Summary of Study ST003151
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001941. The data can be accessed directly via it's Project DOI: 10.21228/M8TM76 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003151 |
| Study Title | BT474 breast cancer cell line grown in 20% D2O-containing RPMI-1640 medium treated with Fasnall and GSK2194069 |
| Study Type | Free fatty acid analysis, D2O tracing |
| Study Summary | BT474 breast cancer cell line grown in 20% D2O-containing medium treated with Fasnall and GSK2194069. Cells were grown for 24 h in RPMI-1640 with 10% dialyzed FBS. |
| Institute | Wistar Institute |
| Department | Molecular and Cellular Oncogenesis Program, Ellen and Ronald Caplan Cancer Center |
| Laboratory | Schug's Lab |
| Last Name | Mukha |
| First Name | Dzmitry |
| Address | 3601 Spruce St, Philadelphia, PA 19104, USA |
| dmukha@wistar.org | |
| Phone | 2154956903 |
| Submit Date | 2024-03-09 |
| Num Groups | 7 |
| Total Subjects | 21 |
| Publications | Submission Pending |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-04-02 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001941 |
| Project DOI: | doi: 10.21228/M8TM76 |
| Project Title: | The shutdown of NADH oxidation via Respiratory Complex I mimics fatty acid biosynthesis inhibition |
| Project Type: | LC-MS Quantitative Analysis |
| Project Summary: | Proliferating cancer cells actively utilize anabolic processes for biomass production, including de novo biosynthesis of amino acids, nucleotides, and fatty acids. The key enzyme of the fatty acid biosynthesis pathway, fatty acid synthase (FASN), is widely recognized as a promising therapeutic target in cancer and other health conditions. Here, we establish a metabolic signature of FASN inhibition using a panel of pharmacological inhibitors (GSK2194069, TVB-2640, TVB-3166, C75, cerulenin, and Fasnall). We find that the activity of some commonly used FASN inhibitors is inconsistent with the metabolic signature of FASN inhibition (accumulation of malonate, succinate, malonyl coenzyme A, succinyl coenzyme A, and other metabolic perturbations). Moreover, we show that one of these putative FASN inhibitors, Fasnall, is a respiratory Complex I inhibitor that mimics FASN inhibition through NADH accumulation and consequent depletion of the tricarboxylic acid cycle metabolites. We demonstrate that Fasnall impairs tumor growth in several oxidative phosphorylation-dependent cancer models, including combination therapy-resistant melanoma patient-derived xenografts. Fasnall administration does not reproduce neurological side effects in mice reported for other Complex I inhibitors. Our results have significant implications for understanding the FASN role in human health and disease and provide evidence of therapeutic potential for Complex I inhibitors with fast systemic clearance. Our findings also highlight the continuing need for validation of small molecule inhibitors to distinguish high-quality chemical probes and to expand the understanding of their application. |
| Institute: | Wistar Institute |
| Department: | Molecular and Cellular Oncogenesis Program, Ellen and Ronald Caplan Cancer Center |
| Laboratory: | Schug's Lab |
| Last Name: | Mukha |
| First Name: | Dzmitry |
| Address: | 3601 Spruce St., Philadelphia, Pennsylvania 19104, USA |
| Email: | dmukha@wistar.org |
| Phone: | +12154956903 |
| Funding Source: | This work was supported by grants from the National Institutes of Health (NIH) National Cancer Institute (NCI) DP2 CA249950-01 (Z.T.S.), NIH NCI P01 CA114046 (Z.T.S.), Melanoma Research Foundation 717173 (Z.T.S.), and Susan G. Komen CCR19608782 (Z.T.S.). |
| Publications: | Submission Pending |
| Contributors: | Dzmitry Mukha, Jena Dessain, Seamus O’Connor, Katherine Pniewski, Fabrizio Bertolazzi, Jeet Patel, Mary Mullins, Zachary T. Schug |
Subject:
| Subject ID: | SU003268 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 60 |
| Gender: | Female |
| Cell Strain Details: | BT-474, breast cancer cell line |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Isotopic Tracer | Drug Treatment |
|---|---|---|---|---|
| SA340953 | 19_FFA_D2O_BT474_GSK0001 | BT-474 cell lipid extract | 20% D2O | 1 nM GSK2194069 |
| SA340954 | 05_FFA_D2O_BT474_GSK0001 | BT-474 cell lipid extract | 20% D2O | 1 nM GSK2194069 |
| SA340955 | 12_FFA_D2O_BT474_GSK0001 | BT-474 cell lipid extract | 20% D2O | 1 nM GSK2194069 |
| SA340956 | 15_FFA_D2O_BT474_Fasnall01 | BT-474 cell lipid extract | 20% D2O | 1 uM Fasnall |
| SA340957 | 22_FFA_D2O_BT474_Fasnall01 | BT-474 cell lipid extract | 20% D2O | 1 uM Fasnall |
| SA340958 | 08_FFA_D2O_BT474_Fasnall01 | BT-474 cell lipid extract | 20% D2O | 1 uM Fasnall |
| SA340959 | 21_FFA_D2O_BT474_GSK1000 | BT-474 cell lipid extract | 20% D2O | 1 uM GSK2194069 |
| SA340960 | 14_FFA_D2O_BT474_GSK1000 | BT-474 cell lipid extract | 20% D2O | 1 uM GSK2194069 |
| SA340961 | 07_FFA_D2O_BT474_GSK1000 | BT-474 cell lipid extract | 20% D2O | 1 uM GSK2194069 |
| SA340962 | 20_FFA_D2O_BT474_GSK0040 | BT-474 cell lipid extract | 20% D2O | 40 nM GSK2194069 |
| SA340963 | 13_FFA_D2O_BT474_GSK0040 | BT-474 cell lipid extract | 20% D2O | 40 nM GSK2194069 |
| SA340964 | 06_FFA_D2O_BT474_GSK0040 | BT-474 cell lipid extract | 20% D2O | 40 nM GSK2194069 |
| SA340965 | 24_FFA_D2O_BT474_Fasnall40 | BT-474 cell lipid extract | 20% D2O | 40 uM Fasnall |
| SA340966 | 10_FFA_D2O_BT474_Fasnall40 | BT-474 cell lipid extract | 20% D2O | 40 uM Fasnall |
| SA340967 | 17_FFA_D2O_BT474_Fasnall40 | BT-474 cell lipid extract | 20% D2O | 40 uM Fasnall |
| SA340968 | 16_FFA_D2O_BT474_Fasnall05 | BT-474 cell lipid extract | 20% D2O | 5 uM Fasnall |
| SA340969 | 09_FFA_D2O_BT474_Fasnall05 | BT-474 cell lipid extract | 20% D2O | 5 uM Fasnall |
| SA340970 | 23_FFA_D2O_BT474_Fasnall05 | BT-474 cell lipid extract | 20% D2O | 5 uM Fasnall |
| SA340971 | 04_FFA_D2O_BT474_Control | BT-474 cell lipid extract | 20% D2O | Vehicle |
| SA340972 | 11_FFA_D2O_BT474_Control | BT-474 cell lipid extract | 20% D2O | Vehicle |
| SA340973 | 18_FFA_D2O_BT474_Control | BT-474 cell lipid extract | 20% D2O | Vehicle |
| SA340974 | 27_FFA_Blank | BT-474 cell lipid extract | NA | NA |
| SA340975 | 02_FFA_Blank | BT-474 cell lipid extract | NA | NA |
| SA340976 | 26_FFA_Blank | BT-474 cell lipid extract | NA | NA |
| SA340977 | 25_FFA_Blank | BT-474 cell lipid extract | NA | NA |
| SA340978 | 01_FFA_Blank | BT-474 cell lipid extract | NA | NA |
| SA340979 | 03_FFA_Blank | BT-474 cell lipid extract | NA | NA |
| Showing results 1 to 27 of 27 |
Collection:
| Collection ID: | CO003261 |
| Collection Summary: | Cells were seeded in 10 cm Petri dishes. At collection, cells were washed with PBS three times. Then, 1 ml of methanol was added and cells were scraped from the surface. All content of the plate was transferred into 13 x 100 mm Pyrex glass tubes. Lipids were extracted by the Folch method. Dried lipids were redissolved in 1 ml of 0.3 M KOH solution in 90% methanol and incubated at 85 °C for 1 h. Then, 100 µl of formic acid were added, followed by 800 µl of hexane for extraction. The hexane phase was transferred to glass LC-MS vials and dried under the stream of nitrogen. Samples were redissolved in 1 ml of 1:1 methanol:isopropanol. |
| Collection Protocol Filename: | DM_free_fatty_acid_analysis_samples.txt |
| Sample Type: | Cultured cells |
| Collection Method: | 100% methanol extraction |
| Volumeoramount Collected: | 1 ml |
| Storage Conditions: | -80℃ |
| Collection Vials: | 1.5 ml plastic centrifuge tubes |
| Storage Vials: | 1.5 ml plastic centrifuge tubes |
Treatment:
| Treatment ID: | TR003277 |
| Treatment Summary: | Cells were grown in 20% D2O-containing RPMI-1640 medium and treated with Fasnall or GSK2194069 at various concentrations. |
| Treatment Compound: | Fasnall, GSK2194069 |
| Treatment Dose: | 1 nM - 40 uM |
| Treatment Doseduration: | 24 h |
| Treatment Vehicle: | DMSO |
| Cell Growth Container: | 6-cm Petri dishes |
| Cell Media: | RPMI-1640 |
| Cell Envir Cond: | 37C, 5% CO2 |
| Cell Pct Confluence: | ~70% |
Sample Preparation:
| Sampleprep ID: | SP003275 |
| Sampleprep Summary: | Sample preparation for LC-MS free fatty acid analysis Cells were seeded in 10 cm Petri dishes. At collection, cells were washed with PBS three times. Then, 1 ml of methanol was added and cells were scraped from the surface. All content of the plate was transferred into 13 x 100 mm Pyrex glass tubes. Lipids were extracted by the Folch method. Dried lipids were redissolved in 1 ml of 0.3 M KOH solution in 90% methanol and incubated at 85 °C for 1 h. Then, 100 µl of formic acid were added, followed by 800 µl of hexane for extraction. The hexane phase was transferred to glass LC-MS vials and dried under the stream of nitrogen. Samples were redissolved in 1 ml of 1:1 methanol:isopropanol. |
| Sampleprep Protocol Filename: | DM_free_fatty_acid_analysis_samples.txt |
| Processing Method: | Lipid saponification in 0.3 M KOH |
| Extraction Method: | 100% methanol |
| Extract Enrichment: | None |
| Extract Cleanup: | None |
| Extract Storage: | -80℃ |
| Sample Resuspension: | None |
| Sample Derivatization: | None |
| Sample Spiking: | None |
| Subcellular Location: | Cellular lipids |
Combined analysis:
| Analysis ID | AN005169 | AN005170 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | Phenomenex Kinetex XB-C18 (150 x 3 mm, 2.6 um) | Phenomenex Kinetex XB-C18 (150 x 3 mm, 2.6 um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF-X Orbitrap | Thermo Q Exactive HF-X Orbitrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | AU | AU |
Chromatography:
| Chromatography ID: | CH003911 |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Phenomenex Kinetex XB-C18 (150 x 3 mm, 2.6 um) |
| Column Temperature: | 65 |
| Flow Gradient: | 0-4.5 min, 15-60% B; 4.5-12 min, 60-82% B; 12-12.75 min, 82-95% B; 12.75-16.5 min, 95-100% B; 16.5-22.5 min, 100% B; 22.5-22.6 min, 100-15% B; 22.6-25 min, 15% B |
| Flow Rate: | 0-22.5 min, 0.333 ml/min; 22.5-22.6 min, 0.333-0.5 ml/min; 22.6-24.9 min, 0.5 ml/min; 24.9-25 min, 0.5-0.333 ml/min |
| Injection Temperature: | 20 |
| Solvent A: | 60% Acetonitrile/40% water; 10 mM ammonium formate |
| Solvent B: | 90% Isopropanol/8% acetonitrile/2% water; 10 mM ammonium formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004904 |
| Analysis ID: | AN005169 |
| Instrument Name: | Thermo Q Exactive HF-X Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The HESI ion source voltage was set to the following parameters: +3,300/-3,500 V, sheath gas 50, auxiliary gas 20, spare gas 0, probe heater 350 °C, capillary temperature 350 °C, S-Lens RF level 80. The mass spectrometer was set to acquire data in polarity-switching mode, one microscan, 60,000 resolution, AGC target 5e6, scan range 130-1950 m/z, IT 200 ms. Data were converted into the mzXML format by ProteoWizard and analyzed in MAVEN. Free cholesterol was measured as a water-loss in-source fragment [M-H2O+H]+. |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 350 |
| Ion Source Temperature: | 350 |
| Ion Spray Voltage: | 3500 |
| Ionization: | Both |
| Source Temperature: | 350 |
| Automatic Gain Control: | 5e6 |
| Dataformat: | RAW |
| MS ID: | MS004905 |
| Analysis ID: | AN005170 |
| Instrument Name: | Thermo Q Exactive HF-X Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The HESI ion source voltage was set to the following parameters: +3,300/-3,500 V, sheath gas 50, auxiliary gas 20, spare gas 0, probe heater 350 °C, capillary temperature 350 °C, S-Lens RF level 80. The mass spectrometer was set to acquire data in polarity-switching mode, one microscan, 60,000 resolution, AGC target 5e6, scan range 130-1950 m/z, IT 200 ms. Data were converted into the mzXML format by ProteoWizard and analyzed in MAVEN. Free cholesterol was measured as a water-loss in-source fragment [M-H2O+H]+. |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 350 |
| Ion Source Temperature: | 350 |
| Ion Spray Voltage: | 3300 |
| Ionization: | Both |
| Source Temperature: | 350 |
| Automatic Gain Control: | 5e6 |
| Dataformat: | RAW |
