New anti-cancer component from murraya paniculata and application of novel anti-cancer component in gastric cancer inhibition

Abstract

The invention discloses a new anti-cancer component from murraya paniculata and application of the new anti-cancer component in gastric cancer inhibition, and belongs to the technical field of anti-tumor. The invention discloses the application of Acerosin ACE (Angiosin) from murraya paniculata in inhibition of gastric cancer for the first time, and clarifies the action mechanism of Acerosin ACE, especially the regulation effect of Acerosin ACE on a methionine metabolic pathway mediated by HIF-1alpha / MAT2A; secondly, verification is carried out in an in-vitro cell experiment, an in-vivo animal experiment and clinical database analysis, and it is proved that the method has a good application prospect. The implementation of the invention not only expands the pharmacological action research of natural components of murraya paniculata, but also provides new candidate drugs and action targets for the treatment of gastric cancer.

Description

Technical Field

[0001] This invention relates to the field of antitumor technology, specifically to a novel anticancer component derived from Murraya paniculata and its application in inhibiting gastric cancer. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors worldwide, characterized by high incidence, high mortality, and poor prognosis. Especially in China, the incidence and mortality rates of gastric cancer rank among the highest, seriously threatening human health. Although surgical resection, radiotherapy, chemotherapy, and targeted therapy are widely used in the comprehensive treatment of gastric cancer, most patients are diagnosed at an advanced stage, with high recurrence and metastasis rates. Existing treatment methods have limited efficacy and are often accompanied by significant adverse reactions. Therefore, there is an urgent need to develop safe and effective new anticancer drugs to improve the survival rate and quality of life of gastric cancer patients.

[0003] Thousand Miles ( Chimonanthus nitens Oliv. Murraya paniculata, a plant belonging to the Calycanthaceae family, is a traditional medicinal plant unique to my country, long used for clearing heat and detoxifying, promoting blood circulation and removing blood stasis, and relieving pain. Modern pharmacological studies have found that Murraya paniculata is rich in flavonoids, phenolic acids, and other natural products, possessing various biological activities such as anti-inflammatory, antioxidant, antibacterial, and antitumor effects. However, to date, research on the modern pharmacological and anticancer mechanisms of Murraya paniculata remains relatively limited, and its specific active ingredients and mechanisms of action have not yet been systematically elucidated.

[0004] Acerosin (ACE) is a natural flavonoid compound isolated from Murraya paniculata. In vitro experiments have shown that ACE exhibits weak inhibitory activity in various tumor cell lines. However, most related studies remain at the preliminary cellular level, lacking systematic research on its molecular mechanisms and in vivo antitumor effects. Particularly in gastric cancer, there are no reports comprehensively revealing the pharmacodynamic characteristics and pathways of action of ACE.

[0005] While existing anti-gastric cancer drugs have made some progress, they generally suffer from poor selectivity, significant toxic side effects, and easy development of drug resistance. Furthermore, research on many natural products has not yet delved into their mechanisms. Therefore, conducting systematic research on the active ingredients derived from Murraya paniculata to explore their efficacy and mechanisms of action in inhibiting gastric cancer will not only help elucidate the pharmacological basis of Murraya paniculata but also potentially provide new candidate compounds and theoretical basis for the development of natural drugs and novel anti-cancer drugs. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a natural component ACE derived from Murraya paniculata and its application in inhibiting gastric cancer, including in vitro inhibition of gastric cancer cell proliferation and apoptosis, in vivo inhibition of xenograft growth, and related molecular mechanism evidence.

[0007] This invention is achieved using the following technical solution: A novel anticancer component derived from Murraya paniculata and its application in inhibiting gastric cancer, characterized in that the component is acetamiprid, abbreviated as ACE, and the inhibition includes inhibition of gastric cancer cell proliferation and / or promotion of gastric cancer cell apoptosis.

[0008] Furthermore, the inhibitory effect on gastric cancer cell viability can be demonstrated through in vitro experiments. In CCK-8 assays, after ACE treatment of gastric cancer cell lines such as MGC-803, cell viability showed a significant decreasing trend with increasing drug concentration, indicating a dose-dependent effect.

[0009] Furthermore, the inhibitory effect on cell proliferation was confirmed by EdU staining. EdU staining results showed that the DNA synthesis rate was significantly reduced in the ACE-treated group, and the proportion of EdU-positive cells decreased. Protein level detection results indicated that ACE could inhibit the protein expression of the proliferation-related protein Ki67. These results suggest that ACE's anti-proliferative effect in reducing the DNA synthesis rate of gastric cancer cells is related to its inhibition of Ki67 protein expression.

[0010] Furthermore, the inhibitory effect of ACE on the colony formation of gastric cancer cells can be confirmed by crystal violet staining assay, and the results show that... ACE (5, 10, 20 μM) significantly inhibited the colony formation of MGC-803 and HGC-27 cells in a dose-dependent manner.

[0011] Furthermore, the effect of promoting apoptosis was confirmed by flow cytometry and molecular-level detection. In Annexin V-FITC / PI double staining flow cytometry experiments, the proportion of apoptotic gastric cancer cells in the ACE-treated group increased significantly, showing a dose-dependent effect. Protein-level detection results showed that ACE upregulated the protein expression of Cleaved-Caspase-3 and Cleaved PARP, and downregulated the protein expression of Bcl-2 and PARP. This result was consistent with the flow cytometry results, further confirming that ACE can induce programmed cell death in gastric cancer cells by regulating the mitochondrial pathway-mediated apoptosis-related molecular pathway.

[0012] Furthermore, the aforementioned inhibition of cell viability is closely related to metabolic regulation. Transcriptomic and metabolomics analyses revealed that the mechanism by which ACE inhibits gastric cancer cells may be related to its regulation of HIF-1α pathway-mediated methionine metabolism, promoting the accumulation of reactive oxygen species (ROS). ACE significantly reduces gastric cancer cell viability, significantly downregulates the expression of HIF-1α and MAT2A, and inhibits methionine metabolism-related pathways, reducing S-adenosylmethionine (SAM) levels. This effect can be antagonized by cobalt chloride (CoCl2). Exogenous SAM supplementation can antagonize the inhibitory effect of ACE on cell viability and the upregulation of ROS in gastric cancer cells by ACE, further indicating that this metabolic pathway is a crucial node in the action of ACE. Therefore, this invention reveals that ACE achieves its anti-proliferative and pro-apoptotic effects by reducing HIF-1α pathway-mediated methionine metabolism and lowering SAM levels in gastric cancer cells, thereby promoting ROS accumulation.

[0013] Furthermore, the inhibitory effect of ACE on gastric cancer cell viability was particularly pronounced in HIF-1α-high expressing gastric cancer cells (MGC803 and HGC27 cells), while it was weaker in HIF-1α-low expressing gastric cancer cells (SGC7901, MKN45, and AGS). The inhibitory effect of ACE on gastric cancer exhibited HIF-1α-high expression selectivity.

[0014] Furthermore, the effective concentration range of ACE in in vitro experiments is 10–80 μM, and the treatment time is 24–48 hours. Within this concentration and time range, ACE stably exhibits inhibitory effects on cell proliferation and induces apoptosis. This range provides experimental basis for subsequent in vivo studies and drug dosage exploration.

[0015] The present invention also provides a pharmaceutical composition with ACE as the main active ingredient.

[0016] Furthermore, the pharmaceutical composition can be formulated into pharmaceutically acceptable dosage forms such as tablets, capsules, injections, and sustained-release preparations, with excipients including commonly used pharmaceutical carriers or excipients. The pharmaceutical composition can be administered orally or by injection to inhibit the occurrence and development of gastric cancer.

[0017] Furthermore, the pharmaceutical composition is suitable for patients with clearly defined molecular subtypes of gastric cancer. Analysis of clinical samples from the TCGA and GEO databases revealed significantly high expression of HIF-1α / MAT2A and methionine metabolism-related genes in gastric cancer tissues, which were associated with poor patient prognosis. Therefore, the pharmaceutical composition of this invention is particularly suitable for high-risk populations with high expression of these molecular markers, thus enabling personalized clinical medication.

[0018] Furthermore, the pharmaceutical composition of the present invention also exhibited good antitumor effects in animal models. In a mouse subcutaneous gastric cancer xenograft model, oral administration of ACE once daily within a dose range of 30–100 mg / kg significantly inhibited tumor growth, manifested as a significant decrease in tumor volume and weight; simultaneously, at the histological level, HE staining showed an increase in the area of ​​cell necrosis, and TUNEL staining indicated an increased proportion of apoptotic cells. These results provide strong experimental evidence for the anticancer effect of ACE in vivo.

[0019] Furthermore, the mechanism of action includes direct regulation of cell proliferation and apoptosis, involving not only HIF-1α / MAT2A-mediated methionine metabolism but also changes in the oxidative stress and Nrf2 antioxidant signaling pathway regulated by the methionine metabolite SAM. In ACE-treated cells, ROS levels were significantly increased, while the expression of the Nrf2 antioxidant pathway was downregulated, an effect antagonized by SAM. This result suggests that ACE may exert its anti-cancer effect by disrupting the redox balance through the HIF-1α / MAT2A / SAM axis-mediated Nrf2 antioxidant pathway in tumor cells, promoting excessive ROS accumulation.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a novel anticancer component derived from Murraya paniculata and its application in inhibiting gastric cancer. It reveals for the first time the application of iridocarotene derived from Murraya paniculata in inhibiting gastric cancer, and clarifies its mechanism of action, particularly its regulatory effect on the HIF-1α / MAT2A-mediated methionine metabolic pathway, reducing the SAM-mediated Nrf2 antioxidant pathway, promoting excessive ROS accumulation, inhibiting gastric cancer cell proliferation, promoting apoptosis, and exerting an inhibitory effect on gastric cancer. This has been validated in in vitro cell experiments, in vivo animal experiments, and clinical database analysis, demonstrating its promising application prospects. The implementation of this invention not only expands the pharmacological research on the natural components of Murraya paniculata but also provides new candidate drugs and targets for the precision treatment of gastric cancer. Attached Figure Description

[0021] Figure 1 The diagram shows the structural formula of ACE and its effects on the viability of human gastric cancer cells at different differentiation levels and normal gastric mucosal epithelial cells. (A) Schematic diagram of the chemical structure of ACE; (B) Effects of ACE on the viability of gastric cancer cells MGC-803 (B), HGC-27 (C), SGC-790 (D), MKN-45 (E), and AGS (F) and their IC50 values; (G) Effects of ACE on the viability of gastric mucosal epithelial cells GES-1 and their IC50 values. Figure 2The effects of ACE on the proliferation of gastric cancer cells; (A) ACE inhibits the proliferation of gastric cancer cells; (B) ACE inhibits the colony formation ability of gastric cancer cells; (C) ACE downregulates the expression of Ki67 protein, a protein related to the proliferation of gastric cancer cells. Figure 3 The effects of ACE on apoptosis in gastric cancer cells; (A) ACE promotes apoptosis in gastric cancer cells; (B) ACE affects the protein expression of apoptosis-related proteins in gastric cancer cells. Figure 4 The inhibitory effect of ACE on gastric cancer xenografts in nude mice; (A) tumor morphology diagrams of each group; (B) tumor volume growth curves; (C) tumor weight statistics. Figure 5 The effects of ACE on pathological changes and apoptosis in xenograft tumor tissues; (A) the effects of ACE on pathological changes and apoptosis in gastric cancer cells; (B) the effects of ACE on the protein expression of proliferative and apoptosis-related proteins in gastric cancer cells. Figure 6 The mechanism of action of ACE in inhibiting gastric cancer; (A) transcriptomic KEGG analysis results; (B) HIF-1α protein expression levels in different gastric cancer cell lines; (C) ACE significantly inhibits HIF-1α mRNA expression in MGC803 cells; (D) ACE downregulates HIF-1α protein expression in gastric cancer cells in a dose-dependent manner. Figure 7 The effects of CoCl2, a hypoxia inducer, on the antagonistic effect of ACE on gastric cancer; (AB) the effects of CoCl2 and ACE on the activity of gastric cancer cells MGC-803 and HGC-27; (C) the effects of CoCl2 and ACE on the expression of HIF-1α protein in gastric cancer cells; Figure 8 The analysis included the differences in metabolites between the ACE-treated group and the control group; (A) volcano plot of differentially metabolites; (B) PCA plot; (C) KEGG pathway classification of differentially metabolites; (D) KEGG pathway functional enrichment analysis of differentially metabolites; (E) changes in methionine content; and (F) changes in L-cysteine ​​content. Figure 9 The effect of CoCl2 on the downregulation of L-methionine and SAM by ACE; (A) PCA diagram; (BC) CoCl2 antagonizes the effect of ACE on downregulating L-methionine and SAM levels in gastric cancer cells MGC-803; Figure 10 The study investigated the effects of ACE on the mRNA expression of methionine metabolism-related genes in gastric cancer cells and the antagonistic effect of CoCl2; where (AK) represent different genes. Figure 11 The effect of ACE on the protein expression of HIF-1α / MAT2A-mediated methionine metabolism-related proteins in gastric cancer cells; Figure 12 The effect of ACE on protein expression of key proteins in the HIF-1α / MAT2A-mediated methionine metabolism pathway in gastric cancer xenograft tissue; Figure 13 The effects of exogenous metabolites arginine (Arg), methionine (Met), and SAM on the inhibitory effect of ACE on gastric cancer cell viability; (A) the effect of Arg on the inhibitory effect of ACE on gastric cancer cell viability; (B) the effect of Met on the inhibitory effect of ACE on gastric cancer cell viability; (CD) the effect of SAM on the inhibitory effect of ACE on gastric cancer cell viability. Figure 14 The effect of exogenous metabolite SAM on the inhibitory effect of ACE on gastric cancer cell proliferation; (A) the effect of SAM on the inhibitory effect of ACE on gastric cancer cell proliferation; (B) the effect of SAM on ACE to downregulate the expression of Ki67, a protein related to gastric cancer cell proliferation. Figure 15 The study investigated the effects of the exogenous metabolite SAM on the apoptosis-inducing effect of ACE on gastric cancer cells. Specifically, (A) SAM antagonized the apoptosis-inducing effect of ACE on MGC-803 cells; (B) SAM antagonized the apoptosis-inducing effect of ACE on HGC-27 cells; (C) SAM antagonized the protein expression regulation of apoptosis-related proteins in MGC-803 cells by ACE; and (D) SAM antagonized the protein expression regulation of apoptosis-related proteins in HGC-27 cells by ACE. Figure 16 SAM, NAC, and CoCl2 antagonized the effect of ACE on ROS upregulation in gastric cancer cells; among them, (AB) ACE promoted ROS accumulation in MGC-803 and HGC-27 cells; (C) the ROS scavenger NAC antagonized the effect of ACE on ROS upregulation in MGC-803 cells; (D) the ROS scavenger NAC antagonized the effect of ACE on inhibiting the viability of MGC-803 cells; (E) NAC antagonized the effect of ACE on ROS upregulation in HGC-27 cells; (F) NAC antagonized the effect of ACE on inhibiting the viability of HGC-27 cells. (GH) The hypoxia mimic CoCl2-induced HIF-1α overexpression antagonized the effect of ACE on ROS upregulation in MGC-803 and HGC-27 cells. (IJ) SAM antagonized the effect of ACE on ROS upregulation in MGC-803 and HGC-27 cells; Figure 17 The study investigated the regulatory effects of SAM antagonism against ACE on the expression of key proteins in the Nrf2 pathway in gastric cancer cells. Specifically, (A) the regulatory effect of SAM antagonism against ACE on the mRNA expression of key proteins in the Nrf2 pathway in gastric cancer cells; (B) the regulatory effect of SAM antagonism against ACE on the protein expression of key proteins in the Nrf2 pathway in MGC-803 cells; and (C) the regulatory effect of SAM antagonism against ACE on the protein expression of key proteins in the Nrf2 pathway in HGC-27 cells. Figure 18 Differential expression analysis of related genes in TCGA and GEO; where (AH) represent different genes; Figure 19 The results of the correlation analysis between HIF-1α and the expression of genes related to methionine metabolism are shown; where (AF) represents different genes. Figure 20 Analysis of methionine metabolism-related genes and gastric cancer survival curves; where (AE) represents different genes. Detailed Implementation

[0022] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0023] Example 1: Effects of ACE on in vitro proliferation and apoptosis of gastric cancer cells. To verify the inhibitory effect of Acerosin (ACE) on gastric cancer cells in vitro, this embodiment conducted cell proliferation and apoptosis experiments in various cell lines.

[0024] (a) CCK-8 assay for cell viability MGC-803, HGC-27, SGC-7901, MKN-45, and GES-1 cells were seeded in 96-well plates, approximately 1 × 10⁶ cells per well. 4 Add 100 μL of complete culture medium to each cell and incubate overnight in an incubator to allow them to adhere. The next day, when the cell density reaches 60–70%, discard the old culture medium and replace it with a medium containing different concentrations of ACE (structure shown in Figure 1). Figure 1 Complete culture medium (as shown in Figure A) was used, with final ACE concentrations of 0, 5, 10, 20, 40, 80, 160, 200, 400, and 600 μM, and six replicates per group. Incubation was continued for 48 hours. After treatment, the drug-containing medium was discarded, and the medium was replaced with working solution containing CCK-8 reagent (CCK-8 and RPMI-1640 at a 1:10 ratio). 100 μL was added to each well, and the cells were incubated in an incubator for 1 hour. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated, and IC50 values ​​were fitted.

[0025]

[0026] After 48 hours of ACE treatment, the IC50 values ​​for MGC-803, HGC-27, SGC-7901, MKN-45, and AGS cells were 8.24, 9.11, 49.57, 63.46, and 284.7 μM, respectively, while the IC50 for normal gastric mucosal epithelial GES-1 cells was 111.7 μM. ACE showed significantly stronger inhibitory effects on gastric cancer cells than on normal cells, exhibiting a clear dose-dependent effect, particularly on the less differentiated MGC-803 and HGC-27 cells. This indicates that ACE possesses a certain degree of selectivity while inhibiting tumor cells.

[0027] (II) Effect of EdU detection on ACE on gastric cancer cell proliferation MGC-803 and HGC-27 cells were seeded in 96-well plates at a density of approximately 6 × 10⁶ cells / well. 4 Cells were cultured at 100 μL / mL, culturing overnight until adherent. The following day, they were treated with 2.5, 5, and 10 μM ACE for 48 hours, respectively. After treatment, the medium was replaced with 50 μM MEdU, and incubation continued for 2 hours. Cells were then washed twice with PBS, fixed in 4% paraformaldehyde for 30 minutes, and stained with Apollo fluorescence according to the kit instructions. Nuclei were counterstained with Hoechst 33342. Finally, the EdU positivity rate was observed under a fluorescence microscope.

[0028] The results are as follows Figure 2 As shown in Figure A, the proportion of EdU-positive cells in the 2.5, 5, and 10 μM ACE treatment groups was significantly lower than that in the control group (p < 0.05; p < 0.01; p < 0.001), indicating that cellular DNA synthesis was significantly inhibited.

[0029] (III) Detection of the effect of crystal violet staining on ACE on the clonal formation of gastric cancer cells MGC-803 and HGC-27 cells were seeded at 500 cells / well in 6-well plates and cultured in 1640 complete medium containing 5, 10, and 20 μMACE for 7–8 days. The supernatant was aspirated, the cells were washed once with PBS, and fixed with 4% paraformaldehyde for 10 min. Each well was stained with 1 mL of 0.1% crystal violet solution for 20 min. After washing twice with PBS, the cells were dried at room temperature and photographed. The number of clones was counted (each clone contained more than 50 cells).

[0030] The results are as follows Figure 2 As shown in Figure B, ACE (5, 10, 20 μM) significantly inhibited the colony formation of MGC-803 and HGC-27 cells in a dose-dependent manner.

[0031] (iv) Western blot detection of proliferation-related protein expression MGC-803 and HGC-27 cells were seeded in 6 cm culture dishes, with 1 × 10⁶ cells per dish. 6 Cells were collected and, after 12 hours of adhesion, were treated with ACE at final concentrations of 5, 10, and 20 μM for 48 hours. Cells were then collected and lysed in RIPA lysis buffer (containing 1% PMSF) on ice for 30 minutes. The supernatant was used to determine protein concentration, and an equal volume of protein (30 μg) was used for SDS-PAGE electrophoresis and transferred to a membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour, then incubated overnight at 4 °C with primary antibody (Ki67, 1:1000). The next day, the membrane was washed three times with TBST for 10 minutes each time, and then incubated for 1 hour at room temperature with HRP-labeled secondary antibody (1:5000). After ECL staining, the membrane was exposed to a gel imaging system, and the gray values ​​of the protein bands were analyzed.

[0032] The results are as follows Figure 2 As shown in Figure C, Ki67 expression was significantly downregulated in the ACE (5, 10, 20 μM) treatment groups (p < 0.01), suggesting that ACE inhibits cell proliferation by regulating Ki67.

[0033] (V) Flow cytometry detection of the effect of ACE on apoptosis of gastric cancer cells MGC-803 and HGC-27 cells were seeded into 6-well plates at a density of 1 × 10⁶ cells per well. 5 Cells were collected and, after adhesion, treated with ACE at final concentrations of 5, 10, and 20 μM for 48 hours. Cells were then collected, washed twice with PBS, and incubated using the Annexin V-FITC / PI double staining kit according to the manufacturer's instructions. Cells were then incubated at room temperature in the dark for 15 minutes before flow cytometry analysis. At least 1 × 10⁶ cells were collected for flow cytometry. 4 We analyzed the proportion of early and late apoptosis in individual cells.

[0034] The results are as follows Figure 3 As shown in Figure A, under the treatment of 5, 10, and 20 μM ACE, the apoptosis rates of MGC-803 cells reached 32.32%, 66.78%, and 82.94%, respectively, while the apoptosis rates of HGC-27 cells were 30.89%, 52.51%, and 60.82%, respectively. This indicates that ACE can significantly induce apoptosis in gastric cancer cells. The apoptosis rate increased significantly with increasing dosage, indicating that the pro-apoptotic effect of ACE is strongly dose-dependent.

[0035] (vi) Western blot detection of apoptosis-related protein expression MGC-803 and HGC-27 cells were seeded in 6 cm culture dishes, with 1 × 10⁶ cells per dish. 6Cells were collected and, after 12 hours of adhesion, were treated with ACE at final concentrations of 5, 10, and 20 μM for 48 hours. Cells were then collected and lysed in RIPA lysis buffer (containing 1% PMSF) on ice for 30 minutes. The supernatant was used to determine protein concentration, and an equal volume of protein (30 μg) was used for SDS-PAGE electrophoresis and transferred to a membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour, then incubated overnight at 4 °C with primary antibodies (Cleaved-Caspase 3, 1:1000; Bax, 1:1000; Bcl-2, 1:1000; β-actin, 1:5000). The next day, the membrane was washed three times with TBST for 10 minutes each time, and then incubated with HRP-labeled secondary antibody (1:5000) at room temperature for 1 hour. After ECL development, the membrane was exposed to a gel imaging system, and the gray values ​​of the protein bands were analyzed.

[0036] The results are as follows Figure 3 As shown in Figure B, the expression of Cleaved-Caspase3 and Cleaved-PARP in MGC-803 and HGC-27 cells treated with ACE (5, 10, 20 μM) was significantly upregulated, while the expression of Bcl-2 and PARP was significantly downregulated (p < 0.01). This suggests that ACE promotes cell apoptosis by regulating the protein expression of apoptosis-related proteins, and its pro-apoptotic mechanism involves the mitochondrial pathway.

[0037] In summary, ACE can dose-dependently inhibit the proliferation of gastric cancer cells in vitro, significantly reduce DNA synthesis levels, and promote apoptosis, manifested by upregulation of pro-apoptotic proteins and downregulation of anti-apoptotic proteins and Ki67. These results collectively demonstrate that ACE possesses significant anti-gastric cancer activity in vitro, providing experimental evidence for its potential role as an anti-cancer component.

[0038] Example 2: Antitumor effect of ACE in a nude mouse xenograft model of gastric cancer To verify the anti-gastric cancer effect of ACE in vivo, this embodiment used MGC-803 cells to construct a nude mouse subcutaneous xenograft model and conducted drug administration experiments.

[0039] (I) Establishment of xenograft model MGC-803 cells in logarithmic growth phase were harvested and their concentration adjusted to 2.5 × 10⁻⁶. 7 Cells / mL: 200 μL of cell suspension was subcutaneously injected into the right axilla of 4-week-old male BALB / c nude mice, with approximately 5 × 10⁶ cells per mouse. 6 Each cell. Tumor growth was continuously monitored after transplantation, and when the tumor volume reached approximately 50 mm... 3 Random grouping begins at that time.

[0040] (II) Grouping and Dosing Nude mice were randomly divided into 5 groups: control group, low-dose ACE group, medium-dose ACE group, high-dose ACE group, and positive control 5-FU group, with 8 mice in each group. The control group was administered an equal volume of 0.5% sodium carboxymethyl cellulose (CMC-Na) orally by gavage; the low-dose ACE group was administered ACE 10 mg / kg; the medium-dose ACE group was administered ACE 30 mg / kg; and the high-dose ACE group was administered ACE 100 mg / kg. All groups were administered the medication once daily by gavage for 21 consecutive days. The 5-FU group was administered 50 mg / kg of 5-fluorouracil (5-FU) intraperitoneally twice a week for 21 days.

[0041] (iii) Measurement of tumor volume and weight During the experiment, the long diameter (a) and short diameter (b) of the tumor were measured every 3 days using calipers, and the result was calculated using the formula V = (a × b). 2 The volume was calculated as () / 2. Animals were sacrificed on day 21 of the experiment, the tumor was removed and weighed. Results are as follows: Figure 4 As shown, the tumor volume growth in the ACE-treated group slowed significantly over time, and at the terminal time point, the average tumor weight in the high-dose group was less than 40% of that in the control group, with a significant difference (p < 0.01). 5-FU also significantly reduced tumor volume and weight, but two animals died during the administration period.

[0042] (iv) Histological examination The dissected tumor tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for HE and TUNEL staining. Results are as follows: Figure 5 As shown in Figure A, HE staining results showed that the cells in the ACE-treated group were loosely arranged and the necrotic area was increased; TUNEL results showed that the proportion of apoptotic cells in the ACE-treated group was significantly higher than that in the control group.

[0043] (v) Protein level detection Total protein was extracted from homogenized tumor tissue, and Western blot was used to detect the expression levels of proteins related to proliferation and apoptosis in gastric cancer xenograft tissue. The results are as follows: Figure 5 As shown in B, ACE can also significantly downregulate the protein expression of Ki67, a protein associated with the proliferation of gastric cancer xenografts, downregulate the protein expression of anti-apoptotic proteins Bcl-2 and PARP, and upregulate the protein expression of pro-apoptotic proteins Cleaved-caspase3 and Cleaved-PARP.

[0044] The above results indicate that ACE can significantly inhibit the growth of gastric cancer xenografts in vivo, manifested by a significant decrease in tumor volume and weight, and an increase in cell necrosis and apoptosis histologically. At the molecular level, ACE downregulates the expression of Ki67 and anti-apoptotic proteins, and upregulates the expression of pro-apoptotic proteins, which are related to mitochondrial pathway-mediated apoptosis, further supporting its potential as a candidate drug for anti-gastric cancer.

[0045] Example 3: Transcriptomics and metabolomics reveal the mechanism by which ACE reduces gastric cancer cell viability by inhibiting HIF-1α-mediated methionine metabolism, promoting ROS accumulation, and reducing cancer cell viability. To explore the molecular mechanism by which ACE inhibits gastric cancer, this embodiment performed transcriptome sequencing and metabolomics analysis on ACE-treated gastric cancer cells, and validated the results using qPCR and Western blot.

[0046] (a) Transcriptomic results suggest that the inhibitory effect of ACE on gastric cancer may be related to its regulation of the HIF-1α signaling pathway. Logarithmic growth phase MGC-803 and HGC-27 cells were seeded in 10 cm culture dishes, with 5 × 10⁶ cells per dish. 6 Cells were collected and adhered to the culture vessel for 12 hours, then divided into a control group (DMSO treatment) and an ACE group (10 μM, final DMSO concentration ≤0.1%), treated for 24 hours. Three biological replicates were prepared for each group. Cells were collected, and total RNA was extracted using TRIzol reagent. RNA quality was assessed using an Agilent 2100 Bioanalyzer, with RIN values ​​greater than 8.0 for all samples. After library construction, sequencing was performed on the Illumina NovaSeq platform. Differential analysis was performed using DESeq2 software, with a screening threshold of |log2FC| ≥ 1.5 and p < 0.05. KEGG pathway enrichment analysis was performed on DEGs.

[0047] The results are as follows Figure 6 As shown in Figure A, a total of 6140 differentially expressed genes were detected in the ACE treatment group, of which 2960 were upregulated and 3180 were downregulated. KEGG enrichment results showed that the differentially expressed genes were mainly enriched in the HIF-1 signaling pathway, indicating that the tumor-suppressing mechanism of ACE may be related to the HIF-1α pathway.

[0048] (II) The inhibitory effect of ACE on gastric cancer is mediated by HIF-1α. MGC-803, HGC-27, SGC-7901, AGS, and MKN-45 cells were seeded in 6-well plates at a density of 2 × 10⁶ cells per well. 5 Cells were deposited and treated under the following conditions: control group (DMSO treatment), ACE group (10 μM), and treatment for 24 hours.

[0049] The Western blot methodology is the same as before, and the results are as follows: Figure 6 As shown in Figure B, HIF-1α expression was significantly higher in MGC-803 and HFC-27 cells, which are more sensitive to ACE, than in other gastric cancer cell lines. This suggests that ACE's inhibitory effect on gastric cancer has HIF-1α specificity and selectivity. Figure 6As shown in Figure D, ACE can significantly inhibit the protein expression of HIF-1α in gastric cancer cells in a dose-dependent manner.

[0050] Total RNA was extracted using an RNA extraction kit, reverse transcribed into cDNA, and HIF-1α was detected using SYBR Green qPCR reagent. β-actin was used as an internal reference gene. Results are as follows: Figure 6 As shown in Figure C, ACE (10 μM) significantly inhibited the mRNA expression of HIF-1α in gastric cancer cells.

[0051] MGC-803 and HGC-27 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were treated under the following conditions after adhesion: ACE (10 μM ACE), CoCl2 group (50, 100, 200 μM CoCl2), and CoCl2+ACE group (50, 100, 200 μM CoCl2 and 10 μM ACE). Treatment lasted 48 hours. Cell viability was assessed using the CCK-8 assay, following the same method as before. MGC-803 and HGC-27 cells were seeded in 6-well plates, with 2 × 10⁶ cells per well. 5 Cells were deposited and treated under the following conditions: ACE (10 μM ACE), CoCl2 group (100 μM CoCl2), and CoCl2+ACE group (100 μM CoCl2 and 10 μM ACE). After 48 hours of treatment, protein expression was detected by Western blot, using the same method as before.

[0052] The results are as follows Figure 7 As shown in AB, the inhibitory effect of ACE on gastric cancer cell viability can be antagonized by CoCl2 (which mimics hypoxia-induced HIF-1α overexpression); Figure 7 As shown in Figure C, the inhibitory effect of ACE on HIF-1α protein expression in gastric cancer cells can be antagonized by CoCl2. These results demonstrate that the inhibitory effect of ACE on gastric cancer is indeed mediated by HIF-1α.

[0053] (III) Metabolomics analysis results suggest that the inhibitory effect of ACE on gastric cancer may be related to its regulation of HIF-1α-mediated methionine. Metabolic related MGC-803 and HGC-27 cells were harvested during the logarithmic growth phase and seeded in 10 cm culture dishes at a density of 5 × 10⁶ cells per dish. 6Cells were collected and treated under the same conditions as in Example 3 after adhesion: control group (DMSO treatment) and ACE group (10 μM); control group (DMSO treatment), ACE group (10 μM), CoCl2 group (100 μM), and ACE+CoCl2 group, treated for 24 hours. Each group had three replicates. After cell collection, metabolites were extracted using a mixture of acetonitrile, methanol, and water (2:2:1, volume ratio) (pre-cooled, -20 °C), vortexed for 30 seconds, repeatedly frozen and thawed in liquid nitrogen, and ground with a tissue homogenizer. The cells were centrifuged at 4 °C (12000 g, 10 min), and the supernatant was collected, concentrated, dried, and reconstituted in a suitable mobile phase for detection. Detection was performed using an LC-MS / MS platform. Data were processed using XCMS software for peak identification, quantification, and standardization. The differential metabolite screening criteria were VIP > 1 and p < 0.05.

[0054] The results are as follows Figure 8 As shown, the metabolic profiles of the ACE-treated group and the control group were significantly separated. Pathway enrichment analysis revealed that the methionine metabolic pathway was significantly inhibited. ACE significantly downregulated the methionine content in gastric cancer cells, but had no significant effect on the L-cysteine ​​content. The inhibitory effect of ACE on gastric cancer may be related to its regulation of methionine metabolism.

[0055] The results are as follows Figure 9 As shown, the four metabolic profiles were significantly separated. ACE significantly downregulated the levels of L-methionine and SAM in MGC-803 cells, an effect antagonized by CoCl2. Therefore, ACE inhibits methionine metabolism in gastric cancer cells via HIF-1α-mediated inhibition. CoCl2 is a hypoxia mimic that induces high expression of HIF-1α.

[0056] (iv) qPCR verification of the HIF-1α-mediated regulation of methionine metabolism-related gene mRNA expression by ACE. MGC-803 cells were seeded into 6-well plates at a density of 2 × 10⁶ cells per well. 5 Cells were deposited and treated under the following conditions after adhesion: control group (DMSO treatment), ACE group (10 μM), CoCl2 group, and ACE+CoCl2 group, for 24 hours. Total RNA was extracted using an RNA extraction kit and reverse transcribed into cDNA. HIF-1α, MAT2A, MTR, and other genes were detected using SYBR Green qPCR reagent, with β-actin as an internal reference gene.

[0057] The results are as follows Figure 10As shown, the mRNA expression levels of methionine metabolism-related genes such as MAT2A, MTR, SLC6A15, MTHFR, MAT2A, DNMT3A, METAP2, and MTAP in gastric cancer cells treated with ACE were significantly decreased, and this effect could be antagonized by CoCl2. CoCl2 is a hypoxia mimic that can induce high expression of HIF-1α.

[0058] (V) Western blot analysis of the regulatory effect of ACE on the protein expression of key proteins in the HIF-1α / MAT2A-mediated methionine metabolism-regulated ROS accumulation pathway in gastric cancer cells. MGC-803 and HGC-27 cells were seeded in 6 cm culture dishes, 1 × 10⁶ cells per dish. 6 Cells were treated under the same conditions as above. Cells and tumor tissues were lysed using RIPA lysis buffer, and total protein (30 μg / sample) was extracted. SDS-PAGE electrophoresis was performed, followed by membrane transfer. After blocking with 5% skim milk at room temperature for 1 hour, primary antibodies (HIF-1α 1:1000, MAT2A 1:1000, SLC6A15 1:2000, Nrf2 1:2000, HO-1 1:2000, NQO1 1:2000, β-actin 1:5000) were added, and the membrane was incubated overnight at 4 °C. The next day, the membrane was washed with TBST, and HRP secondary antibody (1:5000) was added. The membrane was incubated at room temperature for 1 hour, followed by ECL staining and imaging.

[0059] The results are as follows Figure 11-12 As shown, ACE can significantly downregulate the protein expression of MAT2A and SLC6A15, key proteins in the methionine metabolism pathway of gastric cancer cells; ACE can significantly downregulate the protein expression of HIF-1α, MAT2A, SLC6A5, Nrf2, HO-1 and NQO1 in gastric cancer xenograft tissues.

[0060] These results indicate that ACE can exert its anti-gastric cancer effect at the molecular level by regulating the HIF-1α / MAT2A pathway, inhibiting methionine metabolism and the expression of its downstream molecules, promoting ROS accumulation, and ultimately inducing apoptosis and inhibiting the proliferation of gastric cancer cells. This provides important evidence for its mechanism of action.

[0061] Example 4: Experiment on the antagonistic effect of exogenous SAM To verify the key role of SAM in the ACE anticancer mechanism, this embodiment supplemented cells with exogenous SAM and tested its antagonistic effect on ACE.

[0062] (a) Cell viability detection MGC-803 and HGC-27 cells were seeded in 96-well plates at a density of 5 × 10⁶ cells per well. 3Cells were collected and, after adhesion, were grouped and treated as follows: control group (Ctrl); ACE 10 μM; ACE 10 μM + exogenous arginine (0.01-10 mM); ACE 10 μM + exogenous methionine (0.01-30 mM); ACE 10 μM + exogenous SAM (100, 250 μM); exogenous arginine (0.01-10 mM); exogenous methionine (0.01-30 mM); exogenous SAM (100, 250 μM). After 48 hours of incubation, cell viability was assessed using the CCK-8 assay.

[0063] The results are as follows Figure 13 As shown, only SAM significantly reversed the inhibitory effect of ACE on cell viability.

[0064] (ii) EdU staining to detect cell proliferation The method was the same as described above, and the groups were as follows: control group (Ctrl), ACE 10 μM group, SAM 100 μM group, and SAM 100 μM + ACE 10 μM group.

[0065] The results are as follows Figure 14 As shown, SAM can significantly antagonize the inhibitory effect of ACE on the proliferation of gastric cancer cells.

[0066] (III) Annexin V / PI double staining flow cytometry detection of cell apoptosis The method was the same as described above, and the groups were as follows: control group (Ctrl), ACE 10 μM group, SAM 100 μM group, and SAM 100 μM + ACE 10 μM group.

[0067] The results are as follows Figure 15 As shown, SAM can significantly antagonize the apoptosis-promoting effect of ACE on gastric cancer cells, and can also significantly antagonize the protein expression of apoptosis-related proteins in gastric cancer cells regulated by ACE.

[0068] (iv) ROS level detection MGC-803 and HGC27 cells were seeded in 24-well plates at a density of 6 × 10⁶ cells per well. 3 Cells were selected and treated under the following conditions: control group, ACE (5, 10, 20 μM) group, ACE 10 μM + SAM 100 μM group, and SAM 100 μM group. ROS probe was added 24 hours after treatment, and the cells were incubated for 30 minutes before detection using a fluorescence microplate reader.

[0069] The results are as follows Figure 16As shown, ACE dose-dependently increased intracellular ROS levels in MGC-803 and HGC27 cells (P < 0.001), while the well-known ROS scavenger NAC (N-acetylcysteine, 5 mM) completely eliminated ACE-generated ROS (P < 0.001) and antagonized the inhibitory effect of ACE on gastric cancer cell viability (P < 0.001), indicating that ROS is involved in mediating the gastric cancer inhibitory effect of ACE. Similarly, CoCl2-induced HIF-1α overexpression or SAM also antagonized the ROS upregulation effect of ACE on gastric cancer cells and the inhibitory effect of ACE on gastric cancer cell viability (P < 0.001). These results demonstrate that the HIF-1α / SAM / ROS signaling axis is involved in mediating the gastric cancer inhibitory effect of ACE.

[0070] (v) Nrf2 pathway detection MGC-803 and HGC-27 cells were seeded in 6 cm culture dishes, with 1 × 10⁶ cells per dish. 6 Cells were processed under the same conditions as above. Proteins were extracted and Western blot analysis was performed to detect the protein expression of Nrf2 pathway-related proteins.

[0071] The results are as follows Figure 17 As shown, ACE can significantly downregulate the protein expression of Nrf2, HO-1 and NQO1 in gastric cancer cells, and this effect can be antagonized by exogenous SAM.

[0072] The above results indicate that SAM is a crucial metabolic pathway in the action of ACE. Exogenous SAM supplementation can antagonize the antitumor effect of ACE by activating the SAM-mediated Nrf2 pathway and downregulating ROS levels. This further validates the molecular mechanism of ACE.

[0073] Example 5: TCGA and GEO Database Verification To verify the significance of the ACE-targeting pathway in clinical samples, this embodiment utilizes public databases for gene expression and survival analysis.

[0074] (a) Analysis of differences in expression Download the TCGA-STAD and GEO datasets to analyze the expression differences of HIF-1α and methionine metabolism-related genes (MAT2A, MTR, AHCY, SRM, MTAP, MTHFR, MTR, SLC6A15) in gastric cancer tissues and normal tissues.

[0075] The results are as follows Figure 18 As shown, these genes are generally upregulated in gastric cancer tissue.

[0076] (II) Correlation Analysis Further analysis was conducted to determine the expression correlation between HIF-1α and MAT2A, MTR, MTAP, MTHFR, AHCY, and SRM. The results are as follows: Figure 19 As shown, HIF-1α is significantly positively correlated with the expression of the above-mentioned genes.

[0077] (III) Survival Curve Analysis Based on TCGA data, patients were grouped according to expression levels, and survival curves were plotted. Results are as follows: Figure 20 As shown, patients with high expression of MAT2A, MTR, MTAP, MTHFR, and SLC6A15 have significantly shorter overall survival.

[0078] These results indicate that the ACE target is also significant in clinical samples, and the genes downregulated by it are highly expressed in gastric cancer and are associated with poor patient prognosis, further supporting the clinical application potential of this invention.

[0079] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A novel anticancer component derived from Murraya paniculata and its application in inhibiting gastric cancer, characterized in that, The ingredient is acetocin, abbreviated as ACE, and the inhibition includes inhibition of gastric cancer cell proliferation and / or promotion of gastric cancer cell apoptosis.

2. The anticancer component according to claim 1 and its application in inhibiting gastric cancer, characterized in that, The proliferation inhibition is achieved by reducing the viability and DNA synthesis rate of gastric cancer cells in vitro, and the reduction can be characterized by CCK-8 absorbance measurement and a decrease in EdU incorporation rate.

3. The anticancer component according to claim 2 and its application in inhibiting gastric cancer, characterized in that, The reduction in cell viability is achieved by downregulating the HIF-1α / MAT2A-mediated methionine metabolism pathway, inhibiting the intracellular SAM-dependent Nrf2 antioxidant pathway, and promoting excessive ROS accumulation.

4. The anticancer component according to claim 1 and its application in inhibiting gastric cancer, characterized in that, The apoptosis promotion can be detected by Annexin V-FITC / PI flow cytometry, which shows an increase in the proportion of early and late apoptosis, and at the protein level, it is manifested by the upregulation of Cleaved-Caspase-3 and Cleaved-PARP, and the downregulation of Bcl-2 and PARP.

5. The anticancer component according to claim 4 and its application in inhibiting gastric cancer, characterized in that, The ACE can induce apoptosis in gastric cancer cells in a dose-dependent manner.

6. The anticancer component according to claim 1 and its application in inhibiting gastric cancer, characterized in that, The effective concentration range of the ACE for inhibiting gastric cancer is 10–80 μM, and the duration of action is 24–48 hours.

7. A pharmaceutical composition for inhibiting gastric cancer, characterized in that, The main component of the drug is the ACE as described in claim 1.

8. The pharmaceutical composition according to claim 7, characterized in that, The drug is indicated for gastric cancer patients with high expression of HIF-1α / MAT2A or elevated expression of methionine metabolism-related genes, which are associated with poor prognosis.

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