#METABOLOMICS WORKBENCH qiuxu_20240928_063344 DATATRACK_ID:5244 STUDY_ID:ST003509 ANALYSIS_ID:AN005763 PROJECT_ID:PR002155
VERSION             	1
CREATED_ON             	October 2, 2024, 5:29 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:DEPARTMENT                    	Department of Chemical Biology
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                  	Cultured cells
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           	-	C1	Sample source:CT26 colon cancer cells | Factors:Treatment Control	RAW_FILE_NAME(Fid ID)=1
SUBJECT_SAMPLE_FACTORS           	-	C2	Sample source:CT26 colon cancer cells | Factors:Treatment Control	RAW_FILE_NAME(Fid ID)=2
SUBJECT_SAMPLE_FACTORS           	-	C3	Sample source:CT26 colon cancer cells | Factors:Treatment Control	RAW_FILE_NAME(Fid ID)=3
SUBJECT_SAMPLE_FACTORS           	-	C4	Sample source:CT26 colon cancer cells | Factors:Treatment Control	RAW_FILE_NAME(Fid ID)=4
SUBJECT_SAMPLE_FACTORS           	-	C5	Sample source:CT26 colon cancer cells | Factors:Treatment Control	RAW_FILE_NAME(Fid ID)=5
SUBJECT_SAMPLE_FACTORS           	-	C6	Sample source:CT26 colon cancer cells | Factors:Treatment Control	RAW_FILE_NAME(Fid ID)=6
SUBJECT_SAMPLE_FACTORS           	-	C7	Sample source:CT26 colon cancer cells | Factors:Treatment Control	RAW_FILE_NAME(Fid ID)=7
SUBJECT_SAMPLE_FACTORS           	-	C8	Sample source:CT26 colon cancer cells | Factors:Treatment Control	RAW_FILE_NAME(Fid ID)=8
SUBJECT_SAMPLE_FACTORS           	-	C9	Sample source:CT26 colon cancer cells | Factors:Treatment Control	RAW_FILE_NAME(Fid ID)=9
SUBJECT_SAMPLE_FACTORS           	-	C10	Sample source:CT26 colon cancer cells | Factors:Treatment Control	RAW_FILE_NAME(Fid ID)=10
SUBJECT_SAMPLE_FACTORS           	-	5K1	Sample source:CT26 colon cancer cells | Factors:Treatment 5 mM-3HB	RAW_FILE_NAME(Fid ID)=21
SUBJECT_SAMPLE_FACTORS           	-	5K2	Sample source:CT26 colon cancer cells | Factors:Treatment 5 mM-3HB	RAW_FILE_NAME(Fid ID)=22
SUBJECT_SAMPLE_FACTORS           	-	5K3	Sample source:CT26 colon cancer cells | Factors:Treatment 5 mM-3HB	RAW_FILE_NAME(Fid ID)=23
SUBJECT_SAMPLE_FACTORS           	-	5K4	Sample source:CT26 colon cancer cells | Factors:Treatment 5 mM-3HB	RAW_FILE_NAME(Fid ID)=24
SUBJECT_SAMPLE_FACTORS           	-	5K5	Sample source:CT26 colon cancer cells | Factors:Treatment 5 mM-3HB	RAW_FILE_NAME(Fid ID)=25
SUBJECT_SAMPLE_FACTORS           	-	5K6	Sample source:CT26 colon cancer cells | Factors:Treatment 5 mM-3HB	RAW_FILE_NAME(Fid ID)=26
SUBJECT_SAMPLE_FACTORS           	-	5K7	Sample source:CT26 colon cancer cells | Factors:Treatment 5 mM-3HB	RAW_FILE_NAME(Fid ID)=27
SUBJECT_SAMPLE_FACTORS           	-	5K8	Sample source:CT26 colon cancer cells | Factors:Treatment 5 mM-3HB	RAW_FILE_NAME(Fid ID)=28
SUBJECT_SAMPLE_FACTORS           	-	5K9	Sample source:CT26 colon cancer cells | Factors:Treatment 5 mM-3HB	RAW_FILE_NAME(Fid ID)=29
SUBJECT_SAMPLE_FACTORS           	-	5K10	Sample source:CT26 colon cancer cells | Factors:Treatment 5 mM-3HB	RAW_FILE_NAME(Fid ID)=30
SUBJECT_SAMPLE_FACTORS           	-	CAC-C16	Sample source:colon tumor tissue | Factors:Treatment CAC	RAW_FILE_NAME(Fid ID)=34
SUBJECT_SAMPLE_FACTORS           	-	CAC-C12	Sample source:colon tumor tissue | Factors:Treatment CAC	RAW_FILE_NAME(Fid ID)=40
SUBJECT_SAMPLE_FACTORS           	-	CAC-C6	Sample source:colon tumor tissue | Factors:Treatment CAC	RAW_FILE_NAME(Fid ID)=41
SUBJECT_SAMPLE_FACTORS           	-	CAC-C17	Sample source:colon tumor tissue | Factors:Treatment CAC	RAW_FILE_NAME(Fid ID)=46
SUBJECT_SAMPLE_FACTORS           	-	CAC-C11	Sample source:colon tumor tissue | Factors:Treatment CAC	RAW_FILE_NAME(Fid ID)=48
SUBJECT_SAMPLE_FACTORS           	-	CAC-C9	Sample source:colon tumor tissue | Factors:Treatment CAC	RAW_FILE_NAME(Fid ID)=53
SUBJECT_SAMPLE_FACTORS           	-	CAC-C3	Sample source:colon tumor tissue | Factors:Treatment CAC	RAW_FILE_NAME(Fid ID)=54
SUBJECT_SAMPLE_FACTORS           	-	CAC-C1	Sample source:colon tumor tissue | Factors:Treatment CAC	RAW_FILE_NAME(Fid ID)=55
SUBJECT_SAMPLE_FACTORS           	-	CACK-CK18	Sample source:colon tumor tissue | Factors:Treatment CACK	RAW_FILE_NAME(Fid ID)=77
SUBJECT_SAMPLE_FACTORS           	-	CACK-CK6	Sample source:colon tumor tissue | Factors:Treatment CACK	RAW_FILE_NAME(Fid ID)=66
SUBJECT_SAMPLE_FACTORS           	-	CACK-CK7	Sample source:colon tumor tissue | Factors:Treatment CACK	RAW_FILE_NAME(Fid ID)=67
SUBJECT_SAMPLE_FACTORS           	-	CACK-CK8	Sample source:colon tumor tissue | Factors:Treatment CACK	RAW_FILE_NAME(Fid ID)=68
SUBJECT_SAMPLE_FACTORS           	-	CACK-CK9	Sample source:colon tumor tissue | Factors:Treatment CACK	RAW_FILE_NAME(Fid ID)=69
SUBJECT_SAMPLE_FACTORS           	-	CACK-CK11	Sample source:colon tumor tissue | Factors:Treatment CACK	RAW_FILE_NAME(Fid ID)=71
SUBJECT_SAMPLE_FACTORS           	-	CACK-CK13	Sample source:colon tumor tissue | Factors:Treatment CACK	RAW_FILE_NAME(Fid ID)=73
SUBJECT_SAMPLE_FACTORS           	-	CACK-CK14	Sample source:colon tumor tissue | Factors:Treatment CACK	RAW_FILE_NAME(Fid ID)=74
#COLLECTION
CO:COLLECTION_SUMMARY            	Protocols for tissues: Approximately 100 mg of mouse tumor tissue was collected
CO:COLLECTION_SUMMARY            	and pre-cooled methanol, chloroform and water (4:4:2.85, v/v) were added in a
CO:COLLECTION_SUMMARY            	sequential manner. After vortex mixing, the tissue was completely homogenised
CO:COLLECTION_SUMMARY            	using a pre-cooled cryogenic tissue grinder (65 Hz, 3 min). The metabolites were
CO:COLLECTION_SUMMARY            	then 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. Protocols for cells: Cell culture medium
CO:COLLECTION_SUMMARY            	was discarded and cells washed three times with precooled phosphate buffer (PBS,
CO:COLLECTION_SUMMARY            	pH 7.4). The cells were quenched by adding 1 mL of pre-cooled methanol per dish
CO:COLLECTION_SUMMARY            	to stop the metabolic activity of the cells after the PBS was aspirated. The
CO:COLLECTION_SUMMARY            	cells were scraped off with a cell scraper and collected in 15 mL centrifuge
CO:COLLECTION_SUMMARY            	tubes, and the dishes were rinsed twice with 1 mL of methanol to collect the
CO:COLLECTION_SUMMARY            	cells completely. Pre-cooled methanol, chloroform and water (1:1:0.95, v/v) were
CO:COLLECTION_SUMMARY            	added and vortexed for 5 min. The aqueous phase was collected by centrifugation
CO:COLLECTION_SUMMARY            	at 12,000 rpm for 10 min at 4°C. The volatilisation of the methanol from the
CO:COLLECTION_SUMMARY            	aqueous phase was accelerated with nitrogen and the aqueous phase was
CO:COLLECTION_SUMMARY            	transferred to a freeze dryer to remove the water.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Mouse colon cancer CT26 tumor cells were cultured in DMEM (Dulbecco's Modified
TR:TREATMENT_SUMMARY             	Eagle Medium). The cell growth medium was supplemented with 1%
TR:TREATMENT_SUMMARY             	penicillin-streptomycin (PS, Hyclone, USA) and 10% (v/v) fetal bovine serum
TR:TREATMENT_SUMMARY             	(FBS, Corning, USA). Cell culture was performed at 37°C in a carbon dioxide
TR:TREATMENT_SUMMARY             	thermostat with 5% CO2. The medium was renewed every day. For treatment, sodium
TR:TREATMENT_SUMMARY             	(R)-3-hydroxybutyrate (3-HB, Sigma-Aldrich, China) was added to the medium to
TR:TREATMENT_SUMMARY             	assess its effects on the cell proliferation process. For the metabolomic
TR:TREATMENT_SUMMARY             	analysis of CT26 cells, this study was divided into a control group (n = 10) and
TR:TREATMENT_SUMMARY             	a 5 mM-3HB group (n = 10). The control group was CT26 cells cultured without any
TR:TREATMENT_SUMMARY             	intervention and the 5 mM-3HB group was CT26 cells treated with the addition of
TR:TREATMENT_SUMMARY             	5 mM 3-HB for 24 hours. The tumor-bearing mice were randomly assigned to two
TR:TREATMENT_SUMMARY             	groups and subjected to the corresponding interventions. One group of
TR:TREATMENT_SUMMARY             	tumor-bearing mice received a daily intraperitoneal injection of ethyl
TR:TREATMENT_SUMMARY             	3-hydroxybutyrate at a dose of 2 mM/kg per day (CACK, n = 8), and
TR:TREATMENT_SUMMARY             	correspondingly, the other group of tumor-bearing mice (CAC, n = 8), as well as
TR:TREATMENT_SUMMARY             	the control group (NOR, n = 8), was injected daily with an equal volume of PBS.
TR:TREATMENT_SUMMARY             	all mice were subjected to a total of 21 days of the intervention. All mice were
TR:TREATMENT_SUMMARY             	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 (tissues) or 64 (cells) scans, a mixing time of 10 ms, and an
SP:SAMPLEPREP_SUMMARY            	additional relaxation 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 (tissue) or 0.5 mM TSP (cells)
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 (tissue) or 144 (cells)
NM:OFFSET_FREQUENCY              	15.03 ppm
NM:TEMPERATURE                   	25
NM:NUMBER_OF_SCANS               	32 (tissue) or 64 (cells)
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              	colon_tumor.txt UNITS:ppm ct26_cells.txt UNITS:ppm
#FACTORS
NM:NMR_RESULTS_FILE               	ST003509_AN005763_Results.txt	UNITS:ppm
#END