Application of ALDH3A2 as target spot in preparation of medicine for treating gastric cancer

Abstract

The embodiment of the invention discloses application of ALDH3A2 as a target spot in preparation of a medicine for treating gastric cancer. The research clarifies a new effect and a potential mechanism of ALDH3A2 in the aspect of inhibiting gastric cancer progression. Specifically, the ALDH3A2 can be used for down-regulating the expression of SLC47A1, so that the nuclear transfer of NRF2 is prevented, and the activation of UPRmt is inhibited. The damage can aggravate mitochondrial dysfunction, and finally, gastric cancer cell ferroptosis is promoted in a glutathione peroxidase 4 (GPX4) dependent mode. In addition, ferroptosis induced by ALDH3A2 can also promote polarization of macrophages to M1 type, and the progress of gastric cancer cells is inhibited through an IL-1beta signal channel. In conclusion, the effects jointly inhibit proliferation, migration and invasion of gastric cancer cells, and finally inhibition of growth and metastasis of gastric cancer is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of ALDH3A2 as a target in the preparation of drugs for treating gastric cancer. Background Technology

[0002] Gastric cancer, a highly prevalent malignant tumor worldwide, is showing a significant increasing incidence rate. With the growing clinical demand for gastric cancer treatment, the development of related therapeutic drugs has made some progress. The pathogenesis of gastric cancer is complex and diverse; therefore, current clinical treatment largely relies on surgical resection combined with comprehensive treatment strategies such as immunotherapy, targeted therapy, and chemotherapy. However, despite the effectiveness of existing treatments, the treatment of advanced gastric cancer still faces multiple challenges, including low response rates, poor specificity, drug tolerance, and significant toxic side effects. Therefore, we urgently need to conduct in-depth research into the molecular mechanisms of gastric cancer and explore new targeted therapy strategies, especially targeted drugs against tumor-related proteins, in order to provide more effective and personalized treatment options for gastric cancer patients.

[0003] ALDH3A2 is a member of the aldehyde dehydrogenase (ALDH) family, primarily responsible for the metabolism of medium- and long-chain fatty aldehydes. In recent years, its role in various cancers has attracted widespread attention, particularly its aberrant expression and dysfunction in ovarian cancer and clear cell renal cell carcinoma (ccRCC). Studies have found that in ovarian cancer, inhibiting ALDH3A2 significantly increases the sensitivity of cancer cells to ferroptosis, inhibiting ovarian cancer cell survival. This suggests that ALDH3A2 may promote cancer cell survival by regulating iron homeostasis or lipid peroxidation. In ccRCC, miR-1182 activates the PI3K-AK signaling pathway by inhibiting ALDH3A2, thereby promoting tumor progression. Upregulation of ALDH3A2 expression can inhibit the activation of this signaling pathway, reducing tumor cell proliferation and invasion. In gastric cancer, ALDH3A2 has been identified as a novel prognostic biomarker; however, current research is limited to investigating the expression of ALDH3A2 in gastric cancer cells or tissues. There are currently no reports on the role and mechanism of ALDH3A2 in the occurrence and development of gastric cancer. Summary of the Invention

[0004] Therefore, this invention provides an application of ALDH3A2 as a target in the preparation of drugs for treating gastric cancer. This study confirms that ALDH3A2, highly expressed in gastric cancer cells, can exacerbate mitochondrial dysfunction by regulating the NRF2 / UPRmt signaling axis and induce ferroptosis by inhibiting GPX4 expression. These results reveal the specific molecular mechanisms by which ALDH3A2 affects mitochondrial function and ferroptosis, providing a better strategy for clinical treatment of gastric cancer and improving patient prognosis.

[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] According to a first aspect of the present invention, the present invention provides the use of ALDH3A2 as a target in the preparation of a medicament for treating gastric cancer.

[0007] According to a second aspect of the present invention, the present invention provides the use of an ALDH3A2 overexpression reagent in the preparation of a drug for treating gastric cancer.

[0008] Furthermore, the drug inhibits the proliferation, migration, and invasion of gastric cancer cells by inducing ALDH3A2 overexpression, activating the NRF2 / UPRmt axis to aggravate mitochondrial dysfunction, and inhibiting GPX4 expression to promote ferroptosis.

[0009] Furthermore, the ALDH3A2 overexpression reagent includes an ALDH3A2 overexpression plasmid and genistein.

[0010] According to a third aspect of the present invention, the present invention provides a drug for treating gastric cancer, wherein an ALDH3A2 overexpression reagent is used as the active ingredient.

[0011] Furthermore, the ALDH3A2 overexpression reagent includes an ALDH3A2 overexpression plasmid and genistein.

[0012] The embodiments of the present invention have the following advantages:

[0013] This study elucidates a novel role and potential mechanism of ALDH3A2 in inhibiting gastric cancer progression. Specifically, ALDH3A2 downregulates SLC47A1 expression, thereby preventing NRF2 nuclear metastasis and inhibiting UPRmt activation. This disruption exacerbates mitochondrial dysfunction, ultimately promoting ferroptosis in gastric cancer cells in a glutathione peroxidase 4 (GPX4)-dependent manner. Furthermore, ALDH3A2-induced ferroptosis also promotes macrophage polarization towards the M1 type, inhibiting gastric cancer cell progression through the IL-1β signaling pathway. In summary, these effects collectively inhibit the proliferation, migration, and invasion of gastric cancer cells, ultimately achieving inhibition of gastric cancer growth and metastasis.

[0014] By screening potential drugs targeting ALDH3A2 through database analysis, we verified that genistein can significantly upregulate ALDH3A2 expression and inhibit the malignant progression of gastric cancer by inducing ferroptosis, demonstrating good clinical potential as a treatment for gastric cancer.

[0015] In summary, this invention reveals the key role of ALDH3A2 in regulating ferroptosis, mitochondrial homeostasis, and the immune microenvironment, and demonstrates its clinical application value as a potential therapeutic target. Attached Figure Description

[0016] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0017] Figure 1 The ALDH3A2 gene provided for this invention has been identified as a key gene associated with mitochondrial dysfunction and ferroptosis during natural progression.

[0018] Figure 2 The ALDH3A2 overexpression provided by this invention inhibits the invasive progression of gastric cancer cells and induces ferroptosis;

[0019] Figure 3 The GPX4 overexpression provided by this invention reversed the inhibitory effect of ALDH3A2 overexpression on the progression of gastric cancer cells;

[0020] Figure 4 The ALDH3A2 provided by this invention promotes ferroptosis by exacerbating mitochondrial damage in gastric cancer cells;

[0021] Figure 5 The ALDH3A2-mediated inhibition of the mitochondrial unfolded protein response provided by this invention leads to mitochondrial dysfunction and ferroptosis.

[0022] Figure 6 The ALDH3A2 provided by this invention reduces UPRmt and exacerbates ferroptosis in gastric cancer cells, and its mechanism is to prevent NRF2 from entering the cell nucleus;

[0023] Figure 7 The ALDH3A2 provided by this invention prevents NRF2 from entering the nucleus in gastric cancer cells by inhibiting SLC7A1 expression;

[0024] Figure 8The ALDH3A2 overexpression provided by this invention polarizes macrophages to the M1 type, and the IL-6 and IL-1β secreted by M1 tumor-associated macrophages inhibit the migration and invasion of gastric cancer cells;

[0025] Figure 9 The ALDH3A2-induced ferroptosis provided by this invention promotes macrophage polarization to the M1 phenotype through the IL-1β signaling pathway, thereby alleviating the progression of gastric cancer cells;

[0026] Figure 10 The expression of genistein ALDH3A2 provided by this invention inhibits UPRmt, promotes ferroptosis, and inhibits the proliferation and migration of gastric cancer cells;

[0027] Figure 11 The present invention provides that genistein targets ALDH3A2 to inhibit tumor growth and metastasis in vivo;

[0028] Figure 12 The ALDH3A2 overexpression provided by this invention does not affect the total NRF2 protein level, and DMF treatment effectively prevents ALDH3A2-induced NRF2 nuclear translocation inhibition. Detailed Implementation

[0029] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] 1. Experimental Materials and Reagents

[0031] Plasmids (PSPAX2, PMD2.G), Cell Counting Kit-8, ROS fluorescent probe DCFH-DA, and mitochondrial membrane potential fluorescent probe JC-1.

[0032] Rabbit anti-ALDH3A2 antibody, rabbit anti-SLC47A1 antibody, rabbit anti-NRF2 antibody, rabbit anti-LONNP1 antibody, rabbit anti-GPX4 antibody, mouse anti-HSP60 antibody, mouse anti-PD-L1 antibody, mouse anti-β-Actin antibody, mouse anti-β-tubulin, rabbit anti-PCNA, goat anti-rabbit IgG (H+L) secondary antibody Alexa Fluor 488 conjugate, dimethyl fumarate, oligomycin, N-acetylcysteine ​​(NAC).

[0033] 2. Experimental Methods

[0034] 2.1 Cell Culture

[0035] GES-1, MGC-803, HGC-27, and HEK-293T cells were cultured in DMEM containing 10% FBS at 37°C and 5% CO2.

[0036] 2.2 Generation and Transduction of Lentivirals

[0037] HEK-293T cells were co-transfected with packaging plasmid (psPAX2), envelope plasmid (pMD2.G), and PLKO.1-TRC plasmid with a targeting shRNA sequence for knockout, or co-transfected with PLV-EF1a plasmid with the ALDH3A2 gene sequence for overexpression, using Lipo6000™ transfection reagent to generate the corresponding lentiviruses. After culturing the transfected HEK-293T cells for 24 h and 48 h, the supernatant was collected, and viral particles were extracted by centrifugation at 1000 rpm for 5 min. Lentiviral-infected cells expressing the expression genes were then screened in growth medium supplemented with 2 mg / mL puromycin.

[0038] To overexpress ALDH3A2, the ALDH3A2 gene was amplified from the template plasmid PLV-EFla-ALDH3A2 by PCR.

[0039] Forward primer:

[0040] 5'-GCCACCACGCGTCGACACCATGGAGCTCGAAGTCC-3' (SEQ ID NO: 1).

[0041] Reverse primer:

[0042] 5'-AGCTGGGTGCTCTAGTCATCTCTGCTTACTGGACCAACG-3' (SEQ ID NO: 2).

[0043] To construct the SLC47A1 overexpression vector, the SLC47A1 gene was amplified from genomic DNA extracted from cancer cells (MGC-803) using PCR.

[0044] Forward primer:

[0045] 5'-GCCACCACGCGTCGACACCATGGAAGCTCCTGAGGAG-3' (SEQ ID NO: 3).

[0046] Reverse primer:

[0047] 5'-AGCTGGGTGCTCTAGTCACTGAATTCTGACATAGAARCTC-3' (SEQ ID NO: 4).

[0048] An IL-6 short hairpin RNA (shRNA) oligonucleotide that interferes with IL-6 expression was cloned into pLKO.1-TRC. The target sequence for IL-6-shRNA (meaning strand only) is shown below:

[0049] pLKO.1-hIL-6-F:

[0050] 5'-CCGGCAGAACGAATTGACAAACAACTCGAGTTTGTTTGTCAATTCGTTCTGTTTTTTG-3' (SEQ ID NO: 5)'.

[0051] To interfere with IL-6R expression, a short hairpin RNA (shRNA) oligonucleotide targeting IL-6R was cloned into pLKO.1-TRC. The targeting sequence of IL-6R-shRNA (sense strand only) is shown below:

[0052] pLKO.1-hIL-6R-F:

[0053] 5'-CCGGGCAGGCACTTAATACTAATAACTCGAGTTATTAGTAGTAAGTGCCTGCTTTTTG-3' (SEQ ID NO: 6).

[0054] To interfere with IL-1β expression, a short hairpin RNA (shRNA) oligonucleotide targeting IL-1β was cloned into pLKO.1-TRC. The targeting sequence of IL-1β-shRNA (sense strand only) is shown below:

[0055] pLKO.1-h IL-1β-F:

[0056] 5'-CCGGGGGATTTGTCTTCAACAAGATCTCGAGATCTTGAAGACAAATCGCTTTTTG-3' (SEQ ID NO: 7).

[0057] 2.3 Western blot and qRT-PCR

[0058] Cells were lysed using RIPA, protein concentration was measured using the BCA method, and after SDS-PAGE electrophoresis, the cells were transferred to membranes, incubated with primary antibody and HRP-labeled secondary antibody, and then visualized using ECL. Quantification was performed using ImageJ. Total RNA was extracted with Trizol and reverse transcribed. qRT-PCR was performed using GAPDH as an internal control, and relative expression was calculated using 2^-ΔCt.

[0059] 2.4 Clonal Formation and CCK8

[0060] Approximately 500 cells were seeded per well, cultured for 2 weeks, fixed, stained with crystal violet, and clone counted. CCK8 assay was performed at 5 × 10⁶ cells / well. 3 Cells / well were seeded in 96-well plates, and CCK8 was added at 24, 48, and 72 hours. Proliferation capacity was assessed by reading at 450 nm.

[0061] 2.5 Wound Healing and Transwell Migration / Invasion Tests

[0062] Cell monolayers were cultured in DMEM containing 0.2% FBS after scratching, and wound widths were compared by photographing at 0 and 24 hours. In Transwell assays, cells were seeded in the upper cavity coated with Matrigel, and the lower cavity contained 10% FBS medium. After 24 hours, cells were fixed, stained, and migrated / invaded cells were counted.

[0063] 2.6 Cavity-Nucleus Separation and Immunofluorescence

[0064] Cells were lysed with HEPES-containing buffer, and the cytoplasm and nuclear components were separated. After cell fixation and transmembrane transfer, cells were incubated with primary antibody and fluorescent secondary antibody, and the nuclei were stained with DAPI. Confocal microscopy was used to capture images and analyze protein localization.

[0065] 2.7 Detection of ROS and Mitochondrial Membrane Potential

[0066] ROS was detected using DCFH-DA and DAF-FM DA fluorescent probes, and mitochondrial membrane potential was assessed using the JC-1 fluorescent probe. Qualitative observation was performed under a microscope, and quantitative analysis was performed by flow cytometry.

[0067] 2.8 MDA Determination and Animal Experiments

[0068] Cellular MDA levels were detected using a kit. BALB / c nude mice were grouped and subcutaneously injected with MGC-803 cells or an ALDH3A2 overexpressing cell line. In another experiment, genistein (50 or 100 mg / kg) was injected intraperitoneally for 4 weeks, tumor volume was measured, and the mice were ultimately sacrificed and the tumors were collected for subsequent analysis.

[0069] 2.9 Statistical Analysis

[0070] Data are expressed as mean ± standard error. Paired t-tests were used for comparisons between groups, and one-way ANOVA with Bonferroni correction was used for comparisons among multiple groups. *p<0.05, **p<0.01, ***p<0.001 were considered statistically significant.

[0071] Example 1: ALDH3A2 is a gene associated with mitochondrial dysfunction and ferroptosis.

[0072] Bioinformatics analysis results: Three key genes FMDRGs (ALDH3A2, PDK4, and GJA1) were screened using multiple algorithms. In the multivariate Cox regression model, the coefficients for PDK4, ALDH3A2, and GJA1 were 0.0679, 0.2980, and 0.1794, respectively, ultimately identifying the target gene ALDH3A2. qPCR results: Compared with the control group, the mRNA expression level of the target gene ALDH3A2 was significantly downregulated in the experimental group (P<0.05). Western blot results: Compared with the control group, the protein expression level of the target gene ALDH3A2 was significantly downregulated in the experimental group (P<0.05), consistent with the qPCR results. These experimental results indicate that the ALDH3A2 gene plays an important role in ferroptosis and mitochondrial dysfunction, providing a theoretical basis and experimental foundation for further research on the molecular mechanisms of ferroptosis and mitochondrial dysfunction.

[0073] exist Figure 1 A. Volcano plot of differentially expressed genes between gastric cancer tissue and adjacent non-tumor tissue. B. Venn diagram of differentially expressed genes in gastric cancer tissue, ferroptosis-regulating genes from FerrDb, and mitochondrial dysfunction-related genes from MitoProteome. CG. Genes related to overall survival identified by univariate Cox analysis (C), LASSO Cox analysis (DE), Boruta analysis (F), and random forest analysis (G), which affect patient survival. H. Upset plot showing that ALDH3A2, PDK4, and GJA1 are common genes in all machine learning methods. 1. Bar chart showing the coefficients of PDK4, ALDH3A2, and GJA1 in the prognostic model. J. Risk score distribution and patient categorization into high-risk and low-risk groups. K. ROC curves showing the consistency between RiskScore and 1-year, 3-year, and 5-year survival rates. L. Kaplan-Meier survival curves comparing high-risk and low-risk groups. MN. Univariate and multivariate Cox analyses showing the prognostic significance of risk scores. The correlation between ALDH3A2 expression and the characteristics of M1 giant cell polarization.

[0074] Example 2: ALDH3A2 overexpression inhibits gastric cancer cell progression and enhances ferroptosis

[0075] To investigate the effect of ALDH3A2 on ferroptosis and progression in gastric cancer cells, ALDH3A2 was first overexpressed in the MGC803 and HGC27 gastric cancer cell lines, and this was confirmed by qPCR and Western blot analysis. Figure 2 AB). CCK8 and colony formation assays showed that ALDH3A2 overexpression significantly inhibited cell growth and colony formation in both gastric cancer cell lines. Figure 2 CE). To evaluate the effect of ALDH3A2 on gastric cancer metastasis, wound healing and Transwell invasion assays were performed. Notably, ALDH3A2 overexpression significantly inhibited cell migration and invasion, such as ( Figure 2 As shown in FH). Immunoblot analysis showed that overexpression of ALDH3A2 in gastric cancer cells significantly reduced the expression of GPX4 (FH). Figure 2 B), which also promotes lipid peroxidation, manifested as elevated MDA levels ( Figure 2 I). However, compared to the control group, intracellular Fe 2+ The level did not change significantly. Figure 2 These results lead us to infer that ALDH3A2 may inhibit the progression of gastric cancer cells and promote ferroptosis in a GXP4-dependent manner.

[0076] Example 3: ALDH3A2 exerts an inhibitory effect by suppressing the progression of gastric cancer cells and induces ferroptosis in a GPX4-dependent manner.

[0077] To determine whether the inhibitory effect of ALDH3A2 is mediated by GPX4-dependent ferroptosis, we overexpressed GPX4 in ALDH3A2-overexpressing MGC803 and HGC27 cell lines to counteract ferroptosis. As expected, the enhanced ferroptosis induced by ALDH3A2 overexpression was significantly reversed after GPX4 overexpression compared to the ALDH3A2 overexpression group. Figure 3 A), this is due to a decrease in malondialdehyde (MDA) levels ( Figure 3 B) and increased cell viability ( Figure 3 This can be seen in CD. Notably, in MGC-803 and HGC-27 cells, GPX4 overexpression significantly reversed ALDH3A2-mediated cell proliferation (CD). Figure 3 E), Migration ( Figure 3 FG) and invasion ( Figure 3 The decreased ability of ALDH3A2 to induce GPX4-dependent ferroptosis. These findings suggest that ALDH3A2 inhibits the invasive progression of gastric cancer cells by promoting GPX4-dependent ferroptosis.

[0078] Example 4: ALDH3A2 induces ferroptosis in gastric cancer cells by promoting mitochondrial dysfunction.

[0079] DCFH-DA staining results showed that in MGC803 ( Figure 4 A) and HGC27 ( Figure 4 B) In gastric cancer cell lines, overexpression of ALDH3A2 significantly increased the production of reactive oxygen species (ROS). Furthermore, overexpression of ALDH3A2 led to a significant decrease in mitochondrial membrane potential. Figure 4 These results confirm that ALDH3A2 exacerbates mitochondrial dysfunction. To investigate whether ALDH3A2 promotes ferroptosis by inducing mitochondrial dysfunction in gastric cancer cells, we used the ROS scavenger N-acetylcysteine ​​(NAC) to rescue ALDH3A2-induced mitochondrial dysfunction in MGC-803 and HGC-27 cells. As expected, NAC treatment (20 µM, 24 h) significantly reversed the ALDH3A2-induced increase in ROS production (CD). Figure 4 EF) and decrease in mitochondrial membrane potential ( Figure 4 GH), while compared with the ALDH3A2 overexpression group, GPX4 expression increased and MDA level decreased ( Figure 4 The results also showed that the enhancement of ferroptosis was suppressed. These findings suggest that ALDH3A2 promotes ferroptosis by exacerbating mitochondrial dysfunction.

[0080] Example 5: Inhibition of ALDH3A2-mediated mitochondrial unfolded protein response leads to mitochondrial dysfunction and ferroptosis.

[0081] The mitochondrial unfolded protein response (UPRmt) is crucial for maintaining normal mitochondrial function and promoting mitochondrial recovery (MIPRmt and its multiple roles in cellular stress). Impaired UPRmt is closely associated with mitochondrial dysfunction, leading us to hypothesize whether ALDH3A2 exacerbates mitochondrial dysfunction by inhibiting UPRmt in GC cells. Notably, overexpression of ALDH3A2 in MGC-803 and HGC-27 cells significantly reduced the mRNA and protein expression levels of the key UPRmt markers LONP1 and HSP60 compared to the control group. Figure 5 AD). Notably, restoring UPRmt using oligomycin, a known UPRmt activator, prevented ALDH3A2-induced UPRmt inhibition, as evidenced by increased expression of LONP1 and HSP60 in both GC cell lines. Figure 5 AD). Furthermore, oligomycin-mediated UPRmt restoration can reverse ALDH3A2-induced mitochondrial dysfunction and ferroptosis, manifested as reduced ROS accumulation (AD). Figure 5 EF), mitochondrial membrane potential recovery (EF) Figure 5 GH) and GPX4 expression upregulated ( Figure 5 I) and MDA generation reduction (Figure 5 (J). Functionally, oligomycin treatment effectively rescued the ALDH3A2-mediated decline in GC cell viability, proliferation, migration, and invasion. These findings suggest that ALDH3A2 inhibits UPRmt, leading to mitochondrial dysfunction and ferroptosis, thereby suppressing GC progression.

[0082] Example 6: ALDH3A2 promotes mitochondrial dysfunction and ferroptosis by blocking NRF2 nuclear translocation and damaging UPRmt.

[0083] UPRmt is a mitochondrial-specific stress response that can be regulated by a variety of transcription factors (TFs). To elucidate the mechanism by which ALDH3A2 inhibits UPRmt to induce ferroptosis, we attempted to identify potential regulatory TFs by cross-analyzing genes associated with TFs, ferroptosis, and UPRmt. Seven potential TFs were ultimately identified, including NRF2, MYC, TP53, JUN, HSF1, CREB3, and ESR1. Figure 6 A). Bioinformatics analysis of the correlation between ALDH3A2 and identified TF candidates showed that NRF2 was most strongly correlated with ALDH3A2 expression. Figure 6 B). However, subcellular graded immunoblotting analysis showed that ALDH3A2 significantly reduced nuclear translocation of NRF2 in MGC803 and HGC27 cells (B). Figure 6 CE). Immunofluorescence analysis further confirmed these findings, showing reduced NRF2 nuclear translocation in ALDH3A2-overexpressing cells ( Figure 6 F). To further evaluate whether NRF2 restoration could reverse ALDH3A2-induced UPRmt damage and ferroptosis, cells overexpressing ALDH3A2 were treated with dimethyl fumarate (DMF), a known NRF2 activator. Functionally, DMF treatment significantly protected against ALDH3A2-induced UPRmt damage, as evidenced by increased mRNA and protein expression of LONP1 and HSP60. Figure 6 GK). Furthermore, DMF alleviated ferroptosis in ALDH3A2-overexpressing gastric cancer cell lines, manifested by increased GPX4 expression (GK). Figure 6 L) and decreased MDA levels ( Figure 6 These findings suggest that ALDH3A2 suppresses UPRmt by inhibiting NRF2 nuclear translocation.

[0084] Notably, overexpression of ALDH3A2 did not affect the total NRF2 protein level, a result confirmed by Western blotting. Figure 12 AB). DMF treatment effectively prevented ALDH3A2-induced NRF2 nuclear translocation inhibition ( ). Figure 12(CD). Specifically, MGC-803 and HGC-27 cells were transduced using either pLV-EF1a empty lentivirus (control group) or pLV-EF1a-ALDH3A2 lentivirus (EF1a-ALDH3A2 overexpression group), respectively. Gastric cancer cells were treated with either the NRF2 activator dimethyl fumarate (10 µM, 24 h) or DMSO solvent as a control. (A, B) NRF2 protein expression was assessed by Western blotting analysis, and the bar chart on the right shows the protein quantification results. (C, D) NRF2 immunofluorescence staining (green) was performed on MGC-803 and HGC-27 cells, and DAPI (blue) counterstaining was used for nuclear localization. The right panel shows the quantitative analysis of NRF2 signal.

[0085] Example 7: ALDH3A2 prevents NRF2 nuclear transfer by downregulating SLC47A1.

[0086] To determine the status of ALDH3A2-associated genes in different types of cancer, we used a cancer cell metabolic gene database to identify genes associated with ALDH3A2 in head and neck squamous cell carcinoma (HNSC), clear cell renal cell carcinoma (KIRC), lung squamous cell carcinoma (LUSC), and endometrial cancer (UCEC). Wien's intersection analysis showed that solute carrier family 47 member 1 (SLC47A1) was consistently associated with ALDH3A2 in all four cancer types. Figure 7 A). qPCR and Western blot analysis showed that ALDH3A2 overexpression significantly reduced the mRNA and protein expression of SLC47A1 in GC cells (A). Figure 7 To determine whether ALDH3A2 exerts its biological effects by downregulating SLC47A1 expression, we overexpressed SLC47A1 in MGC-803 and HGC-27 cells to counteract the ALDH3A2-induced inhibition of SLC47A1. The results were analyzed by qPCR. Figure 7 D) and immunoblotting ( Figure 7 E) was confirmed. Immunofluorescence analysis further showed that SLC47A1 overexpression significantly restored NRF2 nuclear translocation, which was inhibited by ALDH3A2 overexpression (Figure 7F-G). Furthermore, SLC47A1 overexpression could reverse ALDH3A2-induced UPRmt damage and ferroptosis, as evidenced by increased mRNA and protein levels of LONP1, HSP60, and GPX4, and decreased MDA levels. Figure 7Functionally, overexpression of SLC47A1 restored ALDH3A2-induced inhibition of cell viability, proliferation, migration, and invasion in both gastric cancer cell lines. These findings suggest that ALDH3A2 downregulates SLC47A1, thereby inhibiting NRF2 nuclear metastasis and leading to UPRmt damage and ferroptosis, ultimately resulting in the malignant progression of gastric cancer.

[0087] Example 8: ALDH3A2-induced ferroptosis-induced release of IL-6 promotes macrophage polarization toward the M1 phenotype.

[0088] Tumor-associated macrophages (TAMs) regulate gastric cancer (GC) progression by mediating cell proliferation, invasion, and metastasis, with their mechanism of action involving macrophage polarization. To investigate whether ALDH3A2 partially promotes GC progression by regulating TAM polarization, we performed a correlation analysis between ALDH3A2 and key macrophage polarization-related factors (IFNG, AKT1, NOTCH1, NFKB2, and TNFA) in TCGA STAD patients. Notably, ALDH3A2 expression was significantly correlated with M1 macrophage polarization. Figure 8 A), which leads us to further hypothesize that ALDH3A2-induced ferroptosis may partially promote gastric cancer progression by facilitating TAM polarization through the release of ferroptosis-related molecular factors. To test this hypothesis, we treated THP1-derived M0 macrophages (EF1a-ALDH3A2) with conditioned medium (CM) containing ALDH3A2-overexpressing gastric cancer cells. Figure 8 B). The results showed that conditioned medium in ALDH3A2-overexpressing MGC803 cells (MGC803-CM) and HGC27 cells (HGC27-CM) promoted the polarization of THP1-derived M0 macrophages towards the M1 phenotype, as evidenced by significantly elevated mRNA levels of M1 macrophage markers (IL-6, IL-1α, and IL-1β). Figure 8 C) was observed, while M2 markers (IL-10, Arg1, and TGF-β) were significantly reduced. Figure 8 D). Among them, the upregulation of IL-1β was the most significant. Next, in order to determine the mechanism by which ALDH3A2-induced ferroptosis-associated secretory phenotype (FASP) factors promote macrophage polarization, we performed Venn diagram analysis using the Genecard database to identify protein-coding genes associated with FASP, inflammatory cytokines (ICs), glucocorticoid-associated genes (NRGs), and macrophage reprogramming-associated genes (MRRGs). This analysis identified three candidate factors: IL-6, TGF-β1, and TNF-α ( Figure 8E). ELISA experiments confirmed that ALDH3A2-induced ferroptosis in MGC803 and HGC27 gastric cancer cell lines significantly enhanced IL-6 secretion ( Figure 8 F). To further verify whether IL-6 released by ALDH3A2-induced ferroptosis gastric cancer cells triggers macrophage polarization towards the M1 phenotype, we silenced IL-6 in ALDH3A2-overexpressing gastric cancer cells (F). Figure 8 G) or silencing IL-6 receptor (IL-6R) in THP-1 cells (G) Figure 8 H). Notably, compared with the CM-GC-EF1a-ALDH3A2 group, silencing IL-6 in ALDH3A2-overexpressing gastric cancer cells (CM-GC-(EF1a-ALDH3A2-shIL-6)) or depleting IL-6R in THP-1 cells (CM-GC-EF1a-ALDH3A2+shIL-6R(THP1)) significantly reduced the mRNA expression levels of IL-6, IL-1a, and IL-1β. Figure 9 (I), with the most significant decrease in IL-1β. These findings suggest that ALDH3A2-induced ferroptosis promotes macrophage polarization toward the M1 phenotype through IL-6 release.

[0089] Example 9: ALDH3A2-induced ferroptosis promotes macrophage polarization towards the M1 phenotype through the IL-1β signaling pathway, thereby alleviating the progression of gastric cancer cells.

[0090] We investigated whether the conditioned medium of M1 polarized macrophages regulates gastric cancer progression by releasing IL-1β. We depleted IL-1β in THP1 cells to block the release of IL-1β from polarized M1 macrophages. Figure 9 A). We found that conditioned medium for M1 polarized macrophages induced by ALDH3A2-overexpressing gastric cancer cells (CM-THP1-CM-GC-EF1a-ALDH3A2) significantly inhibited PD-L1 expression. PD-L1 is a key factor in the immunosuppressive tumor microenvironment, and this effect can be prevented by depleting IL-1β in macrophages. Figure 9 BC). Functionally, CM-THP1-CM-GC-ALDH3A2 significantly reduced the cell viability of gastric cancer cell lines (BC). Figure 9 DE), proliferation ( Figure 9 F), migration ( Figure 9 G) and invasion ( Figure 9 H), and these effects can also be reversed by silencing IL-1β in M1-polarized macrophages (H). Figure 9 DH). These data indicate that ALDH3A2 induces (CM-GC-ALDH3A2) ( DH). Figure 9BC). Functionally, CM-THP1-CM-GC-ALDH3A2 significantly reduced the cell viability of gastric cancer cell lines (BC). Figure 9 DE), proliferation ( Figure 9 F), migration ( Figure 10 G) and invasion ( Figure 10 H), and this phenomenon can be reversed by silencing IL-1β in M1 polarized macrophages (CM-THP1-shIL-1β-CM-GC-ALDH3A2). Figure 10 These data suggest that ALDH3A2-induced ferroptosis promotes macrophage polarization toward the M1 phenotype, thereby driving gastric cancer cell progression via the IL-1β signaling pathway.

[0091] Example 10: Gentian isoflavones targeting ALDH3A2 can slow the progression of gastric cancer in vitro and in vivo.

[0092] The data presented to date suggest that targeting ALDH3A2 may be a promising therapeutic strategy for gastric cancer, exerting its effects by promoting ferroptosis. Using pharmacokinetic transcriptomics analysis with the DrugBank database, we identified genistein as a potential candidate drug for upregulating ALDH3A2 expression. We observed that genistein significantly increased ALDH3A2 mRNA levels in gastric cancer cells (…). Figure 10 A). Immunoblot analysis further confirmed a significant increase in ALDH3A2 expression, while the protein levels of LONP1, HSP60, and GPX4 were decreased in both MGC-803 and HGC-27 cell lines. Furthermore, genistein treatment significantly increased malondialdehyde (MDA) levels, indicating enhanced lipid peroxidation in gastric cancer cells. Figure 10 G). DCFH-DA staining showed that the production of reactive oxygen species (ROS) was significantly increased after treatment with genistein. Figure 10 HI). JC-1 staining showed that mitochondrial membrane potential was significantly reduced after treatment with genistein (HI). Figure 10 JK). From a functional perspective, genistein inhibits the proliferation of gastric cancer cells, which is observed in CCK-8 (JK) cells of MGC-803 and HGC-27 cells. Figure 10 LM) and colony formation experiments ( Figure 11 This was confirmed in N). Furthermore, wound healing and Transwell invasion assays showed that genistein significantly reduced the migration of gastric cancer cells (N). Figure 11 OP) and invasion ( Figure 11 QR).

[0093] Example 11: Antitumor activity of genistein in vivo

[0094] To evaluate the antitumor activity of genistein in vivo, we conducted a xenograft experiment by implanting MGC-803 cells into BALB / c nude mice. (For example...) Figure 11 As shown in Figure AB, intraperitoneal administration of genistein produced a dose-dependent inhibitory effect on the growth of MGC-803 tumors, with the 100 mg / kg genistein group showing significantly stronger inhibitory effects than the control group and the 50 mg / kg genistein group. Immunoblotting analysis of the xenograft inhibitory effect on MGC-803 tumor growth showed that the 100 mg / kg genistein group had a significantly better inhibitory effect than the solvent group and the 50 mg / kg genistein group. Immunoblotting analysis of xenograft tumors confirmed that genistein treatment significantly upregulated ALDH3A2 expression while reducing the expression levels of SLC47A1, LONP1, HSP60, and GPX4 proteins. Figure 11 CD). To further verify the role of ALDH3A2 in inhibiting gastric cancer progression in vivo, MGC-803 cells stably transfected with the lentiviral vector EF1A-ALDH3A2 or the empty pLV / EF1A control vector were subcutaneously injected into nude mice. Similarly, compared with control mice, mice injected with ALDH3A2-overexpressing cells showed significantly reduced tumor volume and weight (CD). ​ EF), indicating that ALDH3A2 overexpression effectively inhibited the growth of gastric cancer tumors in vivo. qRT-PCR and Western blot analysis of tumor tissues isolated from implanted mice showed that ALDH3A2 overexpression significantly increased the mRNA and protein levels of ALDH3A2, while significantly decreasing the mRNA levels of SLC47A1, LONP1, and HSP60 (EF). ​ In addition, the protein levels of SLC47A1, LONP1, HSP60, and GPX4 were also measured. ​ These results are consistent with our in vitro experimental results, further confirming the role of ALDH3A2 in inhibiting gastric cancer growth. Overall, our findings provide strong evidence that overexpression of ALDH3A2 promotes ferroptosis through the ALDH3A2-SLC47A1-UPRmt-GPX4 axis, thereby inhibiting gastric cancer development. This highlights the significant potential of genistein as a therapeutic agent for gastric cancer.

[0095] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of ALDH3A2 as a target in the preparation of drugs for the treatment of gastric cancer.

2. Application of ALDH3A2 overexpression reagent in the preparation of drugs for treating gastric cancer.

3. The application according to claim 2, characterized in that, The drug inhibits the proliferation, migration, and invasion of gastric cancer cells by inducing ALDH3A2 overexpression, activating the NRF2 / UPRmt axis to aggravate mitochondrial dysfunction, and inhibiting GPX4 expression to promote ferroptosis.

4. The application according to claim 2, characterized in that, The ALDH3A2 overexpression reagent includes an ALDH3A2 overexpression plasmid and genistein.

5. A drug for treating gastric cancer, characterized in that, The ALDH3A2 overexpression reagent is the active ingredient.

6. The medicament for treating gastric cancer according to claim 5, characterized in that, The ALDH3A2 overexpression reagent includes an ALDH3A2 overexpression plasmid and genistein.

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