Application of traditional Chinese medicine composition in preparation of medicine for reversing pancreatic cancer radiotherapy resistance by destroying redox balance and activating ferroptosis procedure

Abstract

The invention discloses application of a traditional Chinese medicine composition in preparation of a medicine for reversing pancreatic cancer radiotherapy resistance by destroying redox balance and activating a ferroptosis procedure, and belongs to the technical field of medicine. Based on a new mechanism of pancreatic cancer radiotherapy resistance, i.e., tumor cells obtain resistance by up-regulating glutathione S-transferase P1 (GSTP1) to inhibit 5-lipoxygenase (ALOX5)-mediated ferroptosis, the invention provides a new application of a classic prescription dark plum pill or pharmaceutically acceptable extracts, effective parts or effective components thereof. According to the traditional Chinese medicine composition, by targeting a GSTP1 / ALOX5 axis, redox balance reconstructed in radiotherapy resistant cells is destroyed, lipid peroxidation and ferroptosis are reactivated, and therefore the sensitivity of tumor cells to radiotherapy is recovered. The radiotherapy sensitization effect and the mechanism of the dark plum pills are confirmed through systematic network pharmacology, bioinformatics analysis and in-vivo and in-vitro experiments, a brand new target spot and a treatment strategy are provided for overcoming pancreatic cancer radiotherapy resistance, and a scientific foundation is laid for modern development and clinical application of the dark plum pills.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the novel use of a traditional Chinese medicine composition in the preparation of a drug for sensitizing pancreatic cancer radiotherapy and reversing its radiotherapy resistance. The traditional Chinese medicine composition is based on the traditional formula Wumei Pill (WMP) or its pharmaceutically acceptable extract.

[0002] This invention is based on the discovery of a novel mechanism of radioresistance in pancreatic cancer: tumor cells escape ferroptosis and establish radioresistance by upregulating glutathione S-transferase P1 (GSTP1) and inhibiting 5-lipoxygenase (ALOX5)-mediated lipid peroxidation. The traditional Chinese medicine composition provided by this invention can target GSTP1 / ALOX5, disrupt the abnormal redox balance in radioresistant cells, and reactivate lipid peroxidation and ferroptosis, thereby achieving radiosensitization. Background Technology

[0003] Pancreatic cancer (PC) is a highly malignant gastrointestinal tumor with an increasing incidence and mortality rate. Radiotherapy is an important local treatment for pancreatic cancer, but its efficacy is often limited by acquired radioresistance in tumor cells. Studies have shown that radioresistance is closely related to abnormal reactive oxygen species (ROS) metabolism and enhanced DNA damage repair capabilities within tumor cells. In recent years, ferroptosis, an iron-dependent programmed cell death mechanism driven by lipid peroxidation, has been found to play a key role in tumor treatment resistance. Lipid metabolism reprogramming, particularly the imbalance of pathways related to lipid peroxidation, is considered a hallmark of radioresistance in pancreatic cancer.

[0004] Glutathione S-transferase P1 (GSTP1) is a widely expressed phase II metabolic enzyme in the human body with significant antioxidant and anti-inflammatory functions. It has been found to be associated with chemotherapy resistance and ROS resistance in various tumors. 5-Lipooxygenase (ALOX5) is a key enzyme driving lipid peroxidation. Studies suggest that GSTP1 may inhibit ferroptosis by suppressing ALOX5-mediated lipid peroxidation, thereby promoting tumor cell survival. However, the specific roles of GSTP1 and ALOX5 in radiotherapy resistance in pancreatic cancer and their mechanisms of regulating ferroptosis remain unclear. Developing effective radiosensitizers targeting this pathway has significant clinical implications.

[0005] Wumei Wan (WMP) is a classic Chinese herbal formula derived from the *Shanghan Lun* (Treatise on Cold Damage). In clinical practice, it is commonly used to treat complex conditions involving both cold and heat patterns, as well as deficiency and excess. Preliminary clinical observations suggest that Wumei Wan-based formulas combined with radiotherapy may be beneficial for pancreatic cancer patients, but the scientific basis and molecular mechanisms remain unclear.

[0006] Therefore, the purpose of this invention is to provide a new use of Wumei Pill or its active ingredients in the preparation of radiosensitizing drugs for pancreatic cancer, based on the above-mentioned scientific problems and clinical needs, and to elucidate its molecular mechanism of intervening in the GSTP1 / ALOX5 axis, disrupting redox balance, and activating ferroptosis. Summary of the Invention

[0007] The technical problem to be solved by this invention is: addressing the lack of effective sensitization methods for existing pancreatic cancer radiotherapy resistance, and the unclear mechanism of action of Wumei pill in this field, this invention provides a new use for Wumei pill or its pharmaceutically acceptable extracts, effective parts or active ingredients in the preparation of drugs to reverse pancreatic cancer radiotherapy resistance.

[0008] The present invention provides the following technical solution: the application of a traditional Chinese medicine composition in the preparation of a drug for reversing radiotherapy resistance in pancreatic cancer by disrupting redox balance and activating ferroptosis; the traditional Chinese medicine composition is Wumei Wan or its pharmaceutically acceptable extract, effective part or active ingredient.

[0009] Furthermore, the Wumei Pill is made from the following raw materials in parts by weight: 20 parts of dried plum, 3 parts of asarum, 10 parts of dried ginger, 9 parts of cinnamon twig, 9 parts of aconite (decocted first), 6 parts of Sichuan pepper, 9 parts of coptis, 9 parts of ginseng, 6 parts of angelica, and 9 parts of phellodendron; or their conventional pharmaceutically equivalent dosage.

[0010] Furthermore, the traditional Chinese medicine composition exerts its effect of reversing radiotherapy resistance by downregulating the expression and / or activity of glutathione S-transferase P1 (GSTP1) in pancreatic cancer cells, thereby relieving its inhibition of 5-lipoxygenase (ALOX5).

[0011] Furthermore, the traditional Chinese medicine composition inhibits GSTP1, thereby reactivating ALOX5-mediated lipid peroxidation and ultimately inducing ferroptosis, thus achieving radiosensitization.

[0012] Furthermore, the active ingredients that play a role in the traditional Chinese medicine composition include, but are not limited to, kaempferol, quercetin, capsaicin, digitalis flavonoids and their metabolites.

[0013] The beneficial effects of this invention are:

[0014] (1) Clear preclinical evidence was provided: Through systematic network pharmacology, transcriptomics, and in vivo and in vitro functional experiments, it was confirmed for the first time that Wumei Pill has a significant radiosensitizing effect on pancreatic cancer.

[0015] (2) This study reveals a novel target and mechanism of action: For the first time, it elucidates that pancreatic cancer acquires radiotherapy resistance by upregulating GSTP1 to inhibit ALOX5-mediated ferroptosis. Wumei pills, by targeting this GSTP1 / ALOX5 axis, disrupt the redox balance rebuilt after radiotherapy, reigniting lipid peroxidation and ferroptosis, thereby reversing resistance. This provides a completely new scientific perspective and intervention target for overcoming radiotherapy resistance in pancreatic cancer.

[0016] (3) Achieving a modern scientific interpretation of traditional Chinese medicine: The clinical application of the classic formula Wumei Wan is precisely located on the clear modern biological pathway of "GSTP1 / ALOX5-ferroptosis", laying a solid theoretical foundation for its secondary development as a novel radiosensitizer.

[0017] (4) It has broad development prospects: This invention is not limited to the whole formula of Wumei Pill. The discovery of its active ingredients (such as kaempferol, quercetin, etc.) provides a preliminary foundation for the development of modern Chinese medicine innovative drugs or adjuvant therapy products with clear ingredients and controllable quality. Attached Figure Description Figure 1 Network pharmacology and bioinformatics analysis reveals the potential mechanism by which Wumei Pill intervenes in pancreatic cancer by regulating GSTP1 and ALOX5. (A) Wumei Pill "compound-target" network diagram; (B) Venn diagram of overlapping gene sets; (C) Wumei Pill target protein-protein interaction network based on TCMSP database; (D) Key functional modules in the PPI network; (E) Schematic diagram of ligand-target binding interaction, with maximum binding energy (unit: kcal / mol) marked. Figure 2Analysis of the expression, survival correlation, and association between GSTP1 and ALOX5 in pancreatic cancer and their response to radiotherapy and ferroptosis. (A, B) Comparison of transcriptional levels of GSTP1 and ALOX5 in pancreatic ductal adenocarcinoma and adjacent normal tissues (Data source: GSE15471); (C, D) Comparison of transcriptional levels of GSTP1 and ALOX5 in pancreatic cancer and normal tissues (Data source: GSE62452); (E, F) Kaplan-Meier analysis of overall survival in pancreatic cancer patients with high (red) and low (blue) expression of GSTP1 (E) and ALOX5 (F) in the TCGA database, using median expression as the grouping threshold; (G, H) Kaplan-Meier analysis of overall survival in pancreatic cancer patients with high and low expression of GSTP1 (G) and ALOX5 (H) in the GSE62452 dataset, using median expression as the grouping threshold; (I, J) Changes in transcriptional levels of GSTP1 (I) and ALOX5 (J) in patient specimens (GSE225767) before and after stereotactic radiotherapy; (K) (L) Correlation analysis of GSTP1 and ALOX5 expression in the GSE225767 cohort; (M) Correlation analysis of GSTP1 and ALOX5 expression and functional scores in the TCGA-PAAD cohort; (M) Kaplan-Meier analysis of the impact of ferroptosis activity scores (high vs. low, grouped by median) on overall survival in TCGA pancreatic cancer patients. All survival analyses were statistically compared using the Log-rank test, and the corresponding P-values ​​are indicated in the figures. Figure 3 Verification of the radiosensitizing effect of Wumei Pill (WMP) targeting GSTP1 / ALOX5. Figure 3 A: CCK-8 assay was used to detect the inhibition curve and IC50 of WMP on the proliferation of pancreatic cancer cells. Figure 3 BC: Scratch assay verifies the inhibitory effect of WMP on the migration of pancreatic cancer cells. Figure 3 DF: Schematic diagram of animal experiment design and the inhibitory effect of WMP combined with radiotherapy on tumor growth in tumor-bearing mice. Figure 3 GI: Western blotting and transcriptome sequencing were used to verify changes in the levels of GSTP1, ALOX5, and GPX4 proteins and mRNA in tumor tissues. Figure 4 The dynamic effects of radiotherapy on residual pancreatic cancer cells and key protein levels. Figure 4 AC: Changes in cell viability and expression of GSTP1, ALOX5, and GPX4 proteins after different doses of radiotherapy. Figure 4 DF: Kinetic changes in cell viability and expression of the above proteins at different time points after a single radiotherapy session. Figure 5 . Wumei Pills enhance the sensitivity to radiotherapy by regulating ferroptosis-related molecules and lipid metabolism. (A-B) Colony formation assay showing the inhibitory effect on cell proliferation. (C-D) Protein expression levels of GSTP1, ALOX5, and GPX4. (E) Oil Red O staining to evaluate lipid accumulation in tumor tissues. Scale bar: 50 μm. (F) Changes in the transcriptional levels of ferroptosis-related genes. Figure 6 . Wumei Pills promote ferroptosis by activating lipid peroxidation and oxidative stress. (A-C) Intracellular reactive oxygen levels and lipid peroxidation. Scale bar: 50 μm. (D-E) Biochemical indicators of ferroptosis: malondialdehyde content and ferrous ion levels. FPKM: Fragments Per Kilobase of transcript per Million mapped reads. Figure 7 . Schematic diagram of the mechanism of ferroptosis-mediated radiosensitization based on the GSTP1 / ALOX5 axis. Figure 8 . Characterization of active ingredients in serum of mice after Wumei Pills intervention. (A) Total ion chromatogram of LC-MS of the decoction of Wumei Pills in positive ion mode; (B) Total ion chromatogram of LC-MS of the decoction of Wumei Pills in negative ion mode; (C) Chemical structure of isoquercitrin; (D) Chemical structure of quercetin-3-O-glucoside. Figure 9 . Transcriptome profiling and enrichment analysis of Wumei Pills-mediated radiosensitization (Note: CG, sham operation control group; TT, Wumei Pills treatment group; CB, radiotherapy combined with Wumei Pills group). (A) Sample correlation heat map; (B) Principal component analysis; (C) Venn diagram of differentially expressed genes between groups; (D) Cluster analysis of differential transcripts; (E-F) KEGG enrichment analysis of differentially expressed genes in the comparison of RT vs. Sham and RT+WMP vs. WMP; (G-H) KEGG enrichment analysis of differentially expressed genes in the comparison of WMP vs. Sham and RT+WMP vs. RT; (I) KEGG enrichment analysis of differentially expressed genes in the comparison of RT+WMP vs. Sham. Detailed implementation methods

[0018] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are only used to illustrate the present invention and should not be construed as limiting the present invention.

[0019] Example 1: Systematic biological prediction of potential targets and active ingredients of Wumei Pills (WMP) in intervening pancreatic cancer

[0020] (1) Network pharmacology analysis: Chemical components and targets of WMP were retrieved from the TCMSP database, and components with OB ≥ 30% and DL ≥ 0.18 were screened. A "herbal-component-target" network was constructed. The intersection of WMP targets with the set of pancreatic cancer metabolism-related genes and differentially expressed genes obtained in previous studies of our research group was taken to obtain 50 core intersection genes. Through PPI network and MCODE plugin analysis, GSTP1 and ALOX5 were found to be located at key nodes in the network ( Figure 1 ).

[0021] (2) Molecular docking verification: Potential active ingredients (kaempferol, quercetin, capsaicin, and digitalis flavonoids) targeting GSTP1 and ALOX5 were obtained from the network. Molecular docking was performed using AutoDock Vina software. The results showed that the binding energies of the above components to GSTP1 or ALOX5 were all below -5 kcal / mol, indicating stable binding. Figure 1 ).

[0022] (3) LC-MS Validation: LC-MS analysis was performed on the aqueous decoction of WMP and the plasma of mice that had been administered WMP by gavage for 3 consecutive days. Glycoside metabolites of kaempferol and quercetin (such as isoquercitrin) were detected in both. Metabolites such as capsaicin β-D-glucopyranoside (capsaicin-related) and digitalis flavonoids (digitifloxacin-related) were specifically detected in the plasma of the intervention group, confirming the presence of the predicted components in vivo. Figure 8 ).

[0023] Example 2: Validation of the roles of GSTP1 and ALOX5 in radiotherapy resistance in pancreatic cancer

[0024] (1) Bioinformatics analysis: Analysis using the GEO (GSE62452, GSE15471) dataset confirmed that GSTP1 and ALOX5 were significantly overexpressed in pancreatic cancer tissues, and that high expression was associated with poor patient prognosis. Figure 2 ).

[0025] (2) Association analysis with radiotherapy response: The GSE225767 dataset, including samples before and after radiotherapy, was analyzed. It was found that before radiotherapy, GSTP1 / ALOX5 expression was higher in patients who responded to radiotherapy than in those who did not; after radiotherapy, the expression of both decreased in patients who responded to radiotherapy, while it increased in those who did not, showing a "reversal" in expression levels. A significant positive correlation was found between the two expression levels after radiotherapy (R = 0.42, p < 0.05). Figure 2 ).

[0026] (3) Mechanism association analysis: In the TCGA-PAAD cohort, GSTP1 and ALOX5 expression were significantly positively correlated (R = 0.559). Correlation analysis showed that both were most strongly correlated with ferroptosis function scores (correlation coefficients were 0.431 and 0.343, respectively). Patients with high ferroptosis function scores had worse prognoses. Figure 2 ).

[0027] Example 3: In vitro and in vivo verification of the radiosensitizing effect of Wumei Pill on pancreatic cancer.

[0028] (1) Cellular Experiments: Proliferation and Migration: CCK-8 assay showed that WMP inhibited the proliferation of pancreatic cancer cells (Pan02), with an IC50 of 19% ( Figure 3 5). Scratch assay showed that 10% WMP significantly inhibited cell migration. Colony formation: Colony formation assay showed that WMP combined with radiotherapy (4 Gy) significantly inhibited cell colony formation compared to radiotherapy alone or WMP alone. Figure 5 Protein expression: Western blot showed that the expression of GSTP1, ALOX5, and GPX4 was upregulated in the radiotherapy group; while in the WMP combined with radiotherapy group, GSTP1 expression was downregulated compared to the radiotherapy group, and the expression of ALOX5 and GPX4 showed a complex but coordinated pattern of change, indicating that the redox balance was disrupted. Figure 3 ,4).

[0029] (2) Animal Experiments: A subcutaneous pancreatic cancer xenograft model was established in C57BL / 6J mice, divided into a control group, a WMP group, a radiotherapy group, and a combination group. The WMP group received a 16-fold human equivalent dose via gavage, the radiotherapy group received local irradiation (4 Gy / fraction × 3 fractions), and the combination group received WMP + radiotherapy. Results: The combination group showed the highest tumor inhibition rate (64%), significantly better than the single-drug groups (WMP group 36%, radiotherapy group 34%). Tissue Analysis: Western blotting and RNA sequencing (RNA-seq) results were consistent with the cell experiments; the tumor tissue in the combination group exhibited a unique GSTP1 / ALOX5 / GPX4 expression profile. Oil Red O staining showed a significant increase in lipid deposition in the tumors of both the radiotherapy and combination groups, with extensive vacuolation also observed in the combination group. Figure 3 ,5).

[0030] Example 4: Verification of the sensitizing mechanism of Wumei pill and ferroptosis activation

[0031] (1) Detection of key phenotypic features of ferroptosis: ROS and lipid peroxidation: Using H2DCFDA and BODIPY C11 probes, radiotherapy combined with WMP treatment significantly increased intracellular ROS and oxidized lipid levels, with stronger effects than single-agent therapy, and these effects could be partially reversed by the ferroptosis inhibitor Fer-1. Ferrroptosis biomarkers: Detection of intracellular malondialdehyde (MDA) and ferrous ions (Fe2+). 2+ The levels of both were significantly increased in the combined treatment group, further confirming that ferroptosis was strongly activated. Figure 6 ).

[0032] (2) Mechanism verification experiment: In the clonogenesis experiment, the addition of the ferroptosis inducer Erastin can simulate the sensitizing effect of WMP, while the addition of the inhibitor Fer-1 can antagonize the sensitizing effect of WMP. Figure 6 Transcriptome analysis showed that WMP and its co-regulatory group can regulate multiple ferroptosis-related genes (such as downregulating ACSL4 and SLC7A11, and upregulating DHODH), supporting the activation of the ferroptosis pathway at the gene expression level. Figure 9 ).

[0033] In summary, the present invention demonstrates that Wumei Pill targets GSTP1, relieves its inhibition of ALOX5, disrupts the redox balance of radiotherapy-resistant cell remodeling, and reactivates lipid peroxidation and ferroptosis programs, thereby achieving a radiosensitizing effect in pancreatic cancer. Figure 7 This discovery provides a solid experimental basis for the clinical application and secondary development of Wumei Pill as a novel radiosensitizer.

Claims

1. Use of a traditional Chinese medicine composition in the preparation of a medicament for enhancing the radiotherapy sensitivity of pancreatic cancer, characterized in that, The traditional Chinese medicine composition is Wumei pills or a water extract thereof.

2. Use according to claim 1, characterized in that, The Wumei pills are made of Fructus Mume, Szechwan Lovage Rhizome, Dried Ginger, Ramulus Cinnamomi, Aconiti Praeparata, Zanthoxylum, Coptis, Ginseng, Angelica and Phellodendron.

3. Use according to claim 1 or 2, characterized in that, The traditional Chinese medicine composition achieves radiotherapy sensitization by down-regulating the expression of glutathione S-transferase P1 (GSTP1) in pancreatic cancer cells to relieve its inhibition on 5-lipoxygenase (ALOX5), thereby activating ALOX5-mediated lipid peroxidation and up-regulating ferroptosis level.

4. A medicament for enhancing radiotherapy sensitivity of pancreatic cancer, characterized by, It contains the water extract of the Wumei pills of claim 2 as an active ingredient.

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