Summary of Study ST002319

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 PR001485. The data can be accessed directly via it's Project DOI: 10.21228/M8S41S This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST002319
Study TitleMass spectroscopy‑based proteomics and metabolomics analysis of triple‑positive breast cancer cells treated with tamoxifen and trastuzumab
Study SummaryHER2-enriched breast cancer with high levels of hormone receptor expression, known as "triple positive" breast cancer, may represent a new entity with a relatively favourable prognosis against which the combination of chemotherapy, HER-2 inhibition, and endocrine treatment may be considered overtreatment. We explored the effect of the anticancer drugs tamoxifen and trastuzumab, both separately and in combination, on the integrated proteomic and metabolic profile of "triple positive" breast cancer cells (BT-474). Method We employed ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry using a Bruker timsTOF to investigate changes in BT-474 cell line treated with either tamoxifen, trastuzumab or a combination. Differentially abundant metabolites were identified using the Bruker Human Metabolome Database metabolite library and proteins using the Uniprot proteome for Homo sapiens using MetaboScape and MaxQuant, respectively, for identification and quantitation. Results A total of 77 proteins and 85 metabolites were found to significantly differ in abundance in BT-474 treated cells with tamoxifen 5 μM/and or trastuzumab 2.5 μM. Findings suggest that by targeting important cellular signalling pathways which regulate cell growth, apoptosis, proliferation, and chemoresistance, these medicines have a considerable anti-growth effect in BT-474 cells. Pathways enriched for dysregulation include RNA splicing, neutrophil degranulation and activation, cellular redox homeostasis, mitochondrial transmembrane transport, ferroptosis and necroptosis, ABC transporters and central carbon metabolism. Conclusion Our findings in protein and metabolite level research revealed that anti-cancer drug therapy had a significant impact on the key signalling pathways and molecular processes in triple positive BT-474 cell lines.
Institute
University of Sharjah
DepartmentSharjah Institute for Medical Research
LaboratoryBiomarker Discovery Group
Last NameSoares
First NameNelson
AddressM32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah
Emailnsoares@sharjah.ac.ae
Phone065057656
Submit Date2022-10-18
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2022-11-21
Release Version1
Nelson Soares Nelson Soares
https://dx.doi.org/10.21228/M8S41S
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001485
Project DOI:doi: 10.21228/M8S41S
Project Title:Mass spectroscopy‑based proteomics and metabolomics analysis of triple‑positive breast cancer cells treated with tamoxifen and/ or trastuzumab
Project Type:LC-MS/MS
Project Summary:HER2-enriched breast cancer with high levels of hormone receptor expression, known as "triple positive" breast cancer, may represent a new entity with a relatively favourable prognosis against which the combination of chemotherapy, HER-2 inhibition, and endocrine treatment may be considered overtreatment. We explored the effect of the anticancer drugs tamoxifen and trastuzumab, both separately and in combination, on the integrated proteomic and metabolic profile of "triple positive" breast cancer cells (BT-474). Method We employed ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry using a Bruker timsTOF to investigate changes in BT-474 cell line treated with either tamoxifen, trastuzumab or a combination. Differentially abundant metabolites were identified using the Bruker Human Metabolome Database metabolite library and proteins using the Uniprot proteome for Homo sapiens using MetaboScape and MaxQuant, respectively, for identification and quantitation. Results A total of 77 proteins and 85 metabolites were found to significantly differ in abundance in BT-474 treated cells with tamoxifen 5 μM/and or trastuzumab 2.5 μM. Findings suggest that by targeting important cellular signalling pathways which regulate cell growth, apoptosis, proliferation, and chemoresistance, these medicines have a considerable anti-growth effect in BT-474 cells. Pathways enriched for dysregulation include RNA splicing, neutrophil degranulation and activation, cellular redox homeostasis, mitochondrial transmembrane transport, ferroptosis and necroptosis, ABC transporters and central carbon metabolism. Conclusion Our findings in protein and metabolite level research revealed that anti-cancer drug therapy had a significant impact on the key signalling pathways and molecular processes in triple positive BT-474 cell lines.
Institute:Sharjah Institute for Medical Research
Department:Sharjah Institute for Medical Research
Laboratory:Biomarker Discovery Group
Last Name:Facility
First Name:Core
Address:M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, UAE, Sharjah, 000, United Arab Emirates
Email:tims-tof@sharjah.ac.ae
Phone:+971 6 5057656

Subject:

Subject ID:SU002405
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA2272704a_-1_67_1_444COMBINATION
SA2272714a_-2_67_1_445COMBINATION
SA2272724a-2_60_1_443COMBINATION
SA2272734a_-2_68_1_447COMBINATION
SA2272744a-1_60_1_442COMBINATION
SA2272754a_-1_68_1_446COMBINATION
SA2272761A_92_1_327Control
SA2272771A_93_1_331Control
SA2272781A_92_1_330Control
SA2272791A_91_1_326Control
SA2272801A_91_1_329Control
SA2272811A_93_1_328Control
Showing results 1 to 12 of 12

Collection:

Collection ID:CO002398
Collection Summary:The BT-474 BC cell line utilized in this study was cultured as monolayers in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA). All cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2.
Sample Type:Breast cancer cells
Storage Conditions:Described in summary

Treatment:

Treatment ID:TR002417
Treatment Summary:Triplicate flasks were prepared for each treatment condition for each analysis (metabolomic and proteomic) for a total of 24 flasks. Two million cells were seeded in each 75 cm2 tissue culture flask and incubated for 24 h. The cells were then treated with Tamoxifen (5 μM) and/or Trastuzumab (2.5 μM) for 24 h. These concentrations correspond to the IC50 of these compounds with BT-474 cells, as determined by cytotoxicity assays (data not shown). Control cells were treated with vehicle (dimethyl sulfoxide (DMSO) at 0.5% for 24 h. Following the incubation period, cells were collected by trypsinization and washed twice with phosphate-buffered saline solution (PBS) before re-suspending in 1 mL 1 × PBS for further analysis. Finally, cells were collected as pellets by centrifugation at 1200 rounds per minute (rpm) for 10 min at room temperature. To negate the effect of Circadian rhythms on the response of cells to treatment, cells were kept under the same conditions during the entire incubation period and the cell collection was done concurrently for all samples. In addition, the same number of cells were used for each sample to avoid the effect of variation in cell numbers.
Treatment:Drugs
Treatment Compound:Tamoxifen and/or Trastuzumab

Sample Preparation:

Sampleprep ID:SP002411
Sampleprep Summary:Sample metabolite extraction A volume of 1 mL of the extraction solvent (methanol+0.1% formic acid) was added to the cell pellets to quench cells. The cells were then vortexed for 2 min to ensure the quantitative extraction of the metabolites and stored on ice for 1 h, during which the samples were vortexed every 15 min. After this, the insoluble cell matrices were collected and transferred to centrifuge tubes, intermittent ultrasonication using the COPLEY sonicator (QSONICA SONICATOR, USA) under 30% amplifier and for 30 s with an ice bath employed throughout the process. Following that, cells debris were then centrifuged (15,000 rpm, 10 min, − 4 °C) and the sample supernatants were collected and transferred to LC vials for drying in the EZ-2 Plus (GeneVac-Ipswich, UK) at 37±1 °C. Dried samples were resuspended with 200 µL (water+0.1% formic acid), and vortexed for 2 min. Finally, the samples were filtered using a hydrophilic Nylon Syringe Filter of 0.45 µm pore size and analyzed by Q-TOF MS
Processing Storage Conditions:Described in summary
Extract Storage:Described in summary

Combined analysis:

Analysis ID AN003786
Analysis type MS
Chromatography type Reversed phase
Chromatography system Bruker Elute HPG 1300
Column Hamilton Intensity Solo 2 C18
MS Type ESI
MS instrument type QTOF
MS instrument name Bruker timsTOF
Ion Mode POSITIVE
Units AU

Chromatography:

Chromatography ID:CH002800
Chromatography Summary:Samples were chromatographically separated by inline reversed-phase chromatography using the Elute HPG 1300 pumps and Elute Autosampler (Bruker, Darmstadt, Germany) with solvent A 0.1% FA in HPLC grade water and solvent B 0.1% FA in ACN. A Hamilton Intensity Solo 2 C18 column (100 mm × 2.1 mm, 1.8 μm beads) was maintained at 35 ℃ (metabolomics analyses).
Methods ID:.
Methods Filename:.
Chromatography Comments:.
Instrument Name:Bruker Elute HPG 1300
Column Name:Hamilton Intensity Solo 2 C18
Column Pressure:.
Column Temperature:35
Flow Gradient:1%B to 99%B in 15 min
Flow Rate:250 uL/min
Injection Temperature:.
Internal Standard:.
Internal Standard Mt:.
Retention Index:.
Retention Time:.
Sample Injection:.
Sampling Cone:.
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Analytical Time:.
Capillary Voltage:.
Migration Time:.
Oven Temperature:35C
Preconditioning:.
Running Buffer:.
Running Voltage:.
Sheath Liquid:.
Time Program:.
Transferline Temperature:.
Washing Buffer:.
Weak Wash Solvent Name:.
Weak Wash Volume:.
Strong Wash Solvent Name:.
Strong Wash Volume:.
Target Sample Temperature:.
Sample Loop Size:.
Sample Syringe Size:.
Randomization Order:.
Chromatography Type:Reversed phase

MS:

MS ID:MS003529
Analysis ID:AN003786
Instrument Name:Bruker timsTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:The MS analysis was performed using a timsTOF (Bruker, Darmstadt, Germany) with Apollo II electrosprayionization (ESI) source. The drying gas was set to flow at 10 L/min and the drying temperature to 220℃ and the nebulizer pressure to 2.2 bar. The capillary voltage was 4500 V and the end plate offset 500 V. For metabolomics 20–1300 m/z. The instrument was operated in auto-MS/MS mode. For metabolomics the collision energy was set to 20 eV, the cycle time to 0.5 s with a relative minimum intensity threshold of 400 counts per thousand and a target intensity of 20,000. Sodium formate was injected as an external calibrant in the first 0.3 min of each LC–MS/MS run.
Ion Mode:POSITIVE
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