Application of rifamycin in the treatment of colorectal cancer

Abstract

This invention relates to novel uses of pharmaceuticals, particularly the use of rifamycin in the preparation of medicaments for treating colorectal cancer. The application described herein involves rifamycin stabilizing the binding of DHRS4 and LONP2 within the mitochondria of colorectal cancer cells, promoting TFAM degradation, thereby impairing mitochondrial biogenesis and function, and inhibiting mitochondrial activity. In this application, rifamycin is either the sole active ingredient or used in combination with other drugs.

Description

Technical Field

[0001] This invention relates to novel uses of pharmaceuticals, and particularly to the use of rifamycin in the treatment of colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is the second leading cause of cancer death worldwide, and patients are usually diagnosed at an advanced stage. Once CRC has metastasized, it is inoperable, and the main treatment relies on combination chemotherapy, with the addition of targeted drugs based on the identified molecular subtype. Despite these interventions, long-term cancer treatment outcomes remain poor. The metabolic profile of CRC is complex, typically marked by upregulated and enhanced OXPHOS, and current research has shown that OXPHOS inhibition can strongly suppress glycolytic activity and the proliferation of glycolytically deficient CRC cells, as well as CRC tumorigenesis. However, the underlying regulatory mechanisms of mitochondrial metabolism in CRC remain unclear.

[0003] As a key regulator of mitochondrial biology, mitochondrial transcription factor A (TFAM) initiates mitochondrial DNA (mtDNA) replication and maintains mtDNA copy number. This ensures the expression of 13 key mtDNA-coding subunits of the mitochondrial respiratory chain complex, which assemble with nuclear-coding subunits to form OXPHOS complexes I, III, IV, and V. In early-stage CRC, TFAM expression is upregulated with increasing mtDNA copy number. Furthermore, higher mtDNA levels in tumor tissue are significantly associated with clinicopathological features, including larger tumor size, advanced lymph node metastasis, elevated serum carcinoembryonic antigen (CEA) levels, vascular embolism, and liver metastasis. Previous studies have reported aberrant TFAM expression in CRC cells, and its high expression is a representative marker of tumor development in CRC patients.

[0004] Mitochondrial proteomic homeostasis depends on the ATP-dependent proteolytic activity of ionic proteases. The mitochondrial ionic protease LONP1 is an enzyme primarily targeting proteins such as TFAM, which are involved in key metabolic pathways, including the TCA cycle and OXPHOS, thereby maintaining mitochondrial homeostasis. Interestingly, in addition to the mitochondrial isoenzyme LONP1, the Lon peroxidase isoenzyme LONP2 is also present. The 39.6% amino acid identity between LONP2 and LONP1, coupled with the conservation of the ATPase domain, suggests that LONP2 may have similar functions and subcellular localization within mitochondria as LONP1. However, to date, the expression pattern, regulatory mechanisms, and clinical significance of LONP2 in human CRC have not been investigated.

[0005] In summary, this invention utilizes multi-omics analysis, including proteomics and metabolomics, to explore the roles of potential candidate regulators of mitochondrial metabolism.

[0006] Rifamycin is a general term for rifamycin antibiotics. It has broad-spectrum antibacterial activity, showing strong activity against Gram-positive bacteria such as Mycobacterium tuberculosis, Mycobacterium leprae, Streptococcus, and Pneumococcus, especially drug-resistant Staphylococcus aureus, but its activity against Gram-negative bacteria is weaker. Naturally occurring rifamycin antibiotics were initially isolated in Italy. Rifamycin B, in particular, has strong activity and is relatively stable, but its clinical efficacy is not ideal. Later, through various chemical modifications to naturally occurring rifamycin, a series of more potent antibiotics were obtained, known as semi-synthetic rifamycins. For example, rifamycin produced in 1961 was first used clinically. In 1962, rifamycin B diacetamide was developed, possessing better antibacterial efficacy and lower toxicity, replacing rifamycin SV. Rifampin is the most effective and widely used of these antibiotics, not only with a broad antibacterial spectrum, applicable to various bacterial infections, but also without cross-resistance with other drugs. Its efficacy against tuberculosis is particularly outstanding, making it a first-line drug for treating tuberculosis.

[0007] Rifamycin drugs include rifamycin B diacetamide and rifampin. Currently, rifampin, rifapentine, and rifabutin are used clinically.

[0008] This invention uses rifamycin as a potential effective drug component for the treatment of CRC, and explores its effects on DHRS4 and LONP2 proteins in mitochondria, providing valuable insights into the tumor-suppressive effects of rifamycin. Summary of the Invention

[0009] The purpose of this invention is to provide the application of rifamycin in the treatment of colorectal cancer. By applying rifamycin to inhibit mitochondrial activity in CRC cells, CRC growth is inhibited, providing a new method and means for the treatment of CRC.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides the use of rifamycin in the preparation of drugs for treating colorectal cancer.

[0011] The application described in this invention is that rifamycin is used to stabilize the binding of DHRS4 and LONP2 in the mitochondria of colorectal cancer cells, promote TFAM degradation, thereby leading to impaired mitochondrial biogenesis and function, and inhibiting mitochondrial activity.

[0012] The application described in this invention, wherein rifamycin is the sole active ingredient or is used in combination with other drugs.

[0013] The application described in this invention is used to inhibit the proliferation of colorectal cancer cells.

[0014] The application described in this invention is used to enhance the interaction between LONP2 and DHRS4 and TFAM.

[0015] Preferably, the application described in this invention is used to downregulate the levels of MTCO1, MTCO2, and TFAM, inhibit OXPHOS protein and its related pathways, and improve intestinal tumor burden.

[0016] The application described in this invention, wherein the drug comprises rifamycin and medically approved excipients.

[0017] The drug includes various acceptable dosage forms, including injections, pills, capsules, granules, tablets, or oral solutions.

[0018] In the application described in this invention, rifamycin is selected from rifampin, rifapentine, and rifabutin.

[0019] The rifamycin used in this invention has the molecular structure C 37 H 45 NO 12 Its chemical name is (2S,12Z,14E,16S,17S,18R,19R,20R,21S,22R,23S,24E)-5,17,19-trihydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-1,6,9,11-tetraoxo-1,2,6,9-tetrahydro-2,7-(epoxypentadecano[1,11,13]trieneimino)naphtho[2,1-b]furan-21-yl acetate. It is derived from natural fermentation and simple chemical conversion, and its purity is 98.74%.

[0020] The beneficial effects achieved by this invention are as follows: This invention evaluated the inhibitory effect of rifamycin on mitochondrial activity in CRC cells through in vitro cell experiments, in vivo subcutaneous transplantation mouse tumor models, chemically induced colitis-associated colon cancer (CAC) mouse models, and multi-omics analysis. It was found that rifamycin can stabilize the DHRS4-LONP2-TFAM complex in vivo and in vitro, thereby promoting TFAM degradation, inhibiting mitochondrial biogeneration, and thus inhibiting tumor cell growth, achieving the purpose of treating CRC. Attached Figure Description

[0021] Figure 1 shows the protein molecule docking results in this invention (A is the screening results from the natural protein library; B is the structural domain of rifampicin docking with LONP2 and DHRS4 proteins). Figure 2The results of the immunoprecipitation after rifamycin treatment in this invention are as follows (A is the immunoprecipitation result of DHRS4 with LONP2 and TFAM in SW620 cells (n=3); B is the in vitro pull-down analysis result using purified DHRS4 protein (n=3); *P<0.05, **P<0.01, compared with the control group). Figure 3 This is the PLA detection result of LoVo cells after rifamycin treatment in this invention (A shows the interaction between DHRS4-LONP2 and TFAM-LONP2 in LoVo cells and their control group; B shows the quantitative statistical results; red dots represent PLA signals; the blue stained part of DAPI represents the cell nucleus (n=100); ****P<0.0001, intergroup comparison). Figure 4 The results of multi-omics analysis of SW620 cells after rifamycin treatment in this invention are shown in A (enrichment analysis of proteomic maps; bar chart of differentially expressed metabolites). Figure 5 The results are immunoblotting of some proteins in HT29 and SW620 cells and their control group after rifamycin treatment in this invention (A is the immunoblotting analysis results of TFAM, MTCO2 and mitochondrial complex subunits UQCRC2, SDHB, and NDUFB8; B is the quantitative statistical results (n=3); *P<0.05, **P<0.01, ***P<0.001, compared with the control group). Figure 6 The results of observation by laser confocal microscopy after fluorescence staining of SW620 and HT29 cells and their control group after rifamycin treatment in this invention are shown in Figure A (mitochondrial staining results in SW620 and HT29 cells; Figure B (quantitative statistical results, n=100); P < 0.0001, compared with the control group). Figure 7 The results of mitochondrial quality or function detection in HT29 and SW620 cells and their control group after rifamycin treatment in this invention are shown in Figure A (flow cytometry analysis results of mitochondria in HT29 and SW620 cells; Figure B (quantitative statistical results of mitochondrial fluorescence intensity in HT29 and SW620 cells (n=3); P < 0.0001, intergroup comparison). Figure 8The results of respiration rate-related indicators of HT29 and SW620 cells and their control group after rifamycin treatment in this invention are as follows: (A is the statistical results of oxygen consumption rate, basal respiration rate, maximum respiration rate, ATP production rate, and residual respiration capacity of HT29 cells; B is the statistical results of oxygen consumption rate, basal respiration rate, maximum respiration rate, ATP production rate, and residual respiration capacity of SW620 cells; ***P<0.001, ****P<0.0001, compared with the control group). Figure 9 The results show the proliferation of SW620 and HT29 cells and their control group after rifamycin treatment in this invention (A is the cell viability of SW620 and HT29 cells; B is the crystal violet staining results of SW620 and HT29 cells). Figure 10 The results of cell proliferation after rifamycin treatment in this invention are shown in Figure A (cell viability of DLD-1 and SW620 under different treatments; crystal violet staining results of DLD-1 and SW620 cells). Figure 11 The results are flow cytometry apoptosis analysis of SW620 and HT29 cells after rifamycin treatment in this invention (n=3).

[0022] Figure 12 This describes the tumor growth in mice with subcutaneous tumor transplantation after injection of rifamycin (A is a photograph; B is the statistical results of tumor tissue volume and weight (n=8, n=6); ***P<0.001, ****P<0.0001, compared with the control group). Figure 13 These are the proteomic analysis results of tumor tissues obtained from mice with subcutaneous tumor transplantation in this invention after injection of rifampicin (A is a heatmap of mitochondrial-related protein expression levels; B is a diagram of mitochondrial and oxidative phosphorylation-related pathways). Figure 14 The results of immunofluorescence staining of related proteins in tumor tissue obtained from mice with subcutaneous tumor transplantation in this invention after injection of rifamycin are shown in Figure A (immunofluorescence staining results of TFAM, MTCO1, and MTCO2 in tumor tissue; Figure B (quantitative statistical results (n=6)); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the control group). Figure 15 The results of immunoprecipitation analysis of LONP2 and DHRS4, and LONP2 and TFAM in tumor tissue of mice with subcutaneous transplanted tumors in this invention are shown in Figure A (three immunoprecipitations were performed on tumor tissue from three mice; Figure B is the quantitative statistical result (n=3); ****P<0.0001, compared with the control group). Figure 16 These are the results of rifampicin-treated human CRC organoids in this invention (A: bright-field observation of human CRC organoids; B: cell viability of human CRC organoids; C: average length of human CRC organoids). Figure 17 The results are immunofluorescence staining of TFAM and MTCO2 in human CRC organoids after rifamycin treatment in this invention (A is the staining result; B is the quantitative statistical result; ****P<0.0001, compared with the control group).

[0023] Figure 18 This invention describes the intestinal tumor status in a CAC mouse model after rifamycin treatment (A is a photograph, B is the statistical results of tumor tissue quantity and survival rate (n=6); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, intergroup comparison). Figure 19 This image shows the immunofluorescence staining results of mitochondrial-associated proteins MTCO1, MTCO2, and TFAM in CAC mouse intestinal tumor tissue after rifamycin treatment in this invention (A is the staining result; B is the quantitative statistical result; *P<0.05, **P<0.01, ****P<0.0001, compared with the control group). Figure 20 This is a diagram showing the H&E staining results of the heart, liver, spleen, lungs, and kidneys of CAC model mice after rifamycin treatment in this invention (n=6). Detailed Implementation Example

[0024] 1 Experimental Methods 1.1 Protein structure prediction and molecular docking The structures of human DHRS4 and LONP2 proteins were obtained from a protein database (PDB code: 5oji). Protein-protein docking simulations were performed using the ClusPro 2.0 web server, and the obtained docking structures were analyzed using the PyMol molecular graphics system developed by schrdinger, LLC.

[0025] The structure of human DHRS4 was used as the acceptor in molecular docking experiments. The acceptor and drug structure files were converted to pdbqt format using the prepare_receptor4.py and prepare_ligand4.py tools of the AutoDockTools4 program. The AutoDock Vina 1.5.6 program was used for protein-ligand molecular docking simulations. Molecular docking simulations were performed for each chemical molecule combination, including 10 docking runs, followed by further investigation of the optimal match. The binding fraction of a standard drug greater than or equal to -7.0 kcal / mol was set as a threshold. Additionally, molecular docking was performed with LONP2 as the acceptor, and the results showed crossover with previous docking results. Subsequently, interaction analysis was performed on the obtained compounds using BIOVIA Discovery Studio. Hydrogen bonds, hydrophobic contacts, and other non-covalent interactions were identified in each ligand. Molecular visualizations were generated using the PyMol molecular graphics system to observe specific amino acid residues and ligand atoms involved in the interactions, which helped interpret their binding modes.

[0026] 1.2 Cell Culture Process HT29 and SW620 CRC cells were cultured and seeded into wells for various assays. After cell attachment, rifamycin was added for 12 hours. The treated HT29 and SW620 cells were then used in various assays. The rifamycin concentrations were 0, 2.5, and 5 µM, with 0 µM serving as the control.

[0027] Cell culture: Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 humidified incubator. For passage culture, cells were observed under an inverted microscope and passaged when 70-80% confluence was reached. The medium was aspirated, and the cell monolayer was washed twice with phosphate-buffered saline (PBS). Cells were digested and isolated by incubation at 37°C with 0.25% trypsin solution. Digestion was terminated by adding complete medium containing 10% FBS during cell isolation. The cell suspension was collected, centrifuged at 1000 × g for 5 min, and the supernatant was aspirated. The pellet was resuspended in 3 mL of fresh complete medium. For T25 culture flasks, 1 mL of cell suspension was added to 3 mL of preheated medium and mixed well.

[0028] Cell cryopreservation: For cryopreservation, trypsinize and centrifuge the cells as described above. Resuspend the cell pellet in 1 mL of serum-free cryopreservation medium. Transfer the suspension to cryovials, label with cell line identifier and date, and store at -80°C.

[0029] Cell thawing: Thaw the cryovials rapidly with gentle agitation in a 37°C water bath. Aseptically transfer the cell suspension to a 15 mL centrifuge tube containing 2 mL of preheated complete culture medium and centrifuge at 1000 × g for 5 minutes to remove the cryopreservation solution. Aspirate the supernatant and resuspend the cell pellet in 2 mL of fresh culture medium. Transfer the suspension to a culture flask containing an additional 2 mL of culture medium and incubate under standard culture conditions.

[0030] 1.3 Immunoprecipitation For cells: Four SW620 / HT29 cells grown to confluence in four 10 cm culture dishes were washed twice with ice-cold PBS and lysed in 250 μL of lysis buffer containing protease and phosphatase inhibitors. The lysates were centrifuged at 14000 × g for 15 min, and 100 μL of supernatant was reserved. The remaining lysates were aliquoted into two 450 μL aliquots and incubated overnight at 4°C with control IgG or anti-LONP2 antibody. Protein A / G agarose beads (30 μL) were added to each sample and incubated at 4°C for 4 h. The beads were precipitated by centrifugation at 2000 × g for 5 min, washed five times with wash buffer, and resuspended in 40 μL of IP lysis buffer and 10 μL of loading buffer. After heating at 95°C for 5 min, the samples were centrifuged, and the supernatant was analyzed by Western blotting.

[0031] Tumor tissue from mice with subcutaneously transplanted tumors: The extracted subcutaneous xenograft tissue from mice was thoroughly cleaned, homogenized, and lysed in lysis buffer containing protease and phosphatase inhibitors. The lysate was centrifuged at 14000 × g for 20 min, and 50 μL of the supernatant was reserved as input. The remaining lysate was divided into two aliquots and incubated overnight at 4°C with control IgG or anti-LONP2 antibody. Protein A / G agarose beads (30 μL) were added to each sample and incubated at 4°C for 4 h. The beads were precipitated by centrifugation at 2000 × g for 5 min, washed five times with wash buffer, and resuspended in 40 μL of IP lysis buffer and 10 μL of loading buffer. After heating at 95°C for 5 min, the samples were centrifuged, and the supernatant was analyzed by Western blotting.

[0032] 1.4 Protein purification drop-down analysis Purification of recombinant protein: Transfected 293T cell pellet was sonicated, dissolved in buffer, and the supernatant was separated by high-speed centrifugation. A STarm streptoin bead 4FF column was packed with 10 volumes of 1×PBS (pH 7.4). After loading the clarified lysate, the resin was washed, and bound proteins were eluted with buffer containing 250 mM imidazole and 0.3 M NaCl (pH 8.0). Five eluents were collected (one column volume of each). Protein purity and concentration were determined by SDS-PAGE, and endotoxin levels were routinely monitored throughout the process.

[0033] Protein blotting: 1 μg each of purified DHRS4 and LONP2 proteins were mixed in 300 μL of lysis buffer and incubated overnight at 4°C. The mixture was then incubated with 3 μg of DHRS4 antibody at 4°C for 4 hours, followed by the addition of 30 μL of agarose beads and incubation for another 4 hours. After washing five times with wash buffer (2000 × g, 5 minutes each time), the agarose beads were resuspended in 40 μL of IP lysis buffer, mixed with 10 μL of loading buffer, heated at 95°C for 5 minutes, and then centrifuged. The supernatant was collected for Western blot analysis.

[0034] 1.5 Nearest Neighbor Analysis (PLA) The interaction between LONP2-TFAM and DHRS4-LONP2 proteins in LoVo cells was investigated. Protein-protein interactions were detected using the Duolink PLA kit according to the manufacturer's instructions. In short, cells grown on coverslips were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.3% Triton X-100. After blocking, cells were incubated overnight at 4°C with primary antibodies diluted in Duolink antibody dilution buffer.

[0035] The following day, the cells were incubated with species-matched PLA probes (Duolink anti-rabbit positive and anti-mouse negative) at 37°C for 2 hours. Ligation and amplification were performed according to the kit protocol. Then, coverslips were fixed onto slides using DAPI to observe the cell nuclei.

[0036] Images were acquired using a 63× oil immersion objective on a Zeiss LSM 980 confocal microscope. The images were converted to projections of maximum intensity. The PLA signal (red spots) in each cell was quantified using ImageJ software (version 2.10) in at least 100 cells under each condition.

[0037] 1.6 Proteomic Analysis Proteomic data included public datasets from CPTAC and self-tested data generated using data-independent acquisition (DIA). Samples consisted of rifamycin-treated SW620 cells and their control group, including three biological replicates. Proteins with missing values ​​greater than 60% were excluded, and remaining missing values ​​were imputed using half the mean abundance. Differentially expressed proteins were defined as those with |log2FC| > 1.2 and P < 0.05. Functional annotation was performed using GO analysis, and enrichment pathways were assessed using GSEA GO and GSEA KEGG to obtain a cyclic plot of enrichment analysis of the proteomic atlas of rifamycin-treated LoVo cells.

[0038] 1.7 Metabolomics Analysis Energy metabolites in drug-treated cell lines were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Data with missing values ​​greater than 60% were excluded, and the remaining missing values ​​were imputed using half the mean abundance. Differentially expressed metabolites were defined as metabolites with |log2FC|>1.2, P<0.05, and VIP value>1. A bar chart of differentially expressed metabolites in SW620 cells after rifamycin treatment was obtained.

[0039] 1.8 Immunoblot Analysis Protein lysates were extracted from rifamycin-treated HT29 and SW620 cells using RIPA lysis buffer (Beyotime). Protein concentrations were quantified using a BCA protein assay kit (Beyotime). Subsequently, equal volumes of protein lysates were denatured by boiling in SDS loading buffer for 5 minutes. The denatured samples were separated by electrophoresis on 10% or 12.5% ​​SDS-polyacrylamide gels and then transferred to nitrocellulose (NC) membranes (Cytiva) using a wet transfer system. The membranes were blocked with 5% skim milk powder for at least 1 hour at room temperature. After blocking, the membranes were incubated overnight at 4°C with a specific primary antibody, followed by incubation with a suitable horseradish peroxidase (HRP)-conjugated secondary antibody. Protein bands were observed using enhanced chemiluminescence (ECL) substrates, and band grayscale values ​​were quantified using ImageJ software.

[0040] 1.9 Immunofluorescence staining of paraffin sections First, dewaxing and rehydration were performed: sequential treatment with xylene I, II, and III (10 minutes each), followed by gradient hydration with 100%, 95%, 80%, and 75% ethanol (5 minutes each), and washing three times with PBS. Then, endogenous peroxidase was used for blocking for 10 minutes, followed by three washes with PBS. Next, antigen retrieval was performed using EDTA antigen retrieval solution preheated to 100°C for 3 minutes, maintained at 30°C for 15 minutes, cooled to room temperature, and washed three times with PBS. Blocking with 5% BSA at room temperature for 1 hour was then performed. Incubation with primary antibody (diluted with 5% BSA) was then carried out overnight at 4°C. After removal, equilibration was allowed at room temperature for 15 minutes, followed by three washes with PBS (5 minutes each). Fluorescent secondary antibody was added, and incubation was carried out at room temperature in the dark for 1 hour, followed by three washes with PBS (5 minutes each). Finally, the slides were mounted with DAPI-containing mounting medium and observed under a Zeiss LSM 980 laser scanning confocal microscope with 20× objectives.

[0041] 1.10 Confocal Microscope To observe mitochondria, HT29 and SW620 cells grown on coverslips were fluorescently stained with 100 nM MitoTracker Red CMXRos at 37°C for 30 min after rifamycin treatment. Following staining, the cells were washed twice with pre-warmed PBS and fixed with pre-warmed 4% paraformaldehyde for 15 min. The coverslips were then stored overnight in PBS at 4°C. Finally, the coverslips were mounted with a fluorescent mounting medium containing DAPI to counterstain the cell nuclei. Fluorescence images were obtained using a Zeiss LSM 980 laser scanning confocal microscope with a 63× oil immersion objective. MitoTracker Red and DAPI were excited at wavelengths of 579 nm and 405 nm, respectively. Image analysis and quantification were performed using Zeiss ZEN 3.9 software. All immunofluorescence stained samples were observed and photographed using a confocal microscope.

[0042] 1.11 Flow cytometry analysis Mitotracker Red was used to assess mitochondrial quality or function. Briefly, cells were stained with 100 nM MitoTracker Red at 37°C for 30 minutes. The mean fluorescence intensity (MFI) of the stained mitochondria was analyzed by flow cytometry to ensure the accuracy of single-cell mitochondrial assessment.

[0043] Apoptosis was assessed by annexin V-FITC / propidium iodide (PI) staining. Cells were harvested after 12 hours of rifamycin treatment, washed with PBS, and resuspended in 500 μL binding buffer. The cell suspension was then stained with 5 μL annexin V-FITC and 10 μL PI in the dark at room temperature for 5 minutes. The percentage of apoptotic cells was immediately quantified by flow cytometry. Data from both analyses were analyzed using FlowJo v.10 software.

[0044] 1.12 Hippocampal analysis to assess mitochondrial respiration rate Mitochondrial respiration was assessed by measuring oxygen consumption (OCR) using an XFe96 extracellular flow analyzer (Agilent Technologies) and a hippocampus XF Mitochondrial pressure test kit (Agilent, #103015-100). The XFe96 sensor kit was hydrated overnight at 37°C with 200 μL of hippocampus XF calibrator per well in a non-CO2 incubator. Rifampicin-treated CRC cells (HT29 and SW620) were seeded at a density of 10,000 cells per well in XF96 microplates and incubated overnight. One hour prior to assay, the growth medium was replaced with hippocampus XF DMEM medium (pH 7.4) supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate, and the cells were cultured at 37°C in a non-CO2 incubator for 1 h. Mitochondrial stress assays were performed by sequentially injecting mitochondrial inhibitors from ports A, B, and C of the sensor housing to achieve the following final concentrations: 1.5 μM oligomycin, 2 μM FCCP, and 1 μM rotenone / antimycin A. OCR measurements were performed at baseline and after each injection. Data were normalized to the number of cells per well.

[0045] 1.13 Plasmid and shRNA transfection plasmid transfection When the rifampicin-treated cells reached 70-90% confluence, transfection was performed using Lipofectamine 3000. Briefly, for each well of a 6-well plate, 3 μL of Lipofectamine 3000 reagent was diluted in 125 μL of Opti-MEM diluted serum medium (Mix 1). Plasmid DNA (5 μg) and P3000 reagent (10 μL) were mixed separately with 125 μL of Opti-MEM (Mix 2). Mixture 1 and Mixture 2 were then mixed in a 1:1 ratio, gently stirred, and incubated at room temperature for 10 minutes to form a DNA-lipid complex. The complex was added dropwise to the cells. After incubation at 37°C for 12 hours, the medium was replaced with complete medium containing G418 for selecting stable clones. Transfection efficiency was verified by Western blotting analysis.

[0046] shRNA transfection Lentiviral particles encoding shRNA (purchased from GeneChem) were used for gene knockout. Cells treated with rifamycin were infused with 1×10⁶ cells in complete culture medium. 5Cells were seeded at a density of 6-well plates. Based on the predetermined viral titer and desired multiple of infection (MOI), the required viral supernatant volume was calculated using the following formula: Virus volume (μL) = (MOI × cell number) / viral titer (TU / mL). The calculated virus was added to the cell suspension along with 25× HitransGP infection enhancer (40 μL per well). After gentle mixing, the plates were incubated at 37°C for 12 h. The medium was then replaced with fresh complete medium containing puromycin to select stably transfected cells. Knockout efficiency was analyzed by Western blotting.

[0047] 1.14 Proliferation Analysis The proliferation of rifamycin-treated HT29 and SW620 cells, as well as stably transfected DLD-1 and SW620 cells overexpressing or knocked down DHRS4, was assessed using the Cell Counting Kit-8 (CCK-8, SEVEN) assay. Cells were counted at 2 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates. CCK-8 solution was added at different time points, and the plates were incubated at 37°C for 1 hour. The absorbance was then measured at 450 nm using a microplate reader.

[0048] 1.15 Crystal Violet Staining Method Rifampicin-treated HT29 and SW620 cells, as well as stably transfected DLD-1 and SW620 cells overexpressing or knocked down DHRS4, were stained with crystal violet according to standard procedures. Cells were seeded into six-well plates at 5 × 10⁶ cells / well. 5 Cells per well. Once cells have adhered, replace the medium with the appropriate concentration of rifamycin and culture for 1-2 weeks. Discard the collected medium from the six-well plate, wash three times with PBS, discard the liquid, add 1 mL of crystal violet staining solution, let stand for 30 minutes, then discard. Wash three times with PBS, discard the liquid, air dry, and photograph.

[0049] 1.16 Establishment and Treatment of Subcutaneous Transplantation Mouse Tumor Model Male Balb / c Nude mice (4 weeks old) and female BALB / c mice (6 weeks old) were purchased from Vital River Laboratory (Beijing, China). All animal studies were approved by the School of Pharmacy, Harbin Medical University. All mice were provided with free access to food and water in a temperature-controlled environment (22±2 ℃) with a strict 12-hour light and 12-hour dark cycle. Food and water were provided on an ad hoc basis. Mice were randomly assigned to experimental groups. Approximately 5–8 × 10⁶ mice were placed in each group. 6 CRC cells (HT29 and SW620 cells) were subcutaneously injected into the right axilla of each mouse to establish a mouse subcutaneous tumor transplantation model. Tumor growth was monitored every three days, and volume was calculated using the formula: Volume = Length × Width.2 × 0.52 (mm) 3 Seven days after injection, when tumor growth was observed, drug treatment was initiated. Mice were intraperitoneally injected with rifamycin (0, 10, 25 mg / kg) three times a week, while the control group received an equal volume of the carrier. Mice were sacrificed 2-3 weeks after tumor cell injection, and the tumor weight (g) was recorded. The obtained tumor tissue was washed and separated for immunoprecipitation and immunofluorescence staining after paraffin sectioning; the remaining portion was cryopreserved in liquid nitrogen. The 0 mg / kg control group was designated as the control group.

[0050] 1.17 Establishment of a mouse model of colon cancer associated with colitis A standard azomethane (AOM) / sodium dextran sulfate (DSS) was used to induce a CAC mouse model in 6-week-old female Balb / c mice. Briefly, mice (n=20 per group) received a single intraperitoneal injection of AOM (12.5 mg / kg). One week later, mice were given 2.5% DSS as drinking water for 7 days, followed by a 14-day recovery period with standard water; this process was repeated three times. Starting from week 9, mice were given intraperitoneal injections of rifamycin (0, 10, 25 mg / kg) twice weekly, while the control group received an equal volume of the carrier. All mice were euthanized on day 120, and the number and survival rate of tumor tissue were recorded. A portion of the harvested tumor tissue was preserved in liquid nitrogen, and the remainder was paraffin-embedded and sectioned; one portion was used for immunofluorescence treatment, and the other for HE staining. The 0 mg / kg control group received the tumor tissue.

[0051] 1.18 HE staining Paraffin sections of tumor tissue from section 1.17 were dewaxed with xylene I and II (10 minutes each), then dehydrated in a gradient of anhydrous ethanol I and II (5 minutes each), and 95%, 90%, 80%, and 70% ethanol (5 minutes each) to distilled water. Hematoxylin was used to stain the cell nuclei (3-8 minutes), followed by differentiation with 1% hydrochloric acid ethanol, and then blue staining with 0.6% ammonia, followed by eosin staining of the cytoplasm (1-3 minutes). After clearing and dehydration with ethanol and xylene, the sections were mounted with neutral resin and observed and photographed under a microscope.

[0052] 1.19 Patient-derived organoid culture Derived organoids (PDOs) for colorectal cancer were established from five patient individuals (PDO 1–PDO 5). Cancerous colorectal cancer tissue was isolated, washed with antibiotics, cut into 1 mm³ fragments, and digested at 37°C for 30 min–1 h. Cell particles were resuspended in erythrocyte lysis buffer and collected by centrifugation. The cell pellet was mixed 1:1 with Matrigel (BD Biosciences, California, USA) and seeded into 24-well plates for further culture.

[0053] 1.20 Organoid Drug Sensitivity Testing Organic fragments and matrix gel (BD Biosciences, California, USA) were mixed 1:1 and seeded into 96-well plates at 10 µL per well. Rifampicin was used for treatment (0, 2, 4, 6 µM) to allow the organoids to grow to an average diameter of 50 µm. PDO images and average organoid diameters were captured every 3 days, with the average diameter measured in length (µm). After 9 days, cell viability was measured and immunofluorescence staining was performed using the CellTiter Lumi™ Luminescent 3D Cell Viability Assay Kit (Beyotime, Shanghai, China).

[0054] Cell viability measurement: The rifampicin-treated organoids were mixed with 100 µL of CellTiter Glo® 3D reagent, shaken for 5 minutes, and then incubated at room temperature for 30 minutes. The luminescence was measured using a microplate reader.

[0055] 1.21 Data Analysis Statistical analysis: two-sided t-test, two-way matched ANOVA, and ordinary one-way ANOVA; *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001. Data are expressed as mean ± standard deviation.

[0056] 2. Experimental Results Protein docking results showed that virtual screening of the natural product library by molecular docking revealed docking energies of 178 compounds less than -7.0 kcal / mol. By comprehensively considering various factors, including structural diversity and the interaction forces between candidate molecules and amino acids at the docking site, 26 small molecule structures were identified for preliminary screening (Figure 1A).

[0057] The interaction between DHRS4 and LONP2 was analyzed using Cluspro 2.0. It was found that amino acid residues Asp110, Gly157, Lys197, Met198, Val201, and Gln217 of DHRS4 can bind to the Leu247-Phe256 domain of LONP2. To identify the activator of DHRS4, the Ser71-Lys137 domain of DHRS4 was designated as the interface (B in Figure 1).

[0058] Immunoprecipitation assays demonstrated that treatment of SW620 cells with rifampicin enhanced the interaction between DHRS4 and LONP2 and TFAM. Figure 2(A) This effect was validated by pull-down analysis using purified proteins, demonstrating that rifamycin promoted the direct binding of DHRS4 to LONP2 in SW620 cells. Figure 2 C).

[0059] PLA analysis further revealed that rifamycin treatment enhanced the direct interaction between LONP2-DHRS4 and LONP2-TFAM in LoVo cells. Figure 3 Proteomics results showed that rifamycin treatment led to significant inhibition of mitochondrial tissue and transport pathways. Figure 4 A). Metabolomics analysis showed that rifamycin treatment led to a decrease in the levels of key intermediates in the OXPHOS pathway, including pyruvate, phosphoenolpyruvate, and ATP. Figure 4 B).

[0060] TFAM, MTCO2, SDHB, NDUFB8, and UQCRC2 are all important proteins widely involved in mtDNA transcription, replication, and damage repair, maintaining mitochondrial stability. Rifamycin reduced the expression of these mitochondrial-related proteins in CRC cells. Figure 5 Confocal imaging revealed a decrease in mitochondrial fluorescence intensity in CRC cells after rifamycin treatment. Figure 6 Flow cytometry analysis confirmed a significant reduction in mitochondrial quality or function. Figure 7 Hippocampal analysis confirmed impaired mitochondrial respiratory function after rifamycin treatment. Figure 8 ).

[0061] In vitro culture of SW620 and HT29 cells, followed by CCK8 assay, revealed that rifamycin treatment significantly inhibited the proliferation of CRC cells. Figure 9 A), the inhibitory effect of rifamycin on CRC cell growth was further confirmed by crystal violet staining. Figure 9 B). CCK-8 assay and crystal violet staining showed that rifamycin treatment had no anti-proliferative effect in DLD-1 cells lacking DHRS4 expression, while rifamycin significantly inhibited cell proliferation in DLD-1 cells overexpressing DHRS4. Similarly, rifamycin did not show significant anti-proliferative effect in the DHRS4 knockdown SW620 cell line, while rifamycin significantly inhibited cell proliferation in the control group expressing normal DHRS4. Figure 10 Flow cytometry analysis showed that CRC cell apoptosis significantly increased after rifamycin treatment. Figure 11 ).

[0062] In a mouse model of subcutaneous tumor transplantation, rifamycin administration reduced tumor size and weight. Figure 12Multi-omics analysis of tumor tissues showed that after rifamycin treatment, mitochondrial-related proteins such as UQCC2, NDUFB1, and COX7A2 were significantly downregulated, and mitochondrial tissue and transport pathways were significantly inhibited. Figure 13 Immunofluorescence staining analysis of tumor tissue confirmed a significant downregulation of MTCO1, MTCO2, and TFAM after drug treatment. Figure 14 Immunoprecipitation analysis of tumor tissues showed that treatment with rifamycin significantly enhanced the interaction between LONP2 and DHRS4 and TFAM. Figure 15 Patient-derived organoids (PDOs) for colon cancer were established from five patients (PDO1-PDO5) and their functional characterization was performed. The results showed that, compared with the control group, rifamycin treatment resulted in a dose-dependent inhibition of PDO growth and a significant reduction in the average organoid diameter; cell viability decreased accordingly with increasing drug concentration, which corroborated the morphological observations. Figure 16 Furthermore, immunofluorescence analysis of human CRC organoids showed that the expression of mitochondrial proteins TFAM and MTCO2 was significantly reduced after rifamycin treatment, indicating that rifamycin impairs mitochondrial biogenesis. Figure 17 In the CAC mouse model, rifamycin treatment significantly reduced intestinal tumor burden, improved overall survival, and decreased the fluorescence intensity of mitochondrial proteins MTCO1, MTCO2, and TFAM in intestinal tumor tissue. Figure 18-19 The H&E staining results showed that after intraperitoneal injection of rifamycin into CAC mice, rifamycin had no significant damaging effect on major organs (including the heart, liver, spleen, lungs, and kidneys). Figure 20 ).

[0063] In summary, these findings suggest that rifamycin promotes TFAM degradation by enhancing DHRS4-LONP2 interaction, thereby inhibiting tumor growth by impairing mitochondrial function. Rifamycin may serve as a potential treatment for CRC by inhibiting cancer cell growth and thus controlling tumor development.

Claims

1. The application of rifamycin in the preparation of drugs for treating colorectal cancer.

2. The application according to claim 1, characterized in that, Rifamycin is used to stabilize the binding of DHRS4 and LONP2 in mitochondria of colorectal cancer cells, promote TFAM degradation, thereby leading to impaired mitochondrial biogenesis and function, and inhibiting mitochondrial activity.

3. The application according to claim 1, characterized in that, Rifamycin is either the sole active ingredient or used in combination with other drugs.

4. The application according to claim 1, characterized in that, It is used to inhibit the proliferation of colorectal cancer cells.

5. The application according to claim 1, characterized in that, Used to enhance the interaction between LONP2 and DHRS4 and TFAM.

6. The application according to claim 1, characterized in that, It is used to downregulate the levels of MTCO1, MTCO2 and TFAM, inhibit OXPHOS protein and its related pathways, and improve intestinal tumor burden.

7. The application according to claim 1, characterized in that, The drug includes rifamycin and medically approved excipients; the drug includes various acceptable dosage forms.

8. The application according to claim 1, characterized in that, This includes injections, pills, capsules, granules, tablets, or oral liquids.

9. The application according to claim 1, characterized in that, in, Rifamycin is selected from rifampin, rifapentine, and rifabutin.

10. The application according to claim 1, characterized in that, Rifamycin, for example, has a molecular structure of C. 37 H 45 NO 12 Its chemical name is (2S,12Z,14E,16S,17S,18R,19R,20R,21S,22R,23S,24E)-5,17,19-trihydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-1,6,9,11-tetraoxo-1,2,6,9-tetrahydro-2,7-(epoxypentadecano[1,11,13]trieneimino)naphtho[2,1-b]furan-21-yl acetate. It is derived from natural fermentation and simple chemical conversion, and its purity is 98.74%.

Images