#METABOLOMICS WORKBENCH qiuxu_20240928_073436 DATATRACK_ID:5245 STUDY_ID:ST003510 ANALYSIS_ID:AN005764 PROJECT_ID:PR002155 VERSION 1 CREATED_ON October 2, 2024, 10:06 pm #PROJECT PR:PROJECT_TITLE NMR spectra of water-soluble extracts of mouse colon cancer tissue and colon PR:PROJECT_TITLE cancer CT26 cells PR:PROJECT_TYPE NMR PR:PROJECT_SUMMARY In this study, we investigated the role of 3-Hydroxybutyrate (3-HB) in colon PR:PROJECT_SUMMARY cancer cachexia (CAC). We used a CAC mouse model and CT26 colon cancer cells. We PR:PROJECT_SUMMARY used NMR-based metabolomics to investigate 3-HB effects on colon tissue and cell PR:PROJECT_SUMMARY metabolism. PR:INSTITUTE Xiamen University PR:LAST_NAME Qiu PR:FIRST_NAME Xu PR:ADDRESS Xiamen University PR:EMAIL qiuxu@stu.xmu.edu.cn PR:PHONE 13395036603 #STUDY ST:STUDY_TITLE 3-Hydroxybutyrate Suppresses Colon Cancer Growth through Metabolic Reprogramming ST:STUDY_TITLE and Monocarboxylate Transporter-Mediated Lactate Accumulation ST:STUDY_SUMMARY Cancer cachexia (CAC) remains a challenging complication in colon cancer, often ST:STUDY_SUMMARY leading to poor clinical outcomes. This study investigates the therapeutic ST:STUDY_SUMMARY potential of 3-hydroxybutyrate (3-HB) in CAC by exploring its effects on tumor ST:STUDY_SUMMARY growth and cellular metabolism using a colon CAC mouse model and CT26 colon ST:STUDY_SUMMARY cancer cells. Through NMR-based metabolomics and molecular biology approaches, ST:STUDY_SUMMARY we reveal that 3-HB slows tumor growth in CAC mice, potentially by increasing ST:STUDY_SUMMARY lactate accumulation within tumor tissues, modulating key metabolic pathways. ST:STUDY_SUMMARY Additionally, 3-HB induced oxidative stress and apoptosis in CT26 cells, ST:STUDY_SUMMARY characterized by elevated reactive oxygen species (ROS) and caspase-3 ST:STUDY_SUMMARY activation. Mechanistically, 3-HB appears to compete with lactate for ST:STUDY_SUMMARY monocarboxylate transporters (MCTs), leading to intracellular lactate ST:STUDY_SUMMARY accumulation, cellular acidification, and tumor suppression. These findings ST:STUDY_SUMMARY suggest that 3-HB holds promise as a therapeutic agent for colon cancer ST:STUDY_SUMMARY cachexia, offering new insights into metabolic targeting strategies for cancer ST:STUDY_SUMMARY treatment. ST:INSTITUTE Xiamen University ST:LAST_NAME Qiu ST:FIRST_NAME Xu ST:ADDRESS Xiamen University ST:EMAIL qiuxu@stu.xmu.edu.cn ST:PHONE 13395036603 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - CAC-C16 Sample source:colon tumor tissue | Factors:Treatment CAC Subject_ID=-; Fid ID=34 SUBJECT_SAMPLE_FACTORS - CAC-C12 Sample source:colon tumor tissue | Factors:Treatment CAC Subject_ID=-; Fid ID=40 SUBJECT_SAMPLE_FACTORS - CAC-C6 Sample source:colon tumor tissue | Factors:Treatment CAC Subject_ID=-; Fid ID=41 SUBJECT_SAMPLE_FACTORS - CAC-C17 Sample source:colon tumor tissue | Factors:Treatment CAC Subject_ID=-; Fid ID=46 SUBJECT_SAMPLE_FACTORS - CAC-C11 Sample source:colon tumor tissue | Factors:Treatment CAC Subject_ID=-; Fid ID=48 SUBJECT_SAMPLE_FACTORS - CAC-C9 Sample source:colon tumor tissue | Factors:Treatment CAC Subject_ID=-; Fid ID=53 SUBJECT_SAMPLE_FACTORS - CAC-C3 Sample source:colon tumor tissue | Factors:Treatment CAC Subject_ID=-; Fid ID=54 SUBJECT_SAMPLE_FACTORS - CAC-C1 Sample source:colon tumor tissue | Factors:Treatment CAC Subject_ID=-; Fid ID=55 SUBJECT_SAMPLE_FACTORS - CACK-CK18 Sample source:colon tumor tissue | Factors:Treatment CACK Subject_ID=-; Fid ID=77 SUBJECT_SAMPLE_FACTORS - CACK-CK6 Sample source:colon tumor tissue | Factors:Treatment CACK Subject_ID=-; Fid ID=66 SUBJECT_SAMPLE_FACTORS - CACK-CK7 Sample source:colon tumor tissue | Factors:Treatment CACK Subject_ID=-; Fid ID=67 SUBJECT_SAMPLE_FACTORS - CACK-CK8 Sample source:colon tumor tissue | Factors:Treatment CACK Subject_ID=-; Fid ID=68 SUBJECT_SAMPLE_FACTORS - CACK-CK9 Sample source:colon tumor tissue | Factors:Treatment CACK Subject_ID=-; Fid ID=69 SUBJECT_SAMPLE_FACTORS - CACK-CK11 Sample source:colon tumor tissue | Factors:Treatment CACK Subject_ID=-; Fid ID=71 SUBJECT_SAMPLE_FACTORS - CACK-CK13 Sample source:colon tumor tissue | Factors:Treatment CACK Subject_ID=-; Fid ID=73 SUBJECT_SAMPLE_FACTORS - CACK-CK14 Sample source:colon tumor tissue | Factors:Treatment CACK Subject_ID=-; Fid ID=74 #COLLECTION CO:COLLECTION_SUMMARY Approximately 100 mg of mouse tumor tissue was collected and pre-cooled CO:COLLECTION_SUMMARY methanol, chloroform and water (4:4:2.85, v/v) were added in a sequential CO:COLLECTION_SUMMARY manner. After vortex mixing, the tissue was completely homogenised using a CO:COLLECTION_SUMMARY pre-cooled cryogenic tissue grinder (65 Hz, 3 min). The metabolites were then CO:COLLECTION_SUMMARY extracted from the tumor tissue in two phases. The upper aqueous phase was CO:COLLECTION_SUMMARY collected by centrifugation at 4°C and 12,000 rpm for 10 min. Nitrogen was used CO:COLLECTION_SUMMARY to accelerate volatilisation of methanol from the aqueous phase and transfer to CO:COLLECTION_SUMMARY a freeze dryer for removal of water. CO:SAMPLE_TYPE Tumors #TREATMENT TR:TREATMENT_SUMMARY The tumor-bearing mice were randomly assigned to two groups and subjected to the TR:TREATMENT_SUMMARY corresponding interventions. One group of tumor-bearing mice received a daily TR:TREATMENT_SUMMARY intraperitoneal injection of ethyl 3-hydroxybutyrate at a dose of 2 mM/kg per TR:TREATMENT_SUMMARY day (CACK, n = 8), and correspondingly, the other group of tumor-bearing mice TR:TREATMENT_SUMMARY (CAC, n = 8), as well as the control group (NOR, n = 8), was injected daily with TR:TREATMENT_SUMMARY an equal volume of PBS. all mice were subjected to a total of 21 days of the TR:TREATMENT_SUMMARY intervention. All mice were euthanised on Day 28 post-modelling and sampled. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The processed metabolite samples were dissolved in NMR buffer (pH 7.4), prepared SP:SAMPLEPREP_SUMMARY in D2O, 50 mM PO₄³-, 0.05 mM 3-(trimethylsilyl) propionate sodium-2,2,3,3-d4 SP:SAMPLEPREP_SUMMARY (TSP), and subsequently transferred to 5 mm NMR tubes for acquisition of SP:SAMPLEPREP_SUMMARY metabolite NMR spectra on a Bruker Avance III HD 850 MHz spectrometer for SP:SAMPLEPREP_SUMMARY acquisition of metabolite NMR spectra. One-dimensional (1D) 1H-NMR spectrum SP:SAMPLEPREP_SUMMARY acquisition: 1H-NMR spectra were acquired at 298 K on a Bruker Avance III 850 SP:SAMPLEPREP_SUMMARY MHz spectrometer (Bruker BioSpin GmbH, Germany) with the pulse sequence SP:SAMPLEPREP_SUMMARY NOESYGPPR1D [RD-G1-90°-t-90°-τm-G2-90°-ACQ]. The spectral width was set at SP:SAMPLEPREP_SUMMARY 20.00 ppm, with 32 scans, a mixing time of 10 ms, and an additional relaxation SP:SAMPLEPREP_SUMMARY delay time of 4 s. #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker Avance III HD 850 MHz NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:STANDARD_CONCENTRATION 1 mM TSP NM:SPECTROMETER_FREQUENCY 850 MHz NM:NMR_SOLVENT H2O+D2O NM:NMR_TUBE_SIZE 5 mm NM:PULSE_SEQUENCE noesygppr1d [(RD)-90°-t1-90°-τm-90°-ACQ] NM:RECEIVER_GAIN 57 NM:OFFSET_FREQUENCY 15.03 ppm NM:TEMPERATURE 25 NM:NUMBER_OF_SCANS 32 NM:DUMMY_SCANS 4 NM:ACQUISITION_TIME 2 s NM:SPECTRAL_WIDTH 20 ppm NM:NUM_DATA_POINTS_ACQUIRED 64 K NM:LINE_BROADENING 0.3 Hz NM:BASELINE_CORRECTION_METHOD Auto-baseline correction of integral by abs NM:CHEMICAL_SHIFT_REF_STD TSP (0.000 ppm) NM:NMR_RESULTS_FILE ST003510_AN005764_Results.txt UNITS:ppm #END