#METABOLOMICS WORKBENCH TatianaJoao_20231120_025734 DATATRACK_ID:4470 STUDY_ID:ST002982 ANALYSIS_ID:AN004901 PROJECT_ID:PR001307 VERSION 1 CREATED_ON November 20, 2023, 3:56 am #PROJECT PR:PROJECT_TITLE Biochemical Impact of Platinum and Palladium-based Anticancer Agents – PR:PROJECT_TITLE BioIMPACT PR:PROJECT_TYPE NMR-based metabolomics PR:PROJECT_SUMMARY Platinum (Pt(II)) drugs, e.g. cisplatin (cDDP), are some of the most used PR:PROJECT_SUMMARY chemotherapeutic agents, yet tumor acquired resistance and high toxicity are PR:PROJECT_SUMMARY still current drawbacks. Palladium (Pd(II))-complexes are alternatives due to PR:PROJECT_SUMMARY similar metal coordination and promising cytotoxic properties. Metabolomics can PR:PROJECT_SUMMARY measure the metabolic response of both drug-exposed cells and tissues, unveiling PR:PROJECT_SUMMARY insight into drug mechanisms and metabolic markers of drug efficacy, toxicity PR:PROJECT_SUMMARY and resistance. The present 1H NMR metabolomics study aims to (i) describe the PR:PROJECT_SUMMARY polar endometabolome of both MDA-MB-231 cDDP-sensitve and cDDP-resistant cell PR:PROJECT_SUMMARY lines, which are representative of Triple-Negative Breast Cancer, and (ii) PR:PROJECT_SUMMARY characterize the metabolic profile of both cell types exposed to the novel PR:PROJECT_SUMMARY Pd(II)-spermine complex (Pd2Spm), in comparison with cDDP signature, describing PR:PROJECT_SUMMARY possible biomarker patterns of tumor resistance and therapy response. PR:INSTITUTE University of Aveiro PR:DEPARTMENT Department of Chemistry and CICECO-Aveiro Institute of Materials PR:LAST_NAME Carneiro PR:FIRST_NAME Tatiana João PR:ADDRESS Campus Universitário de Santiago, Aveiro, Aveiro, 3810-193, Portugal PR:EMAIL tatiana.joao@ua.pt PR:PHONE +351 234 370 200 PR:FUNDING_SOURCE This work was developed within the CICECO-Aveiro Institute of Materials project PR:FUNDING_SOURCE (UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020) financed by national funds PR:FUNDING_SOURCE through the FCT/MTES (PIDDAC). We also acknowledge funds from POCentro, Portugal PR:FUNDING_SOURCE 2020 and European Community through the FEDER and by the Portuguese Foundation PR:FUNDING_SOURCE for Science and Technology (FCT) through LAQV/REQUIMTE FCT UIDB/50006/2020, PR:FUNDING_SOURCE UIDB/00070/2020 and POCI-01-0145-FEDER-0016786. We are grateful to the PR:FUNDING_SOURCE Portuguese National NMR Network (PTNMR), supported by FCT funds as the NMR PR:FUNDING_SOURCE spectrometer used is part of PTNMR and partially supported by Infrastructure PR:FUNDING_SOURCE Project No. 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL, PR:FUNDING_SOURCE and the FCT through PIDDAC). We also acknowledge FCT for Ph.D. grants PR:FUNDING_SOURCE SFRH/BD/145920/2019 and PD/BD/135460/2017, both grants funded by the European PR:FUNDING_SOURCE Social Fund of the European Union and national FCT/MCTES funds. #STUDY ST:STUDY_TITLE Metabolic characterization of the polar endometabolome of Triple-Negative Breast ST:STUDY_TITLE Cancer parental and cDDP-resistant cells (part 2) ST:STUDY_TYPE NMR-based metabolomics of polar endometabolome of cultured cells ST:STUDY_SUMMARY Platinum (Pt(II)) drugs, e.g. cisplatin (cDDP), are some of the most used ST:STUDY_SUMMARY chemotherapeutic agents, yet tumor acquired resistance and high toxicity are ST:STUDY_SUMMARY still current drawbacks. Metabolomics can measure the metabolic response of ST:STUDY_SUMMARY drug-exposed cells, unveiling insight into drug mechanisms and metabolic markers ST:STUDY_SUMMARY of drug efficacy, toxicity and resistance. The present 1H NMR metabolomics study ST:STUDY_SUMMARY aims to describe the effects of cDDP and Pd2Spm on the polar endometabolome of ST:STUDY_SUMMARY both MDA-MB-231 cDDP-sensitive and cDDP-resistant cell lines, aiming to describe ST:STUDY_SUMMARY metabolic markers of (i) resistance upon cDDP treatment, and (ii) the effect of ST:STUDY_SUMMARY Pd2Spm on the established cDDP-resistant cells. The former observations will ST:STUDY_SUMMARY give helpful insights about the metabolic features of cDDP-resistance during ST:STUDY_SUMMARY treatment, and enlighten on the potential role of Pd2Spm in metabolically ST:STUDY_SUMMARY affecting/tackling cDDP-resistance. ST:INSTITUTE University of Aveiro ST:DEPARTMENT Department of Chemistry and CICECO-Aveiro Institute of Materials ST:LAST_NAME Carneiro ST:FIRST_NAME Tatiana João ST:ADDRESS Campus Universitário de Santiago, Aveiro, Aveiro, 3810-193, Portugal ST:EMAIL tatiana.joao@ua.pt ST:PHONE +351 234 370 200 ST:NUM_GROUPS 14 ST:TOTAL_SUBJECTS 126 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN MDA-MB-231 cells SU:GENDER Not applicable SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC (Manassas, VA, USA); ATCC HTB-26 SU:CELL_STRAIN_DETAILS Epithelial breast cancer cells; absence of estrogen and progesterone receptors, SU:CELL_STRAIN_DETAILS HER2 overexpression SU:CELL_PASSAGE_NUMBER Inferior to 10 SU:CELL_COUNTS 5 M #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 - R_C0h_EA_11_1_2 Treatment_group:Resistant_Controls_0h RAW_FILE_NAME=R_C0h_EA_11_1_2 SUBJECT_SAMPLE_FACTORS - 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S_B24h_EA_21_1_2 Treatment_group:Sensitive_Pd2Spm_treated_24h RAW_FILE_NAME=S_B24h_EA_21_1_2 SUBJECT_SAMPLE_FACTORS - S_B24h_EA_22_1_2 Treatment_group:Sensitive_Pd2Spm_treated_24h RAW_FILE_NAME=S_B24h_EA_22_1_2 SUBJECT_SAMPLE_FACTORS - S_B24h_EA_23_1_2 Treatment_group:Sensitive_Pd2Spm_treated_24h RAW_FILE_NAME=S_B24h_EA_23_1_2 SUBJECT_SAMPLE_FACTORS - S_B24h_EA_31_1_2 Treatment_group:Sensitive_Pd2Spm_treated_24h RAW_FILE_NAME=S_B24h_EA_31_1_2 SUBJECT_SAMPLE_FACTORS - S_B24h_EA_32_1_2 Treatment_group:Sensitive_Pd2Spm_treated_24h RAW_FILE_NAME=S_B24h_EA_32_1_2 SUBJECT_SAMPLE_FACTORS - S_B24h_EA_33_1_2 Treatment_group:Sensitive_Pd2Spm_treated_24h RAW_FILE_NAME=S_B24h_EA_33_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_11_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_11_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_12_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_12_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_13_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_13_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_21_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_21_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_22_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_22_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_23_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_23_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_31_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_31_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_32_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_32_1_2 SUBJECT_SAMPLE_FACTORS - S_C48h_EA_33_1_2 Treatment_group:Sensitive_Controls_48h RAW_FILE_NAME=S_C48h_EA_33_1_2 SUBJECT_SAMPLE_FACTORS - S_A48h_EA_11_1_2 Treatment_group:Sensitive_cDDP_treated_48h RAW_FILE_NAME=S_A48h_EA_11_1_2 SUBJECT_SAMPLE_FACTORS - S_A48h_EA_12_1_2 Treatment_group:Sensitive_cDDP_treated_48h RAW_FILE_NAME=S_A48h_EA_12_1_2 SUBJECT_SAMPLE_FACTORS - S_A48h_EA_13_1_2 Treatment_group:Sensitive_cDDP_treated_48h RAW_FILE_NAME=S_A48h_EA_13_1_2 SUBJECT_SAMPLE_FACTORS - S_A48h_EA_21_1_2 Treatment_group:Sensitive_cDDP_treated_48h RAW_FILE_NAME=S_A48h_EA_21_1_2 SUBJECT_SAMPLE_FACTORS - S_A48h_EA_22_1_2 Treatment_group:Sensitive_cDDP_treated_48h RAW_FILE_NAME=S_A48h_EA_22_1_2 SUBJECT_SAMPLE_FACTORS - S_A48h_EA_23_1_2 Treatment_group:Sensitive_cDDP_treated_48h RAW_FILE_NAME=S_A48h_EA_23_1_2 SUBJECT_SAMPLE_FACTORS - S_A48h_EA_31_1_2 Treatment_group:Sensitive_cDDP_treated_48h RAW_FILE_NAME=S_A48h_EA_31_1_2 SUBJECT_SAMPLE_FACTORS - S_A48h_EA_32_1_2 Treatment_group:Sensitive_cDDP_treated_48h RAW_FILE_NAME=S_A48h_EA_32_1_2 SUBJECT_SAMPLE_FACTORS - S_A48h_EA_33_1_2 Treatment_group:Sensitive_cDDP_treated_48h RAW_FILE_NAME=S_A48h_EA_33_1_2 SUBJECT_SAMPLE_FACTORS - S_B48h_EA_11_1_2 Treatment_group:Sensitive_Pd2Spm_treated_48h RAW_FILE_NAME=S_B48h_EA_11_1_2 SUBJECT_SAMPLE_FACTORS - S_B48h_EA_12_1_2 Treatment_group:Sensitive_Pd2Spm_treated_48h RAW_FILE_NAME=S_B48h_EA_12_1_2 SUBJECT_SAMPLE_FACTORS - S_B48h_EA_13_1_2 Treatment_group:Sensitive_Pd2Spm_treated_48h RAW_FILE_NAME=S_B48h_EA_13_1_2 SUBJECT_SAMPLE_FACTORS - S_B48h_EA_21_1_2 Treatment_group:Sensitive_Pd2Spm_treated_48h RAW_FILE_NAME=S_B48h_EA_21_1_2 SUBJECT_SAMPLE_FACTORS - S_B48h_EA_22_1_2 Treatment_group:Sensitive_Pd2Spm_treated_48h RAW_FILE_NAME=S_B48h_EA_22_1_2 SUBJECT_SAMPLE_FACTORS - S_B48h_EA_23_1_2 Treatment_group:Sensitive_Pd2Spm_treated_48h RAW_FILE_NAME=S_B48h_EA_23_1_2 SUBJECT_SAMPLE_FACTORS - S_B48h_EA_31_1_2 Treatment_group:Sensitive_Pd2Spm_treated_48h RAW_FILE_NAME=S_B48h_EA_31_1_2 SUBJECT_SAMPLE_FACTORS - S_B48h_EA_32_1_2 Treatment_group:Sensitive_Pd2Spm_treated_48h RAW_FILE_NAME=S_B48h_EA_32_1_2 SUBJECT_SAMPLE_FACTORS - S_B48h_EA_33_1_2 Treatment_group:Sensitive_Pd2Spm_treated_48h RAW_FILE_NAME=S_B48h_EA_33_1_2 #COLLECTION CO:COLLECTION_SUMMARY MDA-MB-231 parental and MDA-MB-231/R cDDP-resistant cell lines were seeded at a CO:COLLECTION_SUMMARY density of 3 × 10^4 cells/cm2 onto 13.55 cm diameter Petri dishes, cultured in CO:COLLECTION_SUMMARY a humidified atmosphere of 5% CO2 at 37 ◦C and allowed to adhere for 24 h (t = CO:COLLECTION_SUMMARY 0 h). After this, the experiment was initiated by adding stock solutions of each CO:COLLECTION_SUMMARY drug to achieve the corresponding half of maximal inhibitory effect IC50 values: CO:COLLECTION_SUMMARY 1.0 µM cDDP and 7.9 μM Pd2Spm. Phosphate-buffer saline was used in control CO:COLLECTION_SUMMARY groups. Then, cells were incubated and collected at t = 24 h and t = 48 h, with CO:COLLECTION_SUMMARY basis on the population (25.5 ± 0.9 h and 30.6 ± 1.1 h for MDA-MB-231 and CO:COLLECTION_SUMMARY MDA-MB-231/R cells, respectively). At each time-point, cells were harvested CO:COLLECTION_SUMMARY using a 0.25% (v/v) trypsin-EDTA solution, washed twice with PBS and centrifuged CO:COLLECTION_SUMMARY (300 g, 5 min, 20 ◦C). The cell pellet was directly stored at − 80 ◦C CO:COLLECTION_SUMMARY until analysis. Three independent experiments with triplicates were performed CO:COLLECTION_SUMMARY for each cell type and time-point. CO:SAMPLE_TYPE Epithelial cells CO:COLLECTION_METHOD Trypsinization for cells harvesting followed by pellet collection and storage at CO:COLLECTION_METHOD − 80 ◦C (until extract prepatation) CO:COLLECTION_FREQUENCY One collection per sample replicate CO:COLLECTION_DURATION Between 2 and 5 minutes CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Cells were treated during culture time by adding drugs stock solution to the TR:TREATMENT_SUMMARY growth culture medium. Briefly, after seeding, cells were allowed to adhere for TR:TREATMENT_SUMMARY 24 h (t = 0 h). After this, the experiment was initiated by adding stock TR:TREATMENT_SUMMARY solutions of each drug to achieve the corresponding half of maximal inhibitory TR:TREATMENT_SUMMARY effect IC50 values: 1.0 µM cDDP and 7.9 μM Pd2Spm. Phosphate-buffered saline TR:TREATMENT_SUMMARY (PBS) was used in control groups. Then, cells were incubated and collected at t TR:TREATMENT_SUMMARY = 24 h and t = 48 h, with basis on the population (25.5 ± 0.9 h and 30.6 ± 1.1 TR:TREATMENT_SUMMARY h for MDA-MB-231 and MDA-MB-231/R cells, respectively). TR:TREATMENT_COMPOUND cDDP or Pd2Spm (PBS for controls) TR:TREATMENT_ROUTE Dissolution of drugs stock solutions into the culture medium TR:TREATMENT_DOSE 1.0 µM cDDP and 7.9 μM Pd2Spm TR:TREATMENT_DOSEVOLUME 5.5 mL TR:TREATMENT_DOSEDURATION 24 h and 48 h TR:TREATMENT_VEHICLE Phosphate-buffered saline (PBS) TR:CELL_MEDIA Dulbecco’s Modified Eagle’s Medium—high-glucose cell growth medium TR:CELL_MEDIA (DMEM-HG) TR:CELL_ENVIR_COND Humidified atmosphere of 5% CO2 at 37 ◦C TR:CELL_HARVESTING Tripsinization TR:CELL_PCT_CONFLUENCE ~ 70 to 80% #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The cellular polar extracts were extracted using a biphasic extraction method of SP:SAMPLEPREP_SUMMARY methanol/chloroform/water. Basically, cell pellets were resuspended in 650 µL SP:SAMPLEPREP_SUMMARY of 80% (v/v) methanol-miliQ water solution, transferred to microcentrifuge tubes SP:SAMPLEPREP_SUMMARY with 150 mg of glass beads, and vortexed for 5 min. Subsequently, 260 µL of SP:SAMPLEPREP_SUMMARY 100% chloroform and 260 µL of 100% chloroform plus 220 µL MiliQ water were SP:SAMPLEPREP_SUMMARY added to samples, which were vortexed for 5 min between solvents addition. The SP:SAMPLEPREP_SUMMARY samples were kept at − 20 °C for 10 min and centrifuged. The aqueous phase of SP:SAMPLEPREP_SUMMARY the resulting extract was collected into a new tube, vacuum-dried and stored at SP:SAMPLEPREP_SUMMARY − 80 °C until the NMR analysis. All samples and reagents were kept in ice SP:SAMPLEPREP_SUMMARY during the extraction procedure. Before NMR analysis, the dry aqueous extracts SP:SAMPLEPREP_SUMMARY were suspended in 650 µL of 100 mM sodium phosphate buffer (pH 7.4, in D2O SP:SAMPLEPREP_SUMMARY containing 0.25% 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP) for chemical SP:SAMPLEPREP_SUMMARY shift referencing) and transferred into 5 mm NMR tubes. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACTION_METHOD Biphasic extraction method of methanol/chloroform/water SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Dry aqueous extracts were suspended in 650 µL of 100 mM sodium phosphate buffer SP:SAMPLE_RESUSPENSION (pH 7.4, in D2O containing 0.25% 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid SP:SAMPLE_RESUSPENSION (TSP) for chemical shift referencing) #ANALYSIS AN:ANALYSIS_TYPE NMR AN:OPERATOR_NAME Tatiana João Carneiro AN:SOFTWARE_VERSION Topspin 3.6.5 AN:ACQUISITION_DATE March 2023 #NMR NM:INSTRUMENT_NAME Avance III HD NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:NMR_COMMENTS 1st subfolder contains raw spectra; 2nd subfolder contains manually processed NM:NMR_COMMENTS spectra NM:FIELD_FREQUENCY_LOCK D2O NM:SPECTROMETER_FREQUENCY 500 MHz NM:NMR_PROBE 5 mm TXI probe NM:NMR_SOLVENT 100% D2O NM:NMR_TUBE_SIZE 5mm NM:SHIMMING_METHOD Automatic and manual NM:PULSE_SEQUENCE "Noesypr1d" from Bruker library NM:RECEIVER_GAIN 203 NM:CHEMICAL_SHIFT_REF_CPD TSP NM:TEMPERATURE 298K NM:NUMBER_OF_SCANS 512 NM:DUMMY_SCANS 4 s NM:ACQUISITION_TIME 2.34 s NM:RELAXATION_DELAY 2 s NM:SPECTRAL_WIDTH 7002.801 Hz NM:NUM_DATA_POINTS_ACQUIRED 32 k NM:REAL_DATA_POINTS 64 k NM:LINE_BROADENING 0.3 Hz (multiplication) NM:BASELINE_CORRECTION_METHOD Manual NM:CHEMICAL_SHIFT_REF_STD TSP NM:NMR_RESULTS_FILE ST002982_AN004901_Results.txt UNITS:ppm #END