#METABOLOMICS WORKBENCH teklab_20180925_035957 DATATRACK_ID:1516 STUDY_ID:ST001090 ANALYSIS_ID:AN001775 PROJECT_ID:PR000729 VERSION 1 CREATED_ON November 6, 2018, 11:12 am #PROJECT PR:PROJECT_TITLE NMR metabolomics study of Gemcitabine resistant cancer cells PR:PROJECT_TYPE NMR metabolomics PR:PROJECT_SUMMARY 1D 1H NMR based differential metabolomics study of T3M4 cells, which are PR:PROJECT_SUMMARY gemcitabine sensitive, and gemcitabine resistant T3M4 cells (GemR) were cultured PR:PROJECT_SUMMARY with or without gemcitabine treatment. 1D 1H NMR spectra were collected from the PR:PROJECT_SUMMARY samples and analyzed using MVAPACK software (http://bionmr.unl.edu/mvapack.php). PR:PROJECT_SUMMARY Using multivariate analysis, unique responses to gemcitabine treatment were PR:PROJECT_SUMMARY revealed, which may be useful in the clinical setting for monitoring a PR:PROJECT_SUMMARY patient’s therapeutic response. PR:INSTITUTE University of Nebraska-Lincoln PR:DEPARTMENT Chemistry PR:LABORATORY Robert Powers and Pankaj Singh labs PR:LAST_NAME Gebregiworgis PR:FIRST_NAME Teklab PR:ADDRESS 552 Hamilton Hall, 639 N. 12th Street, Lincoln, NE, 68588, USA PR:EMAIL teklab@huskers.unl.edu PR:PHONE (402) 472-3039 PR:FUNDING_SOURCE NCI, NIGMS, AACR PR:CONTRIBUTORS Teklab Gebregiworgis, Fatema Bhinderwala, Vinee Purohit, Nina V. Chaika, Pankaj PR:CONTRIBUTORS K. Singh, and Robert Powers #STUDY ST:STUDY_TITLE NMR metabolomics study of Gemcitabine resistant cancer cells ST:STUDY_TYPE NMR metabolomics ST:STUDY_SUMMARY 1D 1H NMR spectra were collected from the samples and analyzed using MVAPACK ST:STUDY_SUMMARY software (http://bionmr.unl.edu/mvapack.php). Multivariate analysis was ST:STUDY_SUMMARY conducted to study the metabolic phenotypes of the samples. ST:INSTITUTE University of Nebraska-Lincoln ST:DEPARTMENT Chemistry ST:LABORATORY Dr. Robert Powers and Pankaj Singh labs ST:LAST_NAME Gebregiworgis ST:FIRST_NAME Teklab ST:ADDRESS 552 Hamilton Hall, 639 N. 12th Street, Lincoln, NE, 68588, USA ST:EMAIL teklab@huskers.unl.edu ST:PHONE (402) 472-3039 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_BIOSOURCE_OR_SUPPLIER Gift from Dr. Michael Hollingsworth SU:CELL_STRAIN_DETAILS T3M4 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - T3M4_01 Treatment:No | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_02 Treatment:No | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_03 Treatment:No | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_04 Treatment:No | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_05 Treatment:No | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_06 Treatment:No | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_07 Treatment:No | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_08 Treatment:No | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_09 Treatment:No | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_10 Treatment:Yes | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_11 Treatment:Yes | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_12 Treatment:Yes | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_13 Treatment:Yes | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_14 Treatment:Yes | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_15 Treatment:Yes | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_16 Treatment:Yes | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_17 Treatment:Yes | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_18 Treatment:Yes | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_19 Treatment:Yes | Sensitivity:Sensitive SUBJECT_SAMPLE_FACTORS - T3M4_20 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_21 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_22 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_23 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_24 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_25 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_26 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_27 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_28 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_29 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_30 Treatment:No | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_31 Treatment:Yes | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_32 Treatment:Yes | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_33 Treatment:Yes | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_34 Treatment:Yes | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_35 Treatment:Yes | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_36 Treatment:Yes | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_37 Treatment:Yes | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_38 Treatment:Yes | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_39 Treatment:Yes | Sensitivity:Resistant SUBJECT_SAMPLE_FACTORS - T3M4_40 Treatment:Yes | Sensitivity:Resistant #COLLECTION CO:COLLECTION_SUMMARY For each of the cell culture, 1 x 106 cells were cultured and harvested at CO:COLLECTION_SUMMARY 70-80% confluence in a 100 mm Petri dish. The media was then removed by CO:COLLECTION_SUMMARY aspiration upon harvesting, and the cells were washed twice with PBS at pH 7.2. CO:COLLECTION_SUMMARY The cells were then lysed by the addition of 1 mL of an 80% methanol:water CO:COLLECTION_SUMMARY mixture at -80oC followed by incubation for 15 minutes in a -80oC freezer. The CO:COLLECTION_SUMMARY lysed cells were then transferred to a 1 ml Eppendorf tube using a cell scraper. CO:COLLECTION_SUMMARY The cell lysate was centrifuged at 16,200 g for 5 minutes and the supernatant CO:COLLECTION_SUMMARY was collected and transferred to a clean Eppendorf tube. 250 µL of nanopure CO:COLLECTION_SUMMARY water (Nanopure, Dubuque, IA) was added to the cell debris, the sample was mixed CO:COLLECTION_SUMMARY by pipetting, and then centrifuged as before. The two supernatants were CO:COLLECTION_SUMMARY combined, and the sample was dried by vacuum evaporation (SpeedVac® Plus, CO:COLLECTION_SUMMARY Savant, Thermo Scientific, Waltham, MA) followed by freeze-drying (Labconco, CO:COLLECTION_SUMMARY Kansas City, MO). CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80? #TREATMENT TR:TREATMENT_SUMMARY T3M4 wild type (WT) pancreatic cancer cells were cultured in DMEM with TR:TREATMENT_SUMMARY increasing concentrations of gemcitabine over a period of about 6 months. The TR:TREATMENT_SUMMARY resistance status of the cells at each gemcitabine dose was determined by TR:TREATMENT_SUMMARY calculating an IC50 using the MTT cytotoxicity assays. At the end of the 6-month TR:TREATMENT_SUMMARY treatment, the cell lines generated (GemR) were approximately 500-1000 fold TR:TREATMENT_SUMMARY resistant to gemcitabine as compared to the parental (WT) cells.Wild type T3M4 TR:TREATMENT_SUMMARY cells (WT), which are gemcitabine sensitive, and gemcitabine resistant T3M4 TR:TREATMENT_SUMMARY (GemR) were cultured with or without the addition of gemcitabine for the NMR TR:TREATMENT_SUMMARY metabolomics studies. A total of four cell culture groups were prepared that TR:TREATMENT_SUMMARY consisted of: (1) untreated T3M4 (WT) cells, (2) WT cells treated with 10 nM TR:TREATMENT_SUMMARY gemcitabine for a period of 12 hours (WT+), (3) untreated GemR cells, and (4) TR:TREATMENT_SUMMARY GemR cells treated with 10 nm gemcitabine for 12 hours (GemR+). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The dried metabolite extracts were reconstituted in 600 µL of 50 mM phosphate SP:SAMPLEPREP_SUMMARY buffer in 99.8% D2O (Isotec, St. Louis, MO) at pH 7.2 (uncorrected). 50 µM of SP:SAMPLEPREP_SUMMARY TMSP (3-(tetramethysilane) propionic acid-2,2,3,3-d4) were added for spectral SP:SAMPLEPREP_SUMMARY referencing. The solution was mixed by gentle votexing and centrifuged at 13000 SP:SAMPLEPREP_SUMMARY rpm for 5 min, and then the supernatant were transferred to 5 mm NMR tube. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME Dr. Robert Powers AN:OPERATOR_NAME Teklab Gebregiworgis #NMR NM:INSTRUMENT_NAME Bruker AVANCE DRX NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 500 MHz NM:TEMPERATURE 25 NM:NUMBER_OF_SCANS 128 NM:DUMMY_SCANS 16 NM:SPECTRAL_WIDTH 5483 HZ NM:NUM_DATA_POINTS_ACQUIRED 32000 NM:NMR_RESULTS_FILE ST001090_AN001775_Results.txt UNITS:ppm #END