P<0.01, ***P<0.001.

[0014] Figure 2 Effect of isochordin 7-O-glucuronide on C666-1 cell migration; Left: Scratch map of different treatment groups at 0 h and 48 h; Right: Quantitative statistical graph of scratch healing rate. n=3; *P<0.05, **P<0.01, ***P<0.001.

[0015] Figure 3 The effect of isocoryne 7-O-glucuronide on apoptosis in C666-1 cells (A: Annexin V-FITC / PI double staining flow cytometry was used to detect apoptosis in C666-1 cells treated with different concentrations of isocoryne 7-O-glucuronide at 0, 20, 40, and 80 μM. B: Statistical bar chart of apoptosis rate after treatment with different concentrations of the drug; n=3; *P<0.05, **P<0.01, ***P<0.001).

[0016] Figure 4 The effect of isocoryne 7-O-glucuronide on the cell cycle distribution of C666-1 cells (A: Representative histograms of DNA content distribution after treatment with different concentrations of isocoryne 7-O-glucuronide (0, 20, 40, 80 μM) by flow cytometry. B: Quantitative statistical bar charts of the proportion of cells in each cell cycle phase (G0 / G1, S, G2 / M) after treatment with different concentrations of the drug; n=3; *P<0.05, **P<0.01, ***P<0.001).

[0017] Figure 5 This is a Venn diagram (blue represents nasopharyngeal carcinoma targets, yellow represents isocoryne 7-O-glucuronide targets, and the overlapping part represents the intersection of the two targets).

[0018] Figure 6 This is a network diagram of protein-target interactions (interactions between the potential target of isocorycin 7-O-glucuronide and the target proteins of nasopharyngeal carcinoma. Nodes represent protein targets, lines represent protein interactions, and the core target with the highest degree value is ALB).

[0019] Figure 7 The core target graph of the PPI network is shown (the color depth and size of the nodes are positively correlated with the degree value, among which ALB is the key gene with the highest degree value, followed by HSPA8, MAPK8, CTSS and DHFR).

[0020] Figure 8 KEGG pathway enrichment analysis for isoscrolin 7-O-glucuronide as a potential target for the treatment of nasopharyngeal carcinoma.

[0021] Figure 9 GO functional enrichment analysis of isocoryne 7-O-glucuronide as a potential target in nasopharyngeal carcinoma; GO enrichment bar plots show the top 8 significantly enriched items of the intersection target in the three dimensions of biological processes (BP), cellular components (CC), and molecular functions (MF).

[0022] Figure 10 Molecular docking verification of isocoryne 7-O-glucuronide with the core target (A: ALB, B: MAPK8, C: HSPA8, D: CTSS, E: DHFR).

[0023] Figure 11 This study investigates the regulatory effect of isocorycin 7-O-glucuronide on the expression of core target proteins in C666-1 cells. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0025] In the following experiments, all raw materials used were commercially available products that could be directly purchased, or could be prepared using conventional techniques in the field. Operations or steps not detailed in the experiments can be performed using conventional techniques in the field, and since they are not the innovation of this application, they will not be elaborated upon here.

[0026] 1.1. Cells and reagents.

[0027] Human nasopharyngeal carcinoma cells C666-1; isochoric acid 7-O-glucuronide (purity ≥98%); CCK-8 assay kit, apoptosis and cell cycle assay kit; RPMI 1640 medium, fetal bovine serum; ALB, MAPK8, p-MAPK8, and GAPDH antibodies.

[0028] 1.2. Cell culture.

[0029] C666-1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator, and cells in the logarithmic growth phase were used for experiments.

[0030] 1.3. CCK-8 assay for cell proliferation.

[0031] C666-1 cells in the logarithmic growth phase were collected, counted, and prepared at a concentration of 1 × 10⁻⁶. 5 Cell suspension of cells / mL Cell suspension was seeded into 96-well plates at 100 μL per well, with three replicates per group. The plates were incubated at 37 °C in a 5% CO2 incubator. Cells were collected after 24, 48, and 72 h, the old culture medium was aspirated, and fresh medium was replaced. 10 μL of CCK-8 solution was added to each well, avoiding air bubbles that could affect the absorbance. The plates were then incubated for another 1.5 h for color development (preliminary experiments showed that 1.5 h of incubation yielded the best results). The absorbance at 450 nm was measured using a microplate reader, and the inhibition rate and IC50 were calculated. 50 .

[0032] 1.4. Scratch test to detect migration.

[0033] When cells reached nearly 90% confluence, scribbles were manually made on a monolayer of cells using a 100 μL sterile pipette tip. Serum-free RPMI 1640 medium containing the drug at concentrations of 0, 20, 40, and 80 μM were added, and the cells were incubated at 37°C with 5% CO2 for 48 h. The scribbles were measured and photographed, and the relative migration distance was calculated. The results showed that the drug inhibited cell migration in a concentration-dependent manner.

[0034] 1.5. Flow cytometry detection of apoptosis and cell cycle.

[0035] C666-1 cells in the logarithmic growth phase were collected, digested with trypsin without EDTA, and washed twice with PBS. Cells were resuspended in 500 μL of Binding Buffer, followed by 5 μL of Annexin V-APC, gently pipetting to mix, and then 5 μL of PI staining solution, gently pipetting to mix again. The cells were incubated at room temperature in the dark for 15 min, and analyzed by flow cytometry within 1 h. Results showed that the drug significantly increased the apoptosis rate and arrested the cell cycle at the G0 / G1 phase.

[0036] 1.6. Network pharmacology and molecular docking.

[0037] Drug and disease targets were obtained from databases such as PharmMapper and GeneCards, and 37 intersections were obtained. A PPI network was constructed, and ALB, MAPK8, HSPA8, CTSS, and DHFR were screened as core targets. Molecular docking showed that the binding energies were all <-5.0 kcal / mol, indicating that the binding was stable.

[0038] 1.7. Western blot validation of the target.

[0039] After drug addition, C666-1 cell samples were added to radioimmunoprecipitation (RIPA) lysis buffer. Following homogenization, lysis, and centrifugation, total protein extract from C666-1 cells was obtained, and protein concentration was determined using the Bradford method. Protein electrophoresis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer onto a polyvinylidene fluoride (PVDF) membrane at a constant current of 200 mA for 1 h. The PVDF membrane was blocked in TBS-T blocking buffer containing 5% skim milk powder for 1 h, then primary antibody was added, and the membrane was incubated overnight at 4 °C with shaking. After washing, secondary antibody was added, and the membrane was incubated at 37 °C for 1 h. After washing, chemiluminescence solution was added, the membrane was pressed, exposed, and fixed. The integrated optical density of the protein bands was measured using Fusion image acquisition and analysis software. Internal controls were established using GAPDH or PARK7 protein bands as described in previous articles published by the research group. The results showed that the expression of ALB and MAPK8 / p-MAPK8 exhibited a regulatory trend with increasing drug concentration, validating the role of the core target.

[0040] Note: ALB: 69 kd, MCE antibody (1:2000); MAPK8 44 kd multi-class biological antibody (1:1000) p-MAPK8 multi-class biological antibody (1:1000) This protein is also called JNK, which is a double-banded protein; GAPDH 37 kd Hangzhou Xianzhi (1:1000).

[0041] 2. Results.

[0042] 2.1 Isorhodin 7-O-glucuronide inhibits the proliferation of C666-1 cells.

[0043] Figure 1 The changes in cell viability of C666-1 cells after treatment with isocoryne 7-O-glucuronide for 24 h, 48 h, and 72 h are presented. Figure 1 CCK-8 assay results showed that isocoryne 7-O-glucuronide significantly inhibited the proliferation of C666-1 cells, and the inhibitory effect increased with prolonged treatment time and higher drug concentration. IC50 values ​​were obtained at 24, 48, and 72 h of drug treatment. 50 The concentrations were 152.0 μM, 74.16 μM, and 58.91 μM, respectively, and showed significant time and dose dependence.

[0044] 2.2 Isorhodin 7-O-glucuronide inhibits C666-1 cell migration.

[0045] Figure 2 The effect of isochordin 7-O-glucuronide on the migration of C666-1 cells is shown; Left: Scratch map of different treatment groups at 0 h and 48 h; Right: Quantitative statistical chart of scratch healing rate (the bar chart on the right is a statistical quantification of the chart on the left). Figure 2 The scratch assay results showed that the 0 μM group of cells had a strong scratch healing ability after 48 h, while the scratch healing degree of the 20, 40, and 80 μM drug groups gradually decreased with increasing concentration, and the migration inhibition rate increased significantly (P<0.05), indicating that isocoryne 7-O-glucuronide can inhibit the in vitro migration ability of C666-1 cells in a concentration-dependent manner.

[0046] 2.3 Isorhodin 7-O-glucuronide induces apoptosis in C666-1 cells.

[0047] Figure 3 The effect of isocoryne 7-O-glucuronide on apoptosis in C666-1 cells was presented. A: Annexin V-FITC / PI double staining flow cytometry was used to detect apoptosis in C666-1 cells treated with different concentrations of isocoryne 7-O-glucuronide (0, 20, 40, and 80 μM). B: Statistical quantitative bar chart of apoptosis rate after treatment with different concentrations of the drug. Figure 3 Flow cytometry results showed that, compared with the 0 μM control group, the apoptosis rate of C666-1 cells increased in a concentration-dependent manner after treatment with 20, 40, and 80 μM isocoryne 7-O-glucuronide (P<0.05), with the 80 μM group reaching the peak apoptosis rate, indicating that the drug can effectively induce apoptosis in C666-1 cells.

[0048] 2.4 Isochrysis 7-O-glucuronide arrests the C666-1 cell cycle in the G0 / G1 phase.

[0049] Figure 4 The effects of isocoryne 7-O-glucuronide on the cell cycle distribution of C666-1 cells are presented (A: Representative histograms of DNA content distribution in C666-1 cells after treatment with different concentrations of isocoryne 7-O-glucuronide (0, 20, 40, 80 μM) by flow cytometry; B: Quantitative statistical bar charts of the proportion of cells in each cell cycle phase (G0 / G1, S, G2 / M) after treatment with different concentrations of the drug.). Figure 4 Cell cycle analysis results showed that, compared with the control group, the proportion of cells in the G0 / G1 phase was significantly increased (P<0.05), the proportion of cells in the S phase was significantly decreased (P<0.05), and the proportion of cells in the G2 / M phase was significantly decreased (P<0.05). This indicates that isocoryne 7-O-glucuronide can arrest the cell cycle of C666-1 cells in the G0 / G1 phase, inhibiting cells from entering the S phase from the G0 / G1 phase, thereby inhibiting cell proliferation.

[0050] 2.5 Results of network pharmacology screening. See Table 1.

[0051] Table 1. Online Drug Databases and Software

[0052] 2.5.1 Intersection between potential drug targets and nasopharyngeal carcinoma-related targets.

[0053] Figure 5 Venn diagrams are provided (blue represents nasopharyngeal carcinoma targets, red represents isocoryne 7-O-glucuronide targets, and the overlapping part represents the intersection of the two targets). Figure 5 113 potential targets of isocoryne 7-O-glucuronide were identified by screening the PharmMapper database, and 3092 nasopharyngeal carcinoma-related targets were obtained by integrating six major databases. Venny analysis showed that there were 37 overlapping targets between the two, which are potential targets for drug action against nasopharyngeal carcinoma.

[0054] 2.5.2 PPI network construction and core target screening.

[0055] Figure 6 A network diagram of target-protein interactions is presented (the interaction relationship between the potential target of isocorycin 7-O-glucuronide and the intersection protein of the target of nasopharyngeal carcinoma. The nodes are protein targets, the lines are protein interactions, and the core target with the highest degree value is ALB). Figure 6The PPI network constructed from the STRING database contains 37 nodes and 102 edges. The network was imported into Cytoscape software and analyzed using the CytoNCA plugin. The top 5 core targets were selected based on their Degree values, namely ALB, MAPK8, HSPA8, CTSS, and DHFR. These targets occupy core control positions in the PPI network.

[0056] 2.5.3 GO and KEGG enrichment analysis.

[0057] Figure 7 The core target graph of the PPI network is given (the color depth and size of the nodes are positively correlated with the degree value, among which ALB is the key gene with the highest degree value, followed by HSPA8, MAPK8, CTSS and DHFR). Figure 7 Enrichment analysis of biological processes in GO showed that the overlapping targets were mainly enriched in biological processes such as cell proliferation regulation, apoptosis, oxidative stress response, and signaling pathway regulation.

[0058] Figure 8 We present a KEGG pathway enrichment analysis of isoscrolin 7-O-glucuronide as a potential target for the treatment of nasopharyngeal carcinoma. Figure 8 Enrichment analysis of the KEGG signaling pathway identified the top 20 significantly enriched pathways, mainly involving the tumor necrosis factor signaling pathway, the MAPK signaling pathway, and the cell cycle regulation pathway, all of which are closely related to the occurrence and development of nasopharyngeal carcinoma.

[0059] Figure 9 GO functional enrichment analysis of isosaflavin 7-O-glucuronide as a potential target in nasopharyngeal carcinoma; GO enrichment bar charts show the top 8 significantly enriched items of the intersection targets in the three dimensions of biological processes (BP), cellular components (CC), and molecular functions (MF).

[0060] 2.6 Molecular docking results.

[0061] Table 2 shows the binding energies of isocorycin 7-O-glucuronide to five core targets. From Table 2 and... Figure 10Medium-molecule docking results showed that the binding energies of isocoryne 7-O-glucuronide to all five core targets were below -5.0 kcal / mol, with MAPK8 (-8.61 kcal / mol) and DHFR (-8.67 kcal / mol) having the lowest binding energies, followed by ALB (-7.23 kcal / mol) and HSPA8 (-7.02 kcal / mol). CTSS (-5.82 kcal / mol) was above the lowest threshold, indicating that the drug exhibits good binding activity to all five core targets and can stably bind to form complexes. PyMOL visualization showed that the drug can form hydrogen bonds and hydrophobic interactions with the active pocket amino acid residues of the target proteins.

[0062] Table 2. Binding energies of isocorycin 7-O-glucuronide to 5 core targets.

[0063] 2.7 Validation of expression of core target proteins.

[0064] Figure 11 The regulatory effect of isocoryne 7-O-glucuronide on the expression of core target proteins in C666-1 cells was presented. Figure 11 Western blot results showed that ALB, MAPK8 (JNK, a dual-band protein) and the internal control GAPDH were all clearly expressed in C666-1 cells. Among them, the expression level of MAPK8 / p-MAPK8 protein showed a downward trend with increasing drug concentration, which preliminarily verified the regulatory role of the core target in the drug's anti-nasopharyngeal carcinoma process.

[0065] In summary, the isocoryne 7-O-glucuronide described in this invention can exert anti-nasopharyngeal carcinoma effects by inhibiting cell proliferation and migration, inducing apoptosis, arresting the G0 / G1 phase, and regulating core targets such as ALB and MAPK8, and can be used to prepare anti-nasopharyngeal carcinoma drugs.

Claims

1. The application of an isocoryne 7-O-glucuronide in the preparation of anticancer drugs.

2. The application as described in claim 1, characterized in that, The cancers mentioned include nasopharyngeal carcinoma.

3. The application as described in claim 2, characterized in that, The nasopharyngeal carcinoma includes human nasopharyngeal carcinoma C666-1 cells.

4. The application as described in claim 2, characterized in that, The isocoryne 7-O-glucuronide can significantly inhibit the migration of nasopharyngeal carcinoma cells, induce apoptosis, and arrest the cell cycle in the G0 / G1 phase.

5. The application as described in claim 2, characterized in that, The isocoryne 7-O-glucuronide can regulate the expression of ALB and MAPK8 / p-MAPK8 proteins.

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