#METABOLOMICS WORKBENCH Isaac-Lam-METADATA_1 DATATRACK_ID:197 STUDY_ID:ST000134 ANALYSIS_ID:AN000216 VERSION 1 CREATED_ON 08-08-2023 #PROJECT PR:PROJECT_TITLE Monitoring In Vitro Response of Selenium-Treated Prostate Cells by 1H NMR PR:PROJECT_TITLE Spectroscopy PR:PROJECT_TYPE Metabolomics by 1H NMR PR:PROJECT_SUMMARY 1H NMR data of prostate cells treated with selenium showed a decreasing trend in PR:PROJECT_SUMMARY metabolite levels with the largest change exhibited by creatine mainly due to PR:PROJECT_SUMMARY disrupted energy metabolism, and probably due to loss of structural integrity PR:PROJECT_SUMMARY combined with external dissipation of metabolites. Lactate, choline-containing PR:PROJECT_SUMMARY compounds, and glycine levels increased depending on the type of selenium used PR:PROJECT_SUMMARY and the cell type. Principal component analysis (PCA) showed that SeM-treated PR:PROJECT_SUMMARY cells can be distinguished from SeMSC-treated cells, and DU145 PCa from PNT1A PR:PROJECT_SUMMARY normal cells. PR:INSTITUTE Purdue University PR:DEPARTMENT Biology/Chemistry PR:LABORATORY Biology/Chemistry Dept PR:LAST_NAME Isaac-Lam PR:FIRST_NAME Meden PR:ADDRESS 1401 S US Hwy 421 Westville, Indiana USA PR:EMAIL isaaclam@pnc.edu PR:PHONE 1-219-785-5776 PR:FUNDING_SOURCE Indiana Academy of Science PR:DOI http://dx.doi.org/10.21228/M86C76 #STUDY ST:STUDY_TITLE Monitoring In Vitro Response of Selenium-Treated Prostate Cells by 1H NMR ST:STUDY_TITLE Spectroscopy ST:STUDY_TYPE Metabolite level response after treatment with organoselenium ST:STUDY_SUMMARY Metabolomics analysis was performed on DU145 prostate cancer cells and PNT1A ST:STUDY_SUMMARY non-tumorigenic prostate cells after treatment with selenomethionine and ST:STUDY_SUMMARY Se-methylselenocysteine using 800 MHz Bruker NMR spectrometer on 18 cell ST:STUDY_SUMMARY samples. ST:INSTITUTE Purdue University ST:DEPARTMENT Biology/Chemistry ST:LABORATORY Biology/Chemistry ST:LAST_NAME Isaac-Lam ST:FIRST_NAME Meden ST:ADDRESS 1401 S US Hwy 421, Westville, IN 46391 ST:EMAIL isaaclam@pnc.edu ST:PHONE 219-785-5776 ST:SUBMIT_DATE 2015-01-13 #SUBJECT SU:SUBJECT_TYPE Human cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC, Sigma SU:CELL_STRAIN_DETAILS DU145 prostate cancer, PNT1A prostate nontumor SU:SUBJECT_COMMENTS 10 SU:CELL_PRIMARY_IMMORTALIZED Immortalized SU:CELL_PASSAGE_NUMBER 10 SU:CELL_COUNTS 10 SU:SPECIES_GROUP Human #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - SeM (DU145) Treatment:SeM | Prostate Cell Line:DU145 SUBJECT_SAMPLE_FACTORS - SeM (PNT1A) Treatment:SeM | Prostate Cell Line:PNT1A SUBJECT_SAMPLE_FACTORS - SeMSC (DU145) Treatment:SeMSC | Prostate Cell Line:DU145 SUBJECT_SAMPLE_FACTORS - SeMSC (PNT1A) Treatment:SeMSC | Prostate Cell Line:PNT1A #COLLECTION CO:COLLECTION_SUMMARY After incubation for 24 hrs, cells were washed twice with 0.5 mL PBS in D2O CO:COLLECTION_SUMMARY (Cambridge Isotope Laboratories). D2O (0.7 mL) was again added to each petri CO:COLLECTION_SUMMARY dish and cells were gently scraped from the surface with a sterile cell scraper. CO:COLLECTION_SUMMARY A volume of 0.7 mL of the harvested cell suspension was transferred into a 5 mm CO:COLLECTION_SUMMARY NMR tube and 10 mL of 100 mM TMSP in D2O was added to each tube. CO:SAMPLE_TYPE cell line CO:COLLECTION_METHOD scraping cells from flask CO:COLLECTION_LOCATION lab CO:COLLECTION_FREQUENCY once a week CO:VOLUMEORAMOUNT_COLLECTED 0.7 mL per dish CO:STORAGE_CONDITIONS in ice CO:COLLECTION_TUBE_TEMP in ice #TREATMENT TR:TREATMENT_SUMMARY Cells were grown to 80% confluence in Petri dishes (50 mm in diameter) in 4.5% TR:TREATMENT_SUMMARY CO2 at 37 OC for 5-6 days for full attachment to the substratum. After TR:TREATMENT_SUMMARY aspirating the old growth medium, 3 mL of SeM or SeMSC (300 mM) in fresh TR:TREATMENT_SUMMARY pre-warmed medium at 37 ºC was added to each dish. TR:TREATMENT_COMPOUND Selenomethionine / Se-methylselenocysteine TR:TREATMENT_DOSE 300 mM TR:TREATMENT_DOSEVOLUME 3 mL TR:TREATMENT_DOSEDURATION 24 hrs TR:TREATMENT_VEHICLE dissolved in culture media TR:CELL_STORAGE incubator TR:CELL_GROWTH_CONTAINER petri dish (Corning) TR:CELL_GROWTH_CONFIG monolayer, adherent TR:CELL_GROWTH_RATE 5-6 days TR:CELL_INOC_PROC trypsinized, then subculture TR:CELL_MEDIA RPMI, EMEM TR:CELL_ENVIR_COND 37 C, 4.5% CO2 incubator TR:CELL_HARVESTING after trypsinization TR:CELL_PCT_CONFLUENCE 80% TR:CELL_MEDIA_LASTCHANGED 2-3 days #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were grown to 80% confluence in Petri dishes (50 mm in diameter) in 4.5% SP:SAMPLEPREP_SUMMARY CO2 at 37 OC for 5-6 days for full attachment to the substratum. After SP:SAMPLEPREP_SUMMARY aspirating the old growth medium, 3 mL of SeM or SeMSC (300 mM) in fresh SP:SAMPLEPREP_SUMMARY pre-warmed medium at 37 ºC was added to each dish. After incubation for 24 hrs, SP:SAMPLEPREP_SUMMARY cells were washed twice with 0.5 mL PBS in D2O (Cambridge Isotope Laboratories). SP:SAMPLEPREP_SUMMARY D2O (0.7 mL) was again added to each petri dish and cells were gently scraped SP:SAMPLEPREP_SUMMARY from the surface with a sterile cell scraper. A volume of 0.7 mL of the SP:SAMPLEPREP_SUMMARY harvested cell suspension was transferred into a 5 mm NMR tube and 10 mL of 100 SP:SAMPLEPREP_SUMMARY mM TMSP in D2O was added to each tube. Prior to NMR analysis, tubes were SP:SAMPLEPREP_SUMMARY vortexed to ensure samples were in suspension. Control experiments using SP:SAMPLEPREP_SUMMARY untreated cells were conducted in parallel with treated cells with the same SP:SAMPLEPREP_SUMMARY incubation protocols and sample preparation. SP:SAMPLEPREP_PROTOCOL_FILENAME Prostate_Cells_Metabolomics_Procedure.doc SP:SAMPLE_RESUSPENSION in deuterated water SP:CELL_TYPE prostate #CHROMATOGRAPHY #ANALYSIS AN:LABORATORY_NAME Purdue Chemistry NMR Facility AN:ANALYSIS_TYPE NMR AN:ACQUISITION_DATE May 30, 2014; June 6, 2014 AN:SOFTWARE_VERSION AV-III-800 AN:OPERATOR_NAME Dr. John Harwood AN:DETECTOR_TYPE TX1 #NMR NM:INSTRUMENT_NAME Bruker Avance III NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D 1H NM:FIELD_FREQUENCY_LOCK Deuterium NM:STANDARD_CONCENTRATION 1.43 mM NM:SPECTROMETER_FREQUENCY 800 MHz NM:NMR_PROBE TX1, inverse NM:NMR_SOLVENT D2O NM:NMR_TUBE_SIZE 5 mm x 7 in NM:SHIMMING_METHOD gradient, topshim NM:PULSE_SEQUENCE zg one-pulse NM:WATER_SUPPRESSION presaturation NM:PULSE_WIDTH 8.5 msec NM:POWER_LEVEL 12.25 W NM:RECEIVER_GAIN 912 NM:OFFSET_FREQUENCY 4.8 ppm NM:PRESATURATION_POWER_LEVEL 57 dB NM:CHEMICAL_SHIFT_REF_CPD TMSP NM:TEMPERATURE 295.9 NM:NUMBER_OF_SCANS 256 NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 1.4680564 NM:RELAXATION_DELAY 6.5 msec NM:SPECTRAL_WIDTH 11160 Hz NM:NUM_DATA_POINTS_ACQUIRED 32 K NM:REAL_DATA_POINTS 32 K NM:LINE_BROADENING 1 Hz NM:ZERO_FILLING 16K NM:APODIZATION lorentzian NM:BASELINE_CORRECTION_METHOD polynomial NM:CHEMICAL_SHIFT_REF_STD TMSP #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS % Relative change NMR_METABOLITE_DATA_START Samples SeM (DU145) SeM (PNT1A) SeMSC (DU145) SeMSC (PNT1A) Factors Treatment:SeM | Prostate Cell Line:DU145 Treatment:SeM | Prostate Cell Line:PNT1A Treatment:SeMSC | Prostate Cell Line:DU145 Treatment:SeMSC | Prostate Cell Line:PNT1A Alanine -35.8000 -23.3000 -12.3000 -55.8000 b-Glucose -37.3000 -60.8000 -28.9000 -64.0000 Choline 10.0000 -45.8000 -39.7000 -70.3000 Creatine -25.0000 -56.1000 -35.4000 -80.7000 Glycerophosphocholine 5.4000 -33.3000 -35.2000 -58.3000 Glycine -4.2000 25.4000 -17.5000 -57.5000 Lactate 11.4000 -25.9000 65.6000 -31.9000 Lipids -28.3000 -56.1000 -54.9000 -75.4000 Nicotine adenine dinucleotide phosphate (NADP) -40.0000 -75.0000 -30.0000 Phosphocholine -34.2000 -59.6000 -5.7000 -54.4000 Taurine -14.8000 -10.9000 -29.0000 -19.2000 Uridine 2^-diphospate -33.3000 -35.2000 -61.1000 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant Alanine 5950 C00041 Ala IsaacLam_ID b-Glucose b-Glc IsaacLam_ID Choline 305 C00114 Cho IsaacLam_ID Creatine 586 C00300 Cr IsaacLam_ID Glycerophosphocholine 657272 GPC IsaacLam_ID Glycine 750 C00037 Gly IsaacLam_ID Lactate 107689 C00186 Lac IsaacLam_ID Lipids Lipids IsaacLam_ID Nicotine adenine dinucleotide phosphate (NADP) NADP IsaacLam_ID Phosphocholine 135437 PC IsaacLam_ID Taurine 1123 C00245 Tau IsaacLam_ID Uridine 2'-diphospate UDP IsaacLam_ID METABOLITES_END #END