#METABOLOMICS WORKBENCH VeronicaGhini_20220322_130847 DATATRACK_ID:3133 STUDY_ID:ST002116 ANALYSIS_ID:AN003464 PROJECT_ID:PR001341 VERSION 1 CREATED_ON March 23, 2022, 2:34 am #PROJECT PR:PROJECT_TITLE Comparative NMR metabolomics of the responses of A2780 human ovarian cancer PR:PROJECT_TITLE cells to clinically established Pt based drugs PR:PROJECT_SUMMARY Pt based drugs play a very important role in current cancer treatment; yet their PR:PROJECT_SUMMARY cellular and mechanistic aspects are not fully understood. NMR metabolomics PR:PROJECT_SUMMARY provides a powerful tool to investigate the metabolic perturbations induced by PR:PROJECT_SUMMARY Pt drugs in cancer cells and decipher their meaning in relation to the presumed PR:PROJECT_SUMMARY molecular mechanisms. We have carried out a systematic and comparative NMR PR:PROJECT_SUMMARY metabolomics study to analyze the responses of A2780 human ovarian cancer cells PR:PROJECT_SUMMARY to the main clinically established Pt drugs, i.e. cisplatin, carboplatin and PR:PROJECT_SUMMARY oxaliplatin, with a particular attention for the oxaliplatin/cisplatin PR:PROJECT_SUMMARY comparison in view of recently described mechanistic differences. Notably, NMR PR:PROJECT_SUMMARY analysis revealed some moderate and consistent changes in the metabolomic PR:PROJECT_SUMMARY profiles of A2780 cells treated with the 3 Pt drugs with respect to controls but PR:PROJECT_SUMMARY only very small differences among them. Beyond the expected alterations at the PR:PROJECT_SUMMARY level of the nucleic acids the observed changes highlight in all cases induction PR:PROJECT_SUMMARY of a significant ER stress. Owing to the clinical relevance of platinum PR:PROJECT_SUMMARY resistance the behavior of a cisplatin resistant A2780 cancer cell line upon PR:PROJECT_SUMMARY cisplatin treatment was also evaluated. PR:INSTITUTE University of Florence PR:DEPARTMENT Department of Chemistry PR:LABORATORY Metabolomics PR:LAST_NAME Ghini PR:FIRST_NAME Veronica PR:ADDRESS via Luigi Sacconi 6, 50019, Sesto Fiorentino, Italy PR:EMAIL ghini@cerm.unifi.it PR:PHONE +39 3922800462 #STUDY ST:STUDY_TITLE Comparative NMR metabolomics of the responses of A2780 human ovarian cancer ST:STUDY_TITLE cells to clinically established Pt based drugs ST:STUDY_SUMMARY Pt based drugs play a very important role in current cancer treatment; yet their ST:STUDY_SUMMARY cellular and mechanistic aspects are not fully understood. NMR metabolomics ST:STUDY_SUMMARY provides a powerful tool to investigate the metabolic perturbations induced by ST:STUDY_SUMMARY Pt drugs in cancer cells and decipher their meaning in relation to the presumed ST:STUDY_SUMMARY molecular mechanisms. We have carried out a systematic and comparative NMR ST:STUDY_SUMMARY metabolomics study to analyze the responses of A2780 human ovarian cancer cells ST:STUDY_SUMMARY to the main clinically established Pt drugs, i.e. cisplatin, carboplatin and ST:STUDY_SUMMARY oxaliplatin, with a particular attention for the oxaliplatin/cisplatin ST:STUDY_SUMMARY comparison in view of recently described mechanistic differences. Notably, NMR ST:STUDY_SUMMARY analysis revealed some moderate and consistent changes in the metabolomic ST:STUDY_SUMMARY profiles of A2780 cells treated with the 3 Pt drugs with respect to controls but ST:STUDY_SUMMARY only very small differences among them. Beyond the expected alterations at the ST:STUDY_SUMMARY level of the nucleic acids the observed changes highlight in all cases induction ST:STUDY_SUMMARY of a significant ER stress. Owing to the clinical relevance of platinum ST:STUDY_SUMMARY resistance the behavior of a cisplatin resistant A2780 cancer cell line upon ST:STUDY_SUMMARY cisplatin treatment was also evaluated. ST:INSTITUTE University of Florence ST:DEPARTMENT Department of Chemistry ST:LABORATORY Metabolomics ST:LAST_NAME Ghini ST:FIRST_NAME Veronica ST:ADDRESS via Luigi Sacconi 6 ST:EMAIL turano@cerm.unifi.it ST:PHONE +390554574266 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS A2780 ovarian cancer cells #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 - Cell-Carbo-24h-1 sample_type:lysate | treatment_type:carboplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-Carbo-24h-1 SUBJECT_SAMPLE_FACTORS - Cell-Carbo-24h-2 sample_type:lysate | treatment_type:carboplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-Carbo-24h-2 SUBJECT_SAMPLE_FACTORS - Cell-CARBO-24h-4 sample_type:lysate | treatment_type:carboplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-CARBO-24h-4 SUBJECT_SAMPLE_FACTORS - Cell-Carbo-48h-1 sample_type:lysate | treatment_type:carboplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Carbo-48h-1 SUBJECT_SAMPLE_FACTORS - Cell-Carbo-48h-3 sample_type:lysate | treatment_type:carboplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Carbo-48h-3 SUBJECT_SAMPLE_FACTORS - Cell-Carbo-48h-4 sample_type:lysate | treatment_type:carboplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Carbo-48h-4 SUBJECT_SAMPLE_FACTORS - Cell-Cis-24h-1 sample_type:lysate | treatment_type:cisplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-Cis-24h-1 SUBJECT_SAMPLE_FACTORS - Cell-Cis-24h-2 sample_type:lysate | treatment_type:cisplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-Cis-24h-2 SUBJECT_SAMPLE_FACTORS - Cell-CIS-24h-4 sample_type:lysate | treatment_type:cisplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-CIS-24h-4 SUBJECT_SAMPLE_FACTORS - Cell-Cis-48h-1 sample_type:lysate | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Cis-48h-1 SUBJECT_SAMPLE_FACTORS - Cell-Cis-48h-3 sample_type:lysate | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Cis-48h-3 SUBJECT_SAMPLE_FACTORS - Cell-Cis-48h-4 sample_type:lysate | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Cis-48h-4 SUBJECT_SAMPLE_FACTORS - Cell-Ctr-24h-1 sample_type:lysate | treatment_type:control | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-Ctr-24h-1 SUBJECT_SAMPLE_FACTORS - Cell-Ctr-24h-2 sample_type:lysate | treatment_type:control | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-Ctr-24h-2 SUBJECT_SAMPLE_FACTORS - Cell-Ctr-24h-4 sample_type:lysate | treatment_type:control | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-Ctr-24h-4 SUBJECT_SAMPLE_FACTORS - Cell-Ctr-48h-1 sample_type:lysate | treatment_type:control | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Ctr-48h-1 SUBJECT_SAMPLE_FACTORS - Cell-Ctr-48h-3 sample_type:lysate | treatment_type:control | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Ctr-48h-3 SUBJECT_SAMPLE_FACTORS - Cell-Ctr-48h-4 sample_type:lysate | treatment_type:control | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Ctr-48h-4 SUBJECT_SAMPLE_FACTORS - Cell-Oxa-24h-1 sample_type:lysate | treatment_type:oxaliplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-Oxa-24h-1 SUBJECT_SAMPLE_FACTORS - Cell-Oxa-24h-2 sample_type:lysate | treatment_type:oxaliplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-Oxa-24h-2 SUBJECT_SAMPLE_FACTORS - Cell-OXA-24h-4 sample_type:lysate | treatment_type:oxaliplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=Cell-OXA-24h-4 SUBJECT_SAMPLE_FACTORS - Cell-Oxa-48h-1 sample_type:lysate | treatment_type:oxaliplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Oxa-48h-1 SUBJECT_SAMPLE_FACTORS - Cell-Oxa-48h-3 sample_type:lysate | treatment_type:oxaliplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Oxa-48h-3 SUBJECT_SAMPLE_FACTORS - Cell-Oxa-48h-4 sample_type:lysate | treatment_type:oxaliplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=Cell-Oxa-48h-4 SUBJECT_SAMPLE_FACTORS - Cell-Cis-R-48h-1-1411 sample_type:lysate | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=Cell-Cis-R-48h-1-1411 SUBJECT_SAMPLE_FACTORS - Cell-Cis-R-48h-2-2110 sample_type:lysate | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=Cell-Cis-R-48h-2-2110 SUBJECT_SAMPLE_FACTORS - Cell-Cis-R-48h-3-2810 sample_type:lysate | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=Cell-Cis-R-48h-3-2810 SUBJECT_SAMPLE_FACTORS - Cell-Cont-R-48h-1-1411 sample_type:lysate | treatment_type:control | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=Cell-Cont-R-48h-1-1411 SUBJECT_SAMPLE_FACTORS - Cell-Cont-R-48h-2-2110 sample_type:lysate | treatment_type:control | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=Cell-Cont-R-48h-2-2110 SUBJECT_SAMPLE_FACTORS - Cell-Cont-R-48h-3-2810 sample_type:lysate | treatment_type:control | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=Cell-Cont-R-48h-3-2810 SUBJECT_SAMPLE_FACTORS - SN-CARBO-24h-1 sample_type:medium | treatment_type:carboplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-CARBO-24h-1 SUBJECT_SAMPLE_FACTORS - SN-CARBO-24h-2 sample_type:medium | treatment_type:carboplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-CARBO-24h-2 SUBJECT_SAMPLE_FACTORS - SN-Carbo-24h-4 sample_type:medium | treatment_type:carboplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-Carbo-24h-4 SUBJECT_SAMPLE_FACTORS - SN-CARBO-48h-1 sample_type:medium | treatment_type:carboplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-CARBO-48h-1 SUBJECT_SAMPLE_FACTORS - SN-CARBO-48h-3 sample_type:medium | treatment_type:carboplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-CARBO-48h-3 SUBJECT_SAMPLE_FACTORS - SN-CARBO-48h-4 sample_type:medium | treatment_type:carboplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-CARBO-48h-4 SUBJECT_SAMPLE_FACTORS - SN-CIS-24h-1 sample_type:medium | treatment_type:cisplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-CIS-24h-1 SUBJECT_SAMPLE_FACTORS - SN-CIS-24h-2 sample_type:medium | treatment_type:cisplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-CIS-24h-2 SUBJECT_SAMPLE_FACTORS - SN-Cis-24h-4 sample_type:medium | treatment_type:cisplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-Cis-24h-4 SUBJECT_SAMPLE_FACTORS - SN-CIS-48h-1 sample_type:medium | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-CIS-48h-1 SUBJECT_SAMPLE_FACTORS - SN-CIS-48h-3 sample_type:medium | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-CIS-48h-3 SUBJECT_SAMPLE_FACTORS - SN-CIS-48h-4 sample_type:medium | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-CIS-48h-4 SUBJECT_SAMPLE_FACTORS - SN-CTR-24h-1 sample_type:medium | treatment_type:control | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-CTR-24h-1 SUBJECT_SAMPLE_FACTORS - SN-CTR-24h-2 sample_type:medium | treatment_type:control | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-CTR-24h-2 SUBJECT_SAMPLE_FACTORS - SN-Ctr-24h-4 sample_type:medium | treatment_type:control | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-Ctr-24h-4 SUBJECT_SAMPLE_FACTORS - SN-CTR-48h-1 sample_type:medium | treatment_type:control | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-CTR-48h-1 SUBJECT_SAMPLE_FACTORS - SN-CTR-48h-3 sample_type:medium | treatment_type:control | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-CTR-48h-3 SUBJECT_SAMPLE_FACTORS - SN-CTR-48h-4 sample_type:medium | treatment_type:control | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-CTR-48h-4 SUBJECT_SAMPLE_FACTORS - SN-OXA-24h-1 sample_type:medium | treatment_type:oxaliplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-OXA-24h-1 SUBJECT_SAMPLE_FACTORS - SN-OXA-24h-2 sample_type:medium | treatment_type:oxaliplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-OXA-24h-2 SUBJECT_SAMPLE_FACTORS - SN-Oxa-24h-4 sample_type:medium | treatment_type:oxaliplatin | treatment_time:24 | cell_line:A2780 RAW_FILE_NAME=SN-Oxa-24h-4 SUBJECT_SAMPLE_FACTORS - SN-OXA-48h-1 sample_type:medium | treatment_type:oxaliplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-OXA-48h-1 SUBJECT_SAMPLE_FACTORS - SN-OXA-48h-3 sample_type:medium | treatment_type:oxaliplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-OXA-48h-3 SUBJECT_SAMPLE_FACTORS - SN-OXA-48h-4 sample_type:medium | treatment_type:oxaliplatin | treatment_time:48 | cell_line:A2780 RAW_FILE_NAME=SN-OXA-48h-4 SUBJECT_SAMPLE_FACTORS - SN-CIS-R-48h-1-1411 sample_type:medium | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=SN-CIS-R-48h-1-1411 SUBJECT_SAMPLE_FACTORS - SN-CIS-R-48h-2-2110 sample_type:medium | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=SN-CIS-R-48h-2-2110 SUBJECT_SAMPLE_FACTORS - SN-CIS-R-48h-3-2810 sample_type:medium | treatment_type:cisplatin | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=SN-CIS-R-48h-3-2810 SUBJECT_SAMPLE_FACTORS - SN-Cont-R-48h-1-1411 sample_type:medium | treatment_type:control | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=SN-Cont-R-48h-1-1411 SUBJECT_SAMPLE_FACTORS - SN-Cont-R-48h-2-2110 sample_type:medium | treatment_type:control | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=SN-Cont-R-48h-2-2110 SUBJECT_SAMPLE_FACTORS - SN-Cont-R-48h-3-2810 sample_type:medium | treatment_type:control | treatment_time:48 | cell_line:A2780cp RAW_FILE_NAME=SN-Cont-R-48h-3-2810 #COLLECTION CO:COLLECTION_SUMMARY A2780 cells were purchase from Sigma-Aldrich and derived from the European CO:COLLECTION_SUMMARY Collection of Authenticated Cell Culture (human ovarian carcinoma; catalogue CO:COLLECTION_SUMMARY no.: 93112519; lot no.13J012, passage no.: P + 9). A2780cp cells were kindly CO:COLLECTION_SUMMARY provided by Dr Simona Bracini and were obtained via prolonged exposition to CO:COLLECTION_SUMMARY sub-lethal dose of cisplatin. Cells were maintained in RPMI1640 medium CO:COLLECTION_SUMMARY supplemented with 2 mM glutamine, 10% of FCS and antibiotics at 37 °C in a 5% CO:COLLECTION_SUMMARY CO2 atmosphere and sub-cultured twice weekly. Split 1:5 (3–6 × 10^4 cells per CO:COLLECTION_SUMMARY mL). CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY For drug treatment, 2.7 *10^6 A2780 cells were seeded in p100 plate and after 24 TR:TREATMENT_SUMMARY hours were exposed to a concentration of the three Pt-drugs, i.e. CIS, OXA and TR:TREATMENT_SUMMARY CARBO equal to their 72 h-exposure IC50 value (2.5, 55 and 0.46 uM, TR:TREATMENT_SUMMARY respectively). The drug incubations were stopped at two different time points, TR:TREATMENT_SUMMARY 24 h and 48 h. The A2780cp cells were exposed to a concentration of cisplatin TR:TREATMENT_SUMMARY equal to their 72 h-exposure IC50 value (15 uM). The drug incubation was stopped TR:TREATMENT_SUMMARY after 48 h of treatment. TR:TREATMENT Cisplatin, carboplatin and oxaliplatin TR:CELL_MEDIA RPMI1640 #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen samples were thawed at room temperature and shaken before use. For cell SP:SAMPLEPREP_SUMMARY lysates, 50 μl of 2H2O were added to 450 μl of each lysate sample. In the case SP:SAMPLEPREP_SUMMARY of cell culture media, 300 μl of sodium phosphate buffer (70 mM Na2HPO4; 20% SP:SAMPLEPREP_SUMMARY v/v 2H2O; 4.6 mM TMSP, pH 7.4) was added to 300 μl of each medium sample. The SP:SAMPLEPREP_SUMMARY mixtures were homogenized by vortexing for 30 s and transferred into 5 mm NMR SP:SAMPLEPREP_SUMMARY tubes (Bruker BioSpin srl) for analysis. #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME Metabolomics #NMR NM:INSTRUMENT_NAME Bruker Advance 600 MHZ NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_TUBE_SIZE 5,00 mm NM:WATER_SUPPRESSION water presaturation NM:TEMPERATURE 300 K for lysates and 31o for media NM:NMR_RESULTS_FILE ST002116_AN003464_Results.txt UNITS:ppm #END