Summary of Study ST002703

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 PR001674. The data can be accessed directly via it's Project DOI: 10.21228/M8B14V 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	ST002703
Study Title	Multi-Omics Analysis Revealed a Significant Molecular Changes in Doxorubicin-Resistant Lung Cancer Cells.
Study Type	LC/MS/MS
Study Summary	Lung cancer is the second most common cancer and the leading cause of cancer-related deaths worldwide. Chemotherapy resistance in lung cancer is one of the major characteristics of an aggressive phenotype with poor prognosis. Therefore, there is a critical need to explore the significant molecular changes associated with resistance to conventional chemotherapy, and identify potential therapeutic targets for treatment of resistant lung cancer. In this study, we have performed comprehensive quantitative proteomics and metabolomics analysis of non-small cell lung cancer cells (A549-P) and doxorubicin resistant A549 cells (A549-R), using state-of-the-art Trapped Ion Mobility Spectroscopy, Time-of-Flight Mass Spectrometry (TIMS-TOF-MS). The results revealed 30 dysregulated proteins and 37 significantly altered metabolites in A549-R cells compared to A549-P cells. Among the significantly upregulated proteins are liver carboxylesterase 1, anterior gradient protein 2 homolog and nicotinamide phosphoribosyltransferase. A group of the upregulated proteins are endogenous and xenobiotic-metabolizing enzymes, including UDP-glucuronosyltransferase 1-6, CES1, and epoxide hydrolase 1. While Importin, ATP-citrate synthase and CTP synthase are downregulated. The significantly altered metabolites include sepiapterin, glutathione, glycine, pyridine and niacinamide. The performed multi-omics integrated analysis revealed the involvement of purine and glutathione metabolism, ABC transporters, citric acid cycle in the development of resistance in A549 cells, besides the involvement of energy metabolism, pathways related to cancer progression, invasion and migration, and redox homeostasis. Collectively, this exploratory study effectively revealed the significantly dysregulated proteins and metabolites in doxorubicin resistant A549 cells and shed the light on potential biomarkers for chemotherapy resistant non-small cell lung cancer. In addition, multi-omics integrated analysis elucidates the involved pathways in resistance including pathways related to progression and invasion which would improve prognosis and open the door for new potential therapeutic targets.
Institute	University of Sharjah
Department	Research institute of medical and health science
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
Submit Date	2023-05-04
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2023-11-30
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001674
Project DOI:	doi: 10.21228/M8B14V
Project Title:	Multi-Omics Analysis Revealed a Significant Molecular Changes in Doxorubicin-Resistant Lung Cancer Cells.
Project Type:	LC-MS/MS
Project Summary:	Lung cancer is the second most common cancer and the leading cause of cancer-related deaths worldwide. Chemotherapy resistance in lung cancer is one of the major characteristics of an aggressive phenotype with poor prognosis. Therefore, there is a critical need to explore the significant molecular changes associated with resistance to conventional chemotherapy, and identify potential therapeutic targets for treatment of resistant lung cancer. In this study, we have performed comprehensive quantitative proteomics and metabolomics analysis of non-small cell lung cancer cells (A549-P) and doxorubicin resistant A549 cells (A549-R), using state-of-the-art Trapped Ion Mobility Spectroscopy, Time-of-Flight Mass Spectrometry (TIMS-TOF-MS). The results revealed 30 dysregulated proteins and 37 significantly altered metabolites in A549-R cells compared to A549-P cells. Among the significantly upregulated proteins are liver carboxylesterase 1, anterior gradient protein 2 homolog and nicotinamide phosphoribosyltransferase. A group of the upregulated proteins are endogenous and xenobiotic-metabolizing enzymes, including UDP-glucuronosyltransferase 1-6, CES1, and epoxide hydrolase 1. While Importin, ATP-citrate synthase and CTP synthase are downregulated. The significantly altered metabolites include sepiapterin, glutathione, glycine, pyridine and niacinamide. The performed multi-omics integrated analysis revealed the involvement of purine and glutathione metabolism, ABC transporters, citric acid cycle in the development of resistance in A549 cells, besides the involvement of energy metabolism, pathways related to cancer progression, invasion and migration, and redox homeostasis. Collectively, this exploratory study effectively revealed the significantly dysregulated proteins and metabolites in doxorubicin resistant A549 cells and shed the light on potential biomarkers for chemotherapy resistant non-small cell lung cancer. In addition, multi-omics integrated analysis elucidates the involved pathways in resistance including pathways related to progression and invasion which would improve prognosis and open the door for new potential therapeutic targets.
Institute:	Sharjah Institute for Medical Research
Department:	Research institute of medical and health science
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:	SU002808
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
SA271291	P Control 3A-02_9_1_3222	A549-P
SA271292	P Control 1A-01_7_1_3213	A549-P
SA271293	P Control 3A-01_9_1_3217	A549-P
SA271294	R Control 1A-01_13_1_3229	A549-P
SA271295	P Control 2A-02_8_1_3216	A549-P
SA271296	P Control 1A-02_7_1_3214	A549-P
SA271297	P Control 2A-01_8_1_3215	A549-P
SA271298	R Control 3A-02_15_1_3234	A549-R
SA271299	R Control 3A-01_15_1_3233	A549-R
SA271300	R Control 2A-01_14_1_3231	A549-R
SA271301	R Control 1A-02_13_1_3230	A549-R
SA271302	R Control 2A-02_14_1_3232	A549-R

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO002801
Collection Summary:	The human non-small cell lung cancer cell line (A549) utilized in this study was purchased from AmericanType Culture Collection (ATCC) (Manassas, USA). A doxorubicin-resistant A549 cell line was generated in our laboratory (Drug Design and Discovery Research Group, Pharmacology Laboratory, Sharjah Institute for Medical Research, University of Sharjah). Doxorubicin (Dox) and irinotecan were purchased from Sigma-Aldrich (Darmstadt, Germany) and Abcam (Cambridge, UK), respectively. A Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 (1:1) (DMEM/F-12) medium was purchased from Gibco, Thermo Fisher Scientific (Massachusetts, USA). Fetal bovine serum (FBS), penicillin/streptomycin, trypsin, and trypan blue were purchased from Sigma-Aldrich (Darmstadt, Germany). For cell viability assay: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Darmstadt, Germany). Protease inhibitor cocktail tablets were purchased from Roche (Mannheim, Germany). Methanol (≥99.9%), acetonitrile (ACN) and deionized water, as well as LC-MS CHROMASOLV, were purchased from Honeywell (Seelze, Germany). Trifluoroacetic acid (TFA) was purchased from Sigma-Aldrich (Darmstadt, Germany) and acetonitrile (ACN) was purchased from Honeywell (Charlotte, USA). Lys-C endopeptidase was purchased from Fujifilm Wako Chemicals (Virginia, USA). For western blot analysis: primary antibodies against liver carboxylesterase 1/CES1 and β-actin were obtained from Abcam (Cambridge, UK) and Cell Signaling Technologies (Danvers, USA), respectively. Anti-rabbit IgG, Horseradish Peroxidase (HRP)-linked secondary antibody was purchased from Cell Signaling Technologies (Danvers, USA). Skimmed milk was purchased from Sigma-Aldrich (Darmstadt, Germany). For Enzyme-linked immunosorbent assay (ELISA): carboxylesterase 1 (CES1) specific activity assay kit purchased from Abcam (Cambridge, UK).
Sample Type:	Lung

Treatment:

Treatment ID:	TR002817
Treatment Summary:	The human non-small cell lung cancer cell line (A549) was purchased from AmericanType Culture Collection (ATCC) (Manassas, USA). A doxorubicin-resistant A549 (A549-R) cell line was generated in our laboratory (Drug Design and Discovery Research Group, Pharmacology Laboratory, Sharjah Institute for Medical Research, University of Sharjah). To develop resistant cells from the parental A549 cells (A549-P), A549-P cells were treated with a tenth of Dox's maximal inhibitory concentration (IC10). The surviving population of cells was cultured in a new flask and treated with a gradual increase of Dox concentration for 8 months. Maintenance of A549-R cells resistance was at a dose of 0.02 µg/ml of Dox. A549-P and A549-R cells were cultured in a Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 (1:1) medium (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were incubated in a humidified atmosphere of 5% CO2 at 37 ˚C.

Treatment ID:

TR002817

Treatment Summary:

The human non-small cell lung cancer cell line (A549) was purchased from AmericanType Culture Collection (ATCC) (Manassas, USA). A doxorubicin-resistant A549 (A549-R) cell line was generated in our laboratory (Drug Design and Discovery Research Group, Pharmacology Laboratory, Sharjah Institute for Medical Research, University of Sharjah). To develop resistant cells from the parental A549 cells (A549-P), A549-P cells were treated with a tenth of Dox's maximal inhibitory concentration (IC10). The surviving population of cells was cultured in a new flask and treated with a gradual increase of Dox concentration for 8 months. Maintenance of A549-R cells resistance was at a dose of 0.02 µg/ml of Dox. A549-P and A549-R cells were cultured in a Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 (1:1) medium (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were incubated in a humidified atmosphere of 5% CO2 at 37 ˚C.

Sample Preparation:

Sampleprep ID:	SP002814
Sampleprep Summary:	Two million cells from both cell lines (A549-P and A549-R) were seeded in T75 cell culture flasks. Following culturing the cells, they were collected by trypsin and a pellet of 3x106 cells was stored at -80 ℃ for further analysis. Each cell line was prepared in triplicates for the metabolomics and proteomics studies. Frozen A549-P and A549-R cells pellets were centrifuged at 14000 rpm for 5 min at 4 ℃ to separate them from Phosphate Buffered Saline (PBS) buffer. The pellets were suspended in a lysis buffer (10 mM Tris) and protease inhibitor cocktail tablets. The lysed cells were kept on ice for 10 min, and then each sample was vortexed for 30 s. After that, the samples were sonicated in an ice bath using a Q500 sonicator from QSonica Sonicator (Fairfield, Connecticut, USA) at 30% amp for 30 s. Then, the samples were centrifuged at 14000 rpm for 5 min at 4℃, 400 µL of methanol was added to the supernatant, followed by 300 µL of chloroform and vortexed for 30 seconds, and then centrifuged at 14000 rpm for 5 min at 4 ℃. The resulting aqueous biphasic solution's upper layer was transferred carefully into LC vials without touching the white disk for metabolites analysis. For proteins extraction, the generated white disk and the lower layer were washed with 300 µL of methanol and vortexed vigorously, and then centrifuged at 14000 rpm for 3 min at 4 ℃. After that, the protein pellet was precipitated and allowed to dry by keeping the tubes opened for 5 min. Then a denaturation buffer was used to re-suspend the pellets (10 mM Tris, 6 M Urea, 2 M Thiourea, pH 8) and the concentration of precipitated proteins was determined using the modified Bradford assay. The resulted supernatant from the previous centrifugation, which contains metabolites, was transferred to the previously collected metabolites in the LC vials. The metabolites samples were dried at 45 ℃ using an EZ-2 Plus evaporator from GeneVac (Ipswich, UK), and then the dehydrated extracts were reconstituted in 200 µL 0.1% formic acid (FA) in water and vortexed for 2 min. Finally, the samples were filtered using a hydrophilic syringe filter with a pore size of 0.45 µm and were ready to be analyzed using Q-TOF MS.

Sampleprep ID:

SP002814

Sampleprep Summary:

Two million cells from both cell lines (A549-P and A549-R) were seeded in T75 cell culture flasks. Following culturing the cells, they were collected by trypsin and a pellet of 3x106 cells was stored at -80 ℃ for further analysis. Each cell line was prepared in triplicates for the metabolomics and proteomics studies. Frozen A549-P and A549-R cells pellets were centrifuged at 14000 rpm for 5 min at 4 ℃ to separate them from Phosphate Buffered Saline (PBS) buffer. The pellets were suspended in a lysis buffer (10 mM Tris) and protease inhibitor cocktail tablets. The lysed cells were kept on ice for 10 min, and then each sample was vortexed for 30 s. After that, the samples were sonicated in an ice bath using a Q500 sonicator from QSonica Sonicator (Fairfield, Connecticut, USA) at 30% amp for 30 s. Then, the samples were centrifuged at 14000 rpm for 5 min at 4℃, 400 µL of methanol was added to the supernatant, followed by 300 µL of chloroform and vortexed for 30 seconds, and then centrifuged at 14000 rpm for 5 min at 4 ℃. The resulting aqueous biphasic solution's upper layer was transferred carefully into LC vials without touching the white disk for metabolites analysis. For proteins extraction, the generated white disk and the lower layer were washed with 300 µL of methanol and vortexed vigorously, and then centrifuged at 14000 rpm for 3 min at 4 ℃. After that, the protein pellet was precipitated and allowed to dry by keeping the tubes opened for 5 min. Then a denaturation buffer was used to re-suspend the pellets (10 mM Tris, 6 M Urea, 2 M Thiourea, pH 8) and the concentration of precipitated proteins was determined using the modified Bradford assay. The resulted supernatant from the previous centrifugation, which contains metabolites, was transferred to the previously collected metabolites in the LC vials. The metabolites samples were dried at 45 ℃ using an EZ-2 Plus evaporator from GeneVac (Ipswich, UK), and then the dehydrated extracts were reconstituted in 200 µL 0.1% formic acid (FA) in water and vortexed for 2 min. Finally, the samples were filtered using a hydrophilic syringe filter with a pore size of 0.45 µm and were ready to be analyzed using Q-TOF MS.

Combined analysis:

Analysis ID	AN004383
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Bruker timsTOF
Column	Hamilton Intensity Solo 2 C18(100 x 2.1 mm, 1.8 um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH003288
Chromatography Summary:	Then, liquid chromatography separation was done using Intensity Solo 1.8 C18-2 column (100×2.1 mm, 1.8 μm) from Bruker Daltonik GmbH (Bremen, Germany). The column temperature was kept at 35 ℃ and the flow rate was constant at 0.3 ml/min. The mobile phase composed of 0.1% FA in HPLC grade water (solvent A) and 0.1% FA in ACN (solvent B).
Instrument Name:	Bruker timsTOF
Column Name:	Hamilton Intensity Solo 2 C18(100 x 2.1 mm, 1.8 um)
Column Temperature:	35
Flow Gradient:	1% B was held for 2 min, ramping to 99% B over 15 min, and held at 99% B for 3 min before re-equilibrating to 1% B for 10 min
Flow Rate:	250 uL/min
Solvent A:	Water (0.1% Formic Acid)
Solvent B:	ACN (0.1% Formic Acid)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004132
Analysis ID:	AN004383
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	MetaboScape® 4.0 software from Bruker (Bremen, Germany) was used for data processing. The parameters for the detection were as follows: a minimum of 7 spectra were used for peak detection, while for molecular features detection, the minimum intensity threshold was equivalent to 1000 counts. Mass recalibration was performed within a retention time range between 0-0.3 min. Only those features present in at least 2 of 6 samples were considered. Finally, the detected MS/MS spectra assigned onto the bucket table allowed for better viewing and understanding. The main features included retention time, measured m/z, detected fragments, and molecular weight [12]. Data bucketing parameters were as follows: the retention time range was between 0.3 min and 25 min, whereas the mass range began at 50 m/z and finished at 1,300 m/z. A two-tailed independent student’s t-test was utilized to determine the significantly altered metabolites between the two cell lines. The threshold for significance was p-value <0.05. Functional enrichment analysis was constructed utilizing the MetaboAnalyst 5.0 website (https://www.metaboanalyst.ca).
Ion Mode:	POSITIVE