Combined application of SLC25A6-mediated colorectal cancer cell apoptosis regulation and tumor resistance

Abstract

The invention belongs to the technical field of disease diagnosis and treatment and molecular biology, and particularly relates to combined application of SLC25A6-mediated colorectal cancer cell apoptosis regulation and tumor resistance. Specifically, research finds that SLC25A6 is used as a cancer inhibiting factor for connecting glutamine metabolism and mitochondrial apoptosis, and the SLC25A6 interacts with MIC60 to destroy an MICOS complex, so that excessive mitochondrial division and cell death are caused. Meanwhile, a glutaminase inhibitor CB-839 and a Bcl-2 inhibitor ABT-199 are combined for use, so that a synergistic anti-tumor effect is generated. The invention not only deepens the understanding of a colorectal cancer metabolic stress response mechanism, but also provides a pre-clinical basis for combined metabolic inhibition and apoptosis targeted therapy, thereby having good practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of disease diagnosis and treatment and molecular biology technology, specifically involving the combined application of SLC25A6-mediated apoptosis regulation and anti-tumor activity in colorectal cancer cells. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The high nutritional demands during tumor progression present potential therapeutic opportunities for cancer treatment. Glutamine addiction is a key metabolic characteristic of cancer cells. As a crucial amino acid, glutamine not only participates in energy metabolism through the mitochondrial tricarboxylic acid cycle (TCA cycle) but also serves as a precursor to nucleotides and lipids and maintains cellular redox balance. To maintain glutamine dependence, cancer cells enhance glutamine uptake and upregulate the expression of mitochondrial glutaminase, an enzyme that converts glutamine into glutamate to replenish the TCA cycle. Targeting this metabolic vulnerability by inhibiting glutamine uptake or glutaminase (such as CB-839) can effectively suppress tumor growth. However, clinical trial results for various cancers have shown that monotherapy has limited efficacy.

[0004] Glutamine is the primary fuel for mitochondria, which play a central role in tumorigenesis. Through participation in the TCA cycle and oxidative phosphorylation, mitochondria are core organelles for cellular energy, anabolic metabolism, and signal regulation. Multi-level interactions exist between cellular signaling and mitochondrial function, supporting not only tumorigenesis but also helping cancer cells adapt to environmental stressors and therapeutic interventions. Mitochondria are highly dynamic, with their homeostasis regulated by mitochondrial biosynthesis, autophagy, division, and fusion. Furthermore, mitochondrial function and morphology are closely related to the tumorigenicity, therapeutic resistance, and apoptosis sensitivity of cancer cells.

[0005] Mitochondrial function largely depends on its double-membrane structure. The outer membrane (OMM) is involved in apoptotic signaling, mitochondrial fission and fusion, and autophagy; recruitment of pro-apoptotic proteins Bax and Bak leads to OMM permeability, thereby triggering the apoptotic cascade and activating the key execution enzyme caspase-3. Conversely, anti-apoptotic proteins Bcl-2 and Bcl-xL can bind to Bax / Bak and inhibit their pro-apoptotic activity. The inner membrane (IMM) is characterized by low permeability and abundant cristae, both of which are crucial for energy production. The mitochondrial contact point and cristae organization system (MICOS) complex (with major subunits including MIC60 and MIC19) mediates the contact between the IMM and OMM and plays a key role in the formation and maintenance of mitochondrial cristae. Notably, the opening of the mitochondrial permeability transition pore (mPTP) can lead to membrane potential collapse, inducing various cellular responses ranging from mitophagy to apoptosis. However, the mechanisms by which glutamine metabolic stress regulates mitochondrial dynamics and its molecular targets remain unclear. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a combined application of SLC25A6-mediated apoptosis regulation and anti-tumor activity in colorectal cancer cells. Specifically, this invention compared cell populations with different sensitivities to glutamine metabolism inhibition-induced apoptosis, revealed a novel function of SLC25A6, a member of the adenine nucleotide transporter (ANT) family, in maintaining mitochondrial morphology, and proposed a potential new strategy for cancer treatment. Based on the above research findings, this invention was thus completed.

[0007] Specifically, the present invention relates to the following technical solutions: In a first aspect, the present invention provides the use of reagents for detecting the expression level of the SLC25A6 gene and its expression products in the preparation of colorectal cancer detection products.

[0008] The colorectal cancer detection includes prognostic assessment of colorectal cancer.

[0009] This invention reveals that SLC25A6 mRNA and protein levels are significantly downregulated in CRC clinical samples. Its expression gradually decreases with clinical stage progression, reaching its lowest level in poorly differentiated tumors. In the CRC patient cohort, low SLC25A6 expression is significantly associated with poor prognosis (shortened progression-free survival). Therefore, the expression levels of the SLC25A6 gene and its expression products are negatively correlated with poor prognosis.

[0010] The prognostic assessment includes analysis of clinical stages of colorectal cancer (e.g., stage I, II, III, and IV) and analysis of progression-free survival (PFS) for colorectal cancer.

[0011] The product may be a test kit, a test device and / or equipment, and no specific limitation is made herein.

[0012] The expression product of the SLC25A6 gene can obviously be the SLC25A6 protein.

[0013] A second aspect of the present invention provides a system for detecting colorectal cancer, the system comprising: i) An analysis module, the analysis module comprising: a detection substance selected from the expression level of SLC15A6 in the test sample of the subject; ii) An assessment module, the analysis module comprising: detecting the subject based on the SLC15A6 expression level determined in i).

[0014] The subject can be a colorectal cancer patient, and the sample to be tested can be a colorectal cancer sample, specifically colorectal cancer tissue or cells.

[0015] As described above, the colorectal cancer detection includes prognostic assessment of colorectal cancer. Furthermore, the expression level of the SLC25A6 gene and its expression product is negatively correlated with poor prognosis. The prognostic assessment includes analysis of the clinical stage of colorectal cancer (e.g., stage I, II, III, and IV) and the analysis and assessment of progression-free survival (PFS) for colorectal cancer.

[0016] A third aspect of the invention provides the use of a substance that promotes the expression of the SLC25A6 encoding gene and its expression products and / or enhances their activity in at least one of the following (a1)-(a5); (a1) Prepare products that promote tumor cell apoptosis; (a2) To prepare products that inhibit tumor growth; (a3) Prepare products that promote the division of tumor cell mitochondria; (a4) To prepare products that enhance the antitumor activity of Bcl-2 inhibitors; (a5) Cancer treatment or preparation of cancer treatment products.

[0017] The tumor is a solid tumor, specifically colorectal cancer.

[0018] The product may be a pharmaceutical or a test reagent for non-pharmaceutical purposes, and the test reagent may be used for basic research on colorectal cancer.

[0019] In this invention, the substance that promotes the expression of the SLC25A6 encoding gene and its expression product and / or increases its activity can be a glutaminase inhibitor, and more particularly, CB-839. The Bcl-2 inhibitor may be ABT-199.

[0020] This invention has found that the combination of CB-839 and ABT-199 significantly enhances the anti-tumor effects of various CRC cell lines, producing a synergistic anti-tumor effect. At the same time, in an in vivo xenograft model, the combined treatment significantly inhibits tumor growth and reduces tumor weight. The overall results show that the combination of glutaminase inhibitor and Bcl-2 inhibition can significantly inhibit CRC growth by enhancing apoptosis.

[0021] A fourth aspect of the present invention provides a composition wherein the active ingredient comprises at least a substance that inhibits and / or enhances the expression of the SLC25A6 encoding gene and its expression products, and a Bcl-2 inhibitor.

[0022] In this invention, the substance that promotes the expression of the SLC25A6 encoding gene and its expression product and / or increases its activity can be a glutaminase inhibitor, and more particularly, CB-839. The Bcl-2 inhibitor may be ABT-199.

[0023] The molar ratio of the two is 1-1000:10-10000, and further 10-100:1-100, without specific limitations here.

[0024] A fifth aspect of the invention provides the use of the above-described composition in the preparation of a medicament for treating colorectal cancer.

[0025] As mentioned above, the combination of glutaminase inhibitor CB-839 and Bcl-2 inhibitor ABT-199 produces synergistic anti-tumor effects without significant toxic changes, indicating that this strategy also has good safety.

[0026] In another specific embodiment of the present invention, the medicament may further comprise one or more pharmaceutically or food-grade excipients. The excipients may be solid or liquid. Solid formulations include powders, tablets, dispersible granules, capsules, pills, and suppositories. Powders and tablets may contain about 0.1% to about 99.9% of the active ingredient. Suitable solid excipients may be magnesium carbonate, magnesium stearate, talc, sugar, or lactose. Tablets, powders, pills, and capsules are solid dosage forms suitable for oral administration. Liquid formulations include solutions, suspensions, and emulsions, examples of which are aqueous solutions or water-propylene glycol solutions for non-enteric injection, or oral solutions with added sweeteners and contrast agents. Furthermore, it may be formulated as a small-volume injection, a lyophilized powder for injection, a large-volume infusion, or a small-volume infusion.

[0027] A sixth aspect of the present invention provides a method for treating tumors, the method comprising administering to a subject an effective amount of the substance or the composition thereof that promotes the expression and / or activity of the SLC25A6 encoding gene and its expression products.

[0028] The tumor is a solid tumor, specifically colorectal cancer.

[0029] The subject refers to an animal that is already the object of treatment, observation, or experimentation, and can be a human or non-human mammal, such as a mouse, rat, guinea pig, rabbit, dog, monkey, orangutan, preferably a human. The "effective amount" refers to the amount of the active compound or agent, including the compound of the present invention, that can elicit the biological or medical response in an tissue system, animal, or human sought by the researcher, veterinarian, physician, or other medical professional. This includes the reduction or partial reduction of symptoms of the treated disease, syndrome, symptom, or disorder. It must be recognized that the optimal dosage and interval of the active ingredient described in the present invention are determined by its properties and external conditions such as the form, route, and site of administration, and the specific mammal being treated, and this optimal dosage can be determined using conventional techniques. It must also be recognized that the optimal course of treatment, i.e., the daily dose of the compound within a specified time period, can be determined using methods known in the art.

[0030] The beneficial technical effects of one or more of the above technical solutions: The aforementioned technical approach identified SLC25A6 as a tumor suppressor linking glutamine metabolism and mitochondrial apoptosis. It disrupts the MICOS complex through interaction with MIC60, leading to excessive mitochondrial division and cell death. Simultaneously, the combination of the glutaminase inhibitor CB-839 and the Bcl-2 inhibitor ABT-199 produced a synergistic anti-tumor effect. This approach not only deepens the understanding of the metabolic stress response mechanism in CRC but also provides preclinical evidence for combined metabolic inhibition and apoptosis-targeted therapy, demonstrating significant practical application value. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1. SLC25A6 was identified as a mediator of glutamine metabolism inhibition-induced apoptosis. (A) Schematic diagram of the sorting strategy for HCT116-C3AI cells after glucose deprivation. Cells were divided into high-C3AI (top 30% GFP-positive) and low-C3AI (bottom 30%) populations based on fluorescence intensity. (B–C) Flow cytometry analysis of apoptotic cells in the high-C3AI and low-C3AI populations. (D) Overlap analysis of differentially expressed genes upregulated between groups. (E–F) qPCR validation of SLC25A6 and SLC25A4 expression in the high-C3AI and low-C3AI populations. (G) Caspase-3 activation in HCT116-C3AI cells overexpressing SLC25A6 compared to the vector control group (scale bar: 200 μm). (H) Apoptosis analysis of control and SLC25A6 knockdown (KD) HCT116 cells under glucose deprivation conditions. Data are presented as mean ± standard deviation (n = 3). The error bars represent the standard deviation. The exact P-value is shown in the graph.

[0033] Figure 2. Antitumor function of SLC25A6 in vitro and in vivo. (A–B) Growth curves of control, SLC25A6 overexpressing (OE), and SLC25A6 knockdown (KD) cells as measured using the IncuCyte live-cell imaging system. (C, D) Colony formation assays of control, SLC25A6 overexpressing (OE), and SLC25A6 knockdown (KD) cells. (E) Flow cytometry apoptosis analysis of control and SLC25A6 overexpressing (OE) cells. (F–H) Xenografts from SLC25A6 overexpressing (OE) or vector control cells: representative images (F), tumor volume growth curves (G), and weight (H) (n = 5 mice per group). (I) TUNEL immunohistochemical staining of xenografts (scale bar: 50 μm). (J–L) SLC25A6 knockdown (KD) xenografts: representative images (J), tumor volume growth curves (K), and weight (L) (n = 5 mice per group). (M) TUNEL immunohistochemical staining of knockdown (KD) xenografts (scale bar: 50 μm). Data are presented as mean ± standard deviation (n = 3). Error bars represent standard deviation. Precise p-values ​​are shown in the figures.

[0034] Figure 3. Mitochondrial dysfunction in SLC25A6 overexpression (OE) cells. (A) Representative immunoblot images of cleaved caspase-9, cleaved caspase-3, and Bcl-2 in control and SLC25A6 overexpression (OE) cells. (B) Representative immunoblot images of Bax and Bak in mitochondrial and cytoplasmic components of control and SLC25A6 overexpression (OE) cells. (C) JC-1 staining of mitochondrial membrane potential in control and SLC25A6 overexpression (OE) cells. (D) Quantification of cellular ATP levels. (E) NAD+. + Quantification of the / NADH ratio. (F) Oxygen consumption rate as determined using a Seahorse analyzer. (G) Quantification of mPTP openness. (H–I) Cell viability assays in control and SLC25A6 overexpression (OE) cells after treatment with ABT-199 (H) or astrococcal (I). Data are expressed as mean ± standard deviation (n = 3). Error bars represent standard deviation. Precise P-values ​​are shown in the figures.

[0035] Figure 4. SLC25A6 promotes mitochondrial division (A) Representative immunofluorescence staining images of mitochondrial morphology in control and SLC25A6-overexpressing (OE) cells (scale bar: 5 μm). (B) Representative transmission electron microscopy images of mitochondrial ultrastructure in HCT116 control and SLC25A6-overexpressing (OE) cells (scale bar: 10 μm). (C) Representative immunoblot images of mitochondrial fission proteins DRP1 and MFF in control and SLC25A6-overexpressing (OE) cells. (D) Viability of SLC25A6-overexpressing (OE) cells treated with increasing concentrations of Mdivi. (E, F) Quantification of apoptosis (E) and ATP levels (F) in SLC25A6-overexpressing (OE) cells treated with solvent or Mdivi-1. (G) Representative immunoblot images of mitochondrial fission proteins in colorectal cancer cells cultured under glucose deprivation conditions (with or without SLC25A6 knockdown). Data are expressed as mean ± standard deviation (n = 3). The error bars represent the standard deviation. The graph shows the precise P-value.

[0036] Figure 5. SLC25A6 binds to MIC60 and disrupts the MIC60–MIC19 complex. (A–B) Immunoprecipitation-mass spectrometry analysis of the SLC25A6 interactome, showing enrichment of mitochondrial structural proteins and OXPHOS components. (C) Immunoprecipitation analysis confirming the interaction between SLC25A6 and MIC60. (D) Schematic diagram of SLC25A6 and MIC60 domain truncated mutants. (E–F) Immunoprecipitation of wild-type and truncated mutants, showing the interacting domains. (G) Immunoprecipitation of wild-type SLC25A6 and point mutants with MIC60. (H) Immunoprecipitation of wild-type and T126A mutant SLC25A6 at MIC60 and MIC19. (I) Representative Western blot images of mitochondrial splitting proteins in wild-type and T126A mutant SLC25A6 overexpression (OE) cells. (J) Measurement of cellular ATP levels in wild-type and T126A mutant SLC25A6 overexpression (OE) cells (n = 3). (K) Flow cytometry analysis of apoptosis in wild-type and T126A mutant SLC25A6 overexpression (OE) cells (n = 3). Data are expressed as mean ± standard deviation. Error bars represent standard deviation. Precise p-values ​​are shown in the figures.

[0037] Figure 6. The antitumor effect of glutaminase inhibitors enhancing SLC25A6 synergistically with Bcl-2 inhibition. (A, B) Comparison of SLC25A6 mRNA (A, n = 37 pairs) and protein levels (B, n = 12 pairs) in paired colorectal cancer tissues and adjacent normal tissues. (C) Comparison of SLC25A6 expression in colorectal cancer patients at different clinical stages (Stage I n = 8; Stage II n = 16; Stage III n = 28; Stage IV n = 4). (D) Kaplan-Meier survival analysis of colorectal cancer patients stratified by SLC25A6 expression. (E) Synergistic effect analysis of CB-839 and ABT-199 in HT29 cells (n = 3). (F–H) Representative images (F), tumor growth curves (G), and tumor weights (H) of xenograft tumors in mice treated with solvent, CB-839, ABT-199, or a combination thereof (n = 5 mice per group). (I) Immunohistochemical staining of Ki67, TUNEL, and SLC25A6 in xenograft tumors (n = 5; scale bar: 50 μm). Data are expressed as mean ± standard deviation. Error bars represent standard deviation. Precise P-values ​​are shown in the figure. Detailed Implementation

[0038] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0039] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0040] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0041] Example I. Materials and Methods reagents CB-839 (HY-12248), ABT-199 (HY-15531), oxaliplatin (HY-17371), 5-fluorouracil (HY-90006), Z-VAD-FMK (HY-16658B), and Mdivi (HY-15886) were purchased from MedChemExpress (Shanghai, China). Cell Counting Kit-8 (CK-04) and PK Mito Red (PKMR-1) were purchased from Dojindo (Kumamoto, Japan) and GenVivo (Shanghai, China), respectively.

[0042] Tissue specimens and cell lines Colorectal cancer and adjacent non-tumor tissue were obtained from surgical formalin-fixed paraffin-embedded specimens. All patients were admitted to our center between 2018 and 2019. None of the patients had received neoadjuvant therapy or had other malignancies. This study was approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences (Approval No.: 21 / 105-2776) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients before sample collection.

[0043] Human colorectal cancer cell lines (RKO, DLD1, HCT116, HT29, SW480, and SW620) were purchased from the American Type Culture Collection (Manassas, Virginia, USA). Cells were cultured in DMEM medium (HyClone, Logan, Utah, USA) containing 10% fetal bovine serum (Cell Technologies, Beijing, China) and 1% penicillin / streptomycin (Gibco, Carlsbad, California, USA) and maintained in a humidified incubator at 37°C and 5% CO2.

[0044] Transcriptome sequencing and data analysis Total RNA was extracted from glucose-deprived or CB-839-treated HCT116 cells using TRIzol reagent (Invitrogen, Carlsbad, California, USA). RNA quality and integrity were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA), and samples with an RNA integrity score ≥7.0 were used for subsequent analysis. Sequencing libraries were constructed using the Hieff NGS Ultima Dual-Mode mRNA Library Construction Kit (13335ES, Yeasen Biotechnology, Shanghai, China) and sequenced on the Illumina platform. Bioinformatics analysis of the raw data was performed using the BMKCloud platform (www.biocloud.net). Bioinformatics Analysis We analyzed the expression levels of ANT family members (SLC25A4, SLC25A5, SLC25A6, and SLC25A31) and compared their expression profiles using RNA sequencing data from colorectal adenocarcinoma and rectal adenocarcinoma downloaded from the Cancer Genome Atlas database.

[0045] siRNA transfection The siRNA targeting SLC25A6 was designed and synthesized by SinoGenoMax (Beijing, China). A non-targeted, scrambled siRNA with no homology to any known human gene sequence was used as a negative control (siNC, SinoGenoMax). Transfection was performed using JetPRIME reagent (PolyPlus, Ilkisch, France) according to the manufacturer's instructions. RNA and protein were extracted 48 hours after transfection. The siRNA sequences are as follows: siSLC25A6-1, sense strand: CCUUCGCCAAAGACUUCUUTT (SEQ ID NO.1), antisense strand: AAGAAGUCUUUGGCGAAGGTT (SEQ ID NO.2); siSLC25A6-2, sense strand: GAGACUGCCUGGUGAAGAUTT (SEQ ID NO.3), antisense strand: AUCUUCACCAGGCAGUCUCTT (SEQ ID NO.4).

[0046] Plasmids and the establishment of stable cell lines Expression plasmids (pcDNA3.1-3×FLAG, pcDNA3.1-SLC25A6-3×FLAG, deletion and mutant constructs, and MIC60 plasmid) and lentiviral vectors (pLVX-IRES-Neo-3×FLAG, pLVX-IRES-Neo-3×FLAG-SLC25A6, PLKO.1-EGFP-PURO-shNC, and PLKO.1-EGFP-PURO-shSLC25A6, where the short hairpin RNA shSLC25A6 sequence is derived from the siSLC25A6-2 sequence, with the loop selected as TTCAAGAGA and the terminator using poly-T, i.e., TTTTT, which is the RNA transcription termination signal) were synthesized by Mailgene Biosciences (Beijing, China) and confirmed by sequencing. The plasmid encoding C3AI was kindly provided by Professor Binghui Li. Lentiviral cells were produced by co-transfecting HEK-293FT cells with the transfer vector and packaging plasmids pMD2.G and psPAX.2. Colorectal cancer cells were infected with HiTransGP reagent (GeneChem, Suzhou, China) and stable cell lines were established by selection with puromycin.

[0047] Reverse transcription PCR and quantitative PCR Total RNA was extracted from colorectal cancer cells and tissues using an RNA purification kit (Vazyme Biotech, Nanjing, China). cDNA was synthesized using the HiScript III All-in-One RT SuperMix (Vazyme). The cDNA products were subjected to 40 cycles of PCR. Quantitative PCR was performed using a TB Green Premix Ex Taq (RR420A; Takara Bio, Kusatsu, Japan) on an ABI QuantStudio DX system (Applied Biosystems, Foster City, California, USA). 18S rRNA was used as an internal control, and 2... - The relative mRNA expression level was calculated using the ΔΔCt method.

[0048] IC 50 Measurement Colorectal cancer cells (5 × 10³ cells per well) were seeded in 96-well plates, and cell confluence (%) was monitored every 12 hours using an IncuCyte live cell analysis system (Sartorius, Göttingen, Germany). Dose-response curves were fitted using nonlinear regression (logarithmic [inhibitor] vs. response, variable slope) with GraphPad software (San Diego, California, USA), and IC50 was calculated. 50 value.

[0049] Cell viability assay Colorectal cancer cells (5 × 10³ cells per well) were seeded in 96-well plates, and cell confluence (%) was monitored every 12 hours using the IncuCyte live cell analysis system (Sartorius).

[0050] Immunohistochemistry Paraffin-embedded sections were incubated overnight at 4°C with primary antibody, followed by staining using a Mouse / Rabbit Enhanced Polymer System (PV-9000, ZSGB-BIO, Beijing, China) and DAB (ZLI-9017, ZSGB-BIO). Tissue microarrays were scanned using a NanoZoomer digital scanner (Hamamatsu Photonics, Japan). Immunoreactivity scores were calculated as intensity (0–3) × percentage of positive cells (0–3).

[0051] Mitochondria separated from cytoplasmic components Mitochondrial and cytoplasmic components were separated from the cell pellet using a mitochondrial separation kit (Beyotime, Shanghai, China) following the manufacturer's instructions.

[0052] ATP assay Cellular ATP levels were detected using the CellTiter-Glo luminescence assay (Promega, Madison, Wisconsin, USA). After treatment, CellTiter-Glo reagent was added to each well, and the luminescence signal was detected using a microplate reader.

[0053] Mitochondrial membrane potential measurement Mitochondrial membrane potential was assessed using JC-1 staining (Beyotime). Cells (1 × 10⁻⁶) were then stained with JC-1 stain (Beyotime). 6 (samples) were incubated with JC-1 at 37°C for 15 minutes and analyzed by flow cytometry. Red and green fluorescence were detected using PE and FITC channels, respectively.

[0054] mPTP Open Assessment Using Calcein-AM / Co ion (Co 2+ The mPTP opening was assessed using a quenching-based mPTP detection kit (Beyotime). Cells were co-incubated with Calcein-AM and a cobalt quencher, and residual fluorescence (negatively correlated with mPTP opening) was quantified by flow cytometry.

[0055] Apoptosis detection Apoptosis was detected using the Annexin V FITC / PI (AD10, Dojindo) or Annexin V / 7-AAD (4A Biotech, Shanghai, China) Apoptosis Detection Kit, according to the manufacturer's instructions, followed by flow cytometry analysis.

[0056] Transmission electron microscope Cells were fixed with 3% glutaraldehyde at 4°C for 2 hours or overnight, dehydrated by a gradient of ethanol and propylene oxide, and then embedded in epoxy resin. Ultrathin sections were prepared and observed using a TEM-1400 Plus electron microscope (Talos L120c, Thermo Fisher Scientific, Waltham, Massachusetts, USA).

[0057] Western blot Proteins were extracted using RIPA lysis buffer (C1053, Applygen Technologies, Beijing, China) containing a protease (B14001, Bimake, Shanghai, China) and a phosphatase inhibitor (B15001, Bimake). Concentrations were determined using a BCA protein quantification kit (23225, Thermo Fisher Scientific).

[0058] Immunoprecipitation and mass spectrometry analysis For exogenous immunoprecipitation, HCT116 or HT29 cells were transfected with FLAG-tagged plasmids. Lysis buffer was incubated overnight at 4°C with anti-FLAG magnetic beads (MedChemExpress). The complex was eluted by boiling and analyzed by Western blotting, or separated by SDS-PAGE and then submitted for mass spectrometry analysis (BIOMS, Shanghai, China). For endogenous immunoprecipitation, cell lysate was incubated with specific antibodies and Protein A / G magnetic beads (HY-K0202, MedChemExpress) for 2 hours, followed by overnight rotation at 4°C. The eluted complex was analyzed by Western blotting.

[0059] Molecular docking The 3D structures of MIC60 and ADT3 (SLC25A6) were obtained from the AlphaFold protein structure database (https: / / alphafold.ebi.ac.uk / ). Protein-protein docking was performed using the HDOCK server, and the resulting complexes were visualized using PyMOL (v2.6, Schrödinger, LLC, New York City, NY, USA).

[0060] Seahorse Metabolic Assay HCT116 or HT29 cells (1 × 10⁶ cells per well) 4 Cells were seeded into XF96 plates (Seahorse Bioscience, North Billrica, MA, USA) and cultured overnight. Oxygen consumption rate was measured using a Seahorse XFe-96 analyzer (Agilent Technologies, Santa Clara, CA, USA) after sequential injection of oligomycin (1.5 μM), FCCP (0.5 μM), and rotenone / antimycin A (0.5 μM each). Data were analyzed using WAVE software (Agilent Technologies) and normalized to cell number.

[0061] Fluorescence microscopy examination Cells were cultured on confocal culture dishes, washed with PBS, and stained with PK Mito according to the manufacturer's protocol. Images were acquired using a super-resolution microscope (DeltaVision OMX SR, GE Healthcare, Chicago, Illinois, USA).

[0062] Tumor xenotransplantation model Male BALB / c nude mice (6 weeks old) were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China) and housed under SPF conditions. After acclimatization, 2 × 10⁶ mice were introduced into the rearing system. 6 Mice were injected subcutaneously into the right flank using colorectal cancer cells. Mice were randomly assigned to four groups (n = 5 per group): a solvent control group; an ABT-199 group (50 mg / kg / day, intraperitoneal injection); a CB-839 group (200 mg / kg / day, oral gavage); and a combination therapy group. Tumor volume was measured every 3 days and calculated using the formula (length × width²) / 2. After 12 days, mice were sacrificed by CO2 asphyxiation followed by cervical dislocation to ensure death; the tumors were then dissected, weighed, and subjected to immunohistochemical analysis. All animal studies were approved by the Animal Center of the Cancer Hospital / National Cancer Center, Chinese Academy of Medical Sciences (Approval No.: NCC2021A264).

[0063] Statistical analysis All experiments were independently repeated at least three times. Statistical analysis was performed using GraphPad Prism (v9.0). The Shapiro-Wilk test was used to assess the normality of the data distribution, and the Levene test was used to assess homogeneity of variance. Unpaired Student's t-test was used for comparisons between two groups, and one-way or two-way ANOVA was used for comparisons among multiple groups, followed by Tukey's post-hoc test. Data are expressed as mean ± standard deviation. P < 0.05 was considered statistically significant.

[0064] II. Results SLC25A6-mediated apoptosis induced by glutamine metabolic stress in colorectal cancer cells To investigate the response of colorectal cancer (CRC) cells to glutamine metabolic stress, we first treated different CRC cell lines with glutamine deprivation (GD, i.e., cultured in a glutamine-free medium) or the glutaminase inhibitor CB-839, and selected the moderately sensitive HCT116 cells for subsequent studies. To monitor the process of single-cell apoptosis, we stably transfected HCT116 cells with the caspase-3 activity indicator (C3AI). Based on the C3AI fluorescence intensity, we divided the cells into C3AI-high (top 30%) and C3AI-low (bottom 30%) populations. Figure 1 A). As expected, the apoptosis rate of C3AI-high cells was significantly higher than that of C3AI-low cells, approximately four times higher. Figure 1B, C). RNA sequencing was then performed on both groups of cells, identifying 25 differentially regulated genes that were upregulated under different treatments (fold change > 1.5, P < 0.05), among which SLC25A6, GDF15, SLC25A4, DDIT3, and KRT80 were most significantly upregulated. Figure 1 D). DDIT3 is known to be a key regulator of GD-induced cell death, validating the reliability of the experimental strategy. Notably, the above-mentioned genes include ANT family members SLC25A6 and SLC25A4. qPCR validation showed that SLC25A6 was significantly upregulated in C3AI-high cells ( Figure 1 E, F). Given that its baseline expression is high in both CRC cells and clinical samples ( Figure 1 (E, F) We selected SLC25A6 for in-depth study. The results showed that SLC25A6 overexpression significantly enhanced caspase-3 activation ( Figure 1 G), while knockdown (KD) significantly weakened GD-induced apoptosis (G), Figure 1 These results indicate that SLC25A6 is a key mediator of glutamine metabolic stress-induced apoptosis in HCT116 cells.

[0065] SLC25A6 inhibits CRC progression in vivo and in vitro by inducing apoptosis. qPCR and Western blot analysis showed that SLC25A6 expression was differentially expressed in various CRC cell lines. We established transient overexpression (OE) and knockdown (KD) models in different cell lines. Functional experiments showed that SLC25A6 overexpression significantly inhibited cell growth, while KD promoted cell proliferation. Figure 2 (A–D). Flow cytometry analysis showed that SLC25A6 overexpression significantly increased the apoptosis rate, and this could be reversed in a dose-dependent manner by the broad-spectrum caspase inhibitor Z-VAD.

[0066] In a mouse subcutaneous tumorigenesis model, stable overexpression of SLC25A6 significantly inhibited tumor growth, as evidenced by a reduction in tumor volume and weight. Figure 2 F–H); conversely, stable knockdown of SLC25A6 promotes tumor growth (F–H); Figure 2 J–L). IHC results showed that the number of TUNEL-positive cells in tumors was significantly increased in the SLC25A6 overexpression group, while it was decreased in the KD group (J–L). Figure 2 These results indicate that SLC25A6 exerts a tumor-suppressive effect in CRC by promoting apoptosis.

[0067] SLC25A6 induces mitochondrial dysfunction but does not increase mPTP opening. To elucidate its pro-apoptotic mechanism, we examined key apoptosis molecules. SLC25A6 overexpression upregulated cleaved caspase-9 and caspase-3 (…). Figure 3 A), mitochondrial Bax / Bak levels are elevated, and Bcl-2 expression is decreased ( ). Figure 3 (A, B) This suggests that the endogenous apoptosis pathway is activated. Functional assays showed that SLC25A6 overexpression led to a decrease in mitochondrial membrane potential (JC-1 experiment), reduced ATP production, and decreased NAD+. + The NADH ratio decreased and the oxygen consumption rate decreased. Figure 3 D–F). However, mPTP opening analysis showed a slight increase in fluorescence, suggesting a slight decrease in opening (D–F). Figure 3 G). Furthermore, SLC25A6 enhances the sensitivity of cells to mitochondrial-targeted apoptotic stimuli (such as the Bcl-2 inhibitors ABT-199 and staurosporine). Figure 3 SLC25A6 induces endogenous apoptosis through atypical mechanisms, but has no significant effect on conventional chemotherapy drugs (5-FU, oxaliplatin).

[0068] SLC25A6 mediates GD-related mitochondrial fragmentation by promoting mitochondrial division. Immunofluorescence showed that SLC25A6 overexpression caused mitochondria to change from a reticular structure to a fragmented, dotted morphology. Figure 4 A), TEM showed cristae structure disruption and characteristic "donut-shaped" mitochondria formation ( Figure 4 B). Conversely, mitochondria in KD cells are enlarged and cristae intact. Western blot showed that SLC25A6 selectively upregulated the mitochondrial splitting proteins DRP1 and MFF, while fusion and biosynthesis-related proteins showed no significant changes. Figure 4 C). The cell division inhibitor Mdivi-1 can dose-dependently reverse SLC25A6-induced growth inhibition and apoptosis. Figure 4 D–E) suggests that abnormal cell division is involved in its function. Further experiments found that GD also induces upregulation of mitochondrial fragmentation and cell division markers, while SLC25A6 knockdown significantly alleviated this effect. Figure 4 (G), indicating that SLC25A6 mediates GD-induced mitochondrial division.

[0069] SLC25A6 promotes mitochondrial division by disrupting the MIC60–MIC19 interaction. Immunoprecipitation-mass spectrometry analysis revealed that SLC25A6 primarily interacts with mitochondrial structural regulatory proteins (MIC60, PHB, PHB2) and OXPHOS components (NDUFS2, NDUFS3, UQCRC2). Figure 5A, B). This further verifies the direct combination of SLC25A6 and MIC60 ( Figure 5 C). Truncation mutation experiments showed that deletion of the second domain (amino acids 110–211) of SLC25A6 or deletion of the N-terminus of MIC60 both disrupted their binding. Figure 5 EF). Molecular docking results indicate that the T126 residue of SLC25A6 is a key binding site; mutation of T126A prevents binding to MIC60 (EF). Figure 5 G).

[0070] MIC60–MIC19 binding is crucial for the stability of the MICOS complex (20, 21). Experiments showed that SLC25A6 overexpression weakens MIC60–MIC19 binding, while the T126A mutation loses this effect and no longer induces mitochondrial division, ATP depletion, or apoptosis. Figure 5 H–K). This indicates that SLC25A6 binds to MIC60 through the T126 residue and disrupts the MIC60-MIC19 interaction, thereby inducing mitochondrial structural disorder and apoptosis.

[0071] Low expression of SLC25A6 is associated with poor prognosis in colorectal cancer. In CRC clinical samples, SLC25A6 mRNA and protein levels were significantly downregulated (reduced in 70% and 75% of tumor tissues, respectively, compared to adjacent tissues). Figure 6 A, B). Its expression gradually decreases with the progression of clinical stage, and is lowest in poorly differentiated tumors (A, B). Figure 6 C). In a cohort of 163 CRC patients, low SLC25A6 expression was significantly associated with poor prognosis (C). Figure 6 (D), suggesting that it has potential prognostic biomarker value.

[0072] The combined effect of glutamine metabolism inhibition and Bcl-2 blockade produces a synergistic antitumor effect. Because inhibition of glutamine metabolism can upregulate SLC25A6 expression ( Figure 1 We hypothesize that the glutaminase inhibitor CB-839 and the Bcl-2 inhibitor ABT-199 may have a synergistic effect. In vitro experiments showed that the combination of these two inhibitors significantly enhanced the antitumor effects of various CRC cell lines (HT29 synergistic score 18, HCT116 12.48). Figure 6 E). In an in vivo xenograft model, the combination therapy significantly inhibited tumor growth and reduced tumor weight (E). Figure 6 F–H). IHC showed that CB-839 alone upregulated SLC25A6, while the combination group had the lowest Ki67 and the highest TUNEL positivity (F–H). Figure 6I). No significant toxic changes were observed in mouse body weight and major organs, indicating that this strategy has good safety. Overall results show that the combination of GD and Bcl-2 inhibition significantly inhibited CRC growth by enhancing apoptosis.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of reagents for detecting the expression level of SLC25A6 gene and its expression products in the preparation of colorectal cancer detection products.

2. The application as described in claim 1, characterized in that, The colorectal cancer detection includes prognostic assessment of colorectal cancer.

3. The application as described in claim 2, characterized in that, The expression levels of the SLC25A6 gene and its expression products were negatively correlated with poor prognosis. The prognostic assessment includes analysis of the clinical stage of colorectal cancer (including stage I, II, III and IV) and analysis of progression-free survival in colorectal cancer.

4. A system for detecting colorectal cancer, characterized in that, The system includes: i) An analysis module, the analysis module comprising: a detection substance selected from the expression level of SLC15A6 in the test sample of the subject; ii) An assessment module, the analysis module comprising: detecting the subject based on the SLC15A6 expression level determined in i).

5. The system as described in claim 4, characterized in that, The subjects were colorectal cancer patients, and the samples to be tested were colorectal cancer samples, specifically colorectal cancer tissue or cells.

6. The application of substances that promote the expression of the SLC25A6 encoding gene and its expression products and / or enhance their activity in at least one of the following (a1)-(a5); (a1) Prepare products that promote tumor cell apoptosis; (a2) To prepare products that inhibit tumor growth; (a3) Prepare products that promote the division of tumor cell mitochondria; (a4) To prepare products that enhance the antitumor activity of Bcl-2 inhibitors; (a5) Cancer treatment or preparation of cancer treatment products.

7. The application as described in claim 6, characterized in that, The tumor is a solid tumor, specifically colorectal cancer; The product is a drug or a test reagent for non-medical purposes, and the test reagent is used for basic research on colorectal cancer.

8. The application as described in claim 6, characterized in that, The substance that promotes the expression of the SLC25A6 encoding gene and its expression products and / or increases its activity is a glutaminase inhibitor, and may further be CB-839; The Bcl-2 inhibitor is ABT-199.

9. A composition, characterized in that, The active ingredient of the composition includes at least a substance that inhibits and / or enhances the expression of the SLC25A6 encoding gene and its expression products, as well as a Bcl-2 inhibitor. The substance that promotes the expression of the SLC25A6 encoding gene and its expression products and / or increases its activity is a glutaminase inhibitor, and may further be CB-839; The Bcl-2 inhibitor is ABT-199; The molar ratio of the two is 1-1000:10-10000, and further to 10-100:1-100.

10. Use of the composition of claim 9 in the preparation of a medicament for treating colorectal cancer.

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