#METABOLOMICS WORKBENCH antogar_20240508_085543 DATATRACK_ID:4822 STUDY_ID:ST003195 ANALYSIS_ID:AN005244 PROJECT_ID:PR001991 VERSION 1 CREATED_ON May 8, 2024, 10:39 am #PROJECT PR:PROJECT_TITLE Unveiling cellular changes in leukaemia cell line K-562 after cannabidiol PR:PROJECT_TITLE treatment through lipidomics PR:PROJECT_SUMMARY The present study was aimed at revealing the metabolic changes that occurred in PR:PROJECT_SUMMARY the cellular lipid pattern of acute and chronic myeloid leukaemia cells PR:PROJECT_SUMMARY following treatment with cannabidiol (CBD). CBD is a non-psychoactive compound PR:PROJECT_SUMMARY present in Cannabis sativa L., which has shown an antiproliferative action in PR:PROJECT_SUMMARY these type of cancer cells, to determine significant alterations of the cell PR:PROJECT_SUMMARY metabolism attributable to the induction of apoptosis, previously observed from PR:PROJECT_SUMMARY in vitro studies. Control and treated cells of chronic myeloid leukaemia (K562) PR:PROJECT_SUMMARY were studied through an untargeted lipidomics approach. Treatment was carried PR:PROJECT_SUMMARY out with CBD at a concentration of 23 µM CBD for 48 h. After the extraction of PR:PROJECT_SUMMARY the lipid content from cell lysates, the samples were analysed by PR:PROJECT_SUMMARY UHPLC-QTOF-MS/MS both in the positive and the negative ionization modes. PR:INSTITUTE Universidad CEU- San Pablo PR:LABORATORY CEMBIO PR:LAST_NAME Garcia Fernández PR:FIRST_NAME Antonia PR:ADDRESS Urb. Montepríncipe., Alcorcón, Madrid, 28925, Spain PR:EMAIL antogar@ceu.es PR:PHONE (+34) 91 372 47 69 #STUDY ST:STUDY_TITLE Unveiling cellular changes in leukaemia cell line K-562 after cannabidiol ST:STUDY_TITLE treatment through lipidomics ST:STUDY_SUMMARY The present study was aimed at revealing the metabolic changes that occurred in ST:STUDY_SUMMARY the cellular lipid pattern of acute and chronic myeloid leukaemia cells ST:STUDY_SUMMARY following treatment with cannabidiol (CBD). CBD is a non-psychoactive compound ST:STUDY_SUMMARY present in Cannabis sativa L., which has shown an antiproliferative action in ST:STUDY_SUMMARY these type of cancer cells, to determine significant alterations of the cell ST:STUDY_SUMMARY metabolism attributable to the induction of apoptosis, previously observed from ST:STUDY_SUMMARY in vitro studies. Control and treated cells of chronic myeloid leukaemia (K562) ST:STUDY_SUMMARY were studied through an untargeted lipidomics approach. Treatment was carried ST:STUDY_SUMMARY out with CBD at a concentration of 23 µM CBD for 48 h. After the extraction of ST:STUDY_SUMMARY the lipid content from cell lysates, the samples were analysed by ST:STUDY_SUMMARY UHPLC-QTOF-MS/MS both in the positive and the negative ionization modes. ST:INSTITUTE Universidad CEU- San Pablo ST:LABORATORY CEMBIO ST:LAST_NAME Garcia Fernández ST:FIRST_NAME Antonia ST:ADDRESS Urb. Montepríncipe., Alcorcón, Madrid, 28925, Spain ST:EMAIL antogar@ceu.es ST:PHONE (+34) 91 372 47 69 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 10 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Not applicable #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 - K562_CBD1 Sample source:Leukemia cell line K-562 | Treatment:CBD RAW_FILE_NAME(Raw file name LCMSPOS)=K562_CBD1_POS; RAW_FILE_NAME(Raw file name LCMSNEG)=K562_CBD1_NEG SUBJECT_SAMPLE_FACTORS - K562_CBD2 Sample source:Leukemia cell line K-562 | Treatment:CBD RAW_FILE_NAME(Raw file name LCMSPOS)=K562_CBD2_POS; RAW_FILE_NAME(Raw file name LCMSNEG)=K562_CBD2_NEG SUBJECT_SAMPLE_FACTORS - K562_CBD3 Sample source:Leukemia cell line K-562 | Treatment:CBD RAW_FILE_NAME(Raw file name LCMSPOS)=K562_CBD3_POS; RAW_FILE_NAME(Raw file name LCMSNEG)=K562_CBD3_NEG SUBJECT_SAMPLE_FACTORS - K562_CBD4 Sample source:Leukemia cell line K-562 | Treatment:CBD RAW_FILE_NAME(Raw file name LCMSPOS)=K562_CBD4_POS; RAW_FILE_NAME(Raw file name LCMSNEG)=K562_CBD4_NEG SUBJECT_SAMPLE_FACTORS - K562_CBD5 Sample source:Leukemia cell line K-562 | Treatment:CBD RAW_FILE_NAME(Raw file name LCMSPOS)=K562_CBD5_POS; RAW_FILE_NAME(Raw file name LCMSNEG)=K562_CBD5_NEG SUBJECT_SAMPLE_FACTORS - K562_CTR1 Sample source:Leukemia cell line K-562 | Treatment:CTR RAW_FILE_NAME(Raw file name LCMSPOS)=K562_CTR1_POS; RAW_FILE_NAME(Raw file name LCMSNEG)=K562_CTR1_NEG SUBJECT_SAMPLE_FACTORS - K562_CTR2 Sample source:Leukemia cell line K-562 | Treatment:CTR RAW_FILE_NAME(Raw file name LCMSPOS)=K562_CTR2_POS; RAW_FILE_NAME(Raw file name LCMSNEG)=K562_CTR2_NEG SUBJECT_SAMPLE_FACTORS - K562_CTR3 Sample source:Leukemia cell line K-562 | Treatment:CTR RAW_FILE_NAME(Raw file name LCMSPOS)=K562_CTR3_POS; RAW_FILE_NAME(Raw file name LCMSNEG)=K562_CTR3_NEG SUBJECT_SAMPLE_FACTORS - K562_CTR4 Sample source:Leukemia cell line K-562 | Treatment:CTR RAW_FILE_NAME(Raw file name LCMSPOS)=K562_CTR4_POS; RAW_FILE_NAME(Raw file name LCMSNEG)=K562_CTR4_NEG SUBJECT_SAMPLE_FACTORS - K562_CTR5 Sample source:Leukemia cell line K-562 | Treatment:CTR RAW_FILE_NAME(Raw file name LCMSPOS)=K562_CTR5_POS; RAW_FILE_NAME(Raw file name LCMSNEG)=K562_CTR5_NEG #COLLECTION CO:COLLECTION_SUMMARY K-562 cells were grown in RPMI medium supplemented with 10% FBS, 2 mM CO:COLLECTION_SUMMARY L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were CO:COLLECTION_SUMMARY cultured in a humidified incubator at 37 °C with 95% humidity and 5% CO2.Cells CO:COLLECTION_SUMMARY were treated with CBD. Cells were washed with cold PBS, centrifuged and the CO:COLLECTION_SUMMARY pellets were stored at - 80°C until the day of the analysis. CO:SAMPLE_TYPE Leukemia cells #TREATMENT TR:TREATMENT_SUMMARY Cells were treated with 23 µM of CBD #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The samples were vortex-mixed for 2 min and 100 µL of cold MeOH (- 20°C) were SP:SAMPLEPREP_SUMMARY added to each replicate for deproteinization. Ultrasound probe (UP 200 S Dr. SP:SAMPLEPREP_SUMMARY Hielscher Ultrasonic Gmbh) was used (0.5 cycles; 80 amplitude x 16 times) to SP:SAMPLEPREP_SUMMARY lyse the cells. Cell lysis was verified using a microscope. Twenty µL of the SP:SAMPLEPREP_SUMMARY working solution of 20 mg/L of C17 sphinganine in methanol were added to 100 µL SP:SAMPLEPREP_SUMMARY of each replicate. After that, 284 µL of methyl tert-butyl ether (MTBE) was SP:SAMPLEPREP_SUMMARY added to each sample to extract hydrophobic compounds. Each replicate were SP:SAMPLEPREP_SUMMARY vortex-mixed for 1 h (room temperature, 10,000 xg). Then, 71 µL of water was SP:SAMPLEPREP_SUMMARY added to each sample and vortex-mixed for 1 min (25°C, 10,000 xg). Samples were SP:SAMPLEPREP_SUMMARY centrifuged for 10 min at 16,000 xg at 4 °C. After centrifugation, 90 µL of SP:SAMPLEPREP_SUMMARY the supernatant were transferred to vials for the analysis by LC-MS in the SP:SAMPLEPREP_SUMMARY positive and in the negative ionization mode #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) CH:SOLVENT_A 90% Water, 10% Methanol; 10 mM ammonium acetate, 0.2 mM ammonium fluoride CH:SOLVENT_B 50% Isopropanol, 30% Methanol, 20% Acetonitrile; 10 mM ammonium acetate, 0.2 mM CH:SOLVENT_B ammonium fluoride CH:FLOW_GRADIENT Started at 70% of B at 0-1 min, 86% B at 3.5-10 min, 100% B at 11-17 min. The CH:FLOW_GRADIENT starting conditions were recovered by minute 17. CH:FLOW_RATE 0.6 mL/min CH:COLUMN_TEMPERATURE 50 °C CH:INTERNAL_STANDARD Sphinganine (D17:0) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The Agilent 6545 QTOF mass spectrometer equipped with a dual AJS ESI ion source MS:MS_COMMENTS was set with the following parameters: 3000 V in the positive ionization mode, MS:MS_COMMENTS 150 V fragment voltage, 50 psi nebulizer gas pressure, 10 L/min at 300°C drying MS:MS_COMMENTS gas flow rate in negative ionization mode, 750 V octopole radio frequency MS:MS_COMMENTS voltage and 65 V skimmer. Data were collected in positive ESI mode, operated in MS:MS_COMMENTS full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. The MS:MS_COMMENTS reference solution contained purine (C5H4N4) at m/z 119.0363 and HP-0921+ MS:MS_COMMENTS acetate (C18H18O6N3P3F24) at m/z 980.0163. These masses were continuously MS:MS_COMMENTS infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split MS:MS_COMMENTS ratio 1:100) to provide a constant mass correction. Data was acquired using MS:MS_COMMENTS Agilent MassHunter Workstation Software LC/MS Data Acquisition for 6200 series MS:MS_COMMENTS TOF/6500 series Q-TOF B 9.0.9044.0 (Agilent Technologies). The raw data were MS:MS_COMMENTS processed using Agilent Technologies MassHunter Profinder B.10.0.2.162 (Santa MS:MS_COMMENTS Clara, United States) to clean the background noise and unrelated ions. MS:MS_RESULTS_FILE ST003195_AN005244_Results.txt UNITS:Corrected areas Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END