Summary of Study ST002273

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 PR001454. The data can be accessed directly via it's Project DOI: 10.21228/M8S71T 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	ST002273
Study Title	Multi-Omics analysis revealed a significant alteration of critical metabolic pathways due to sorafenib-resistance in Hep3B cell lines
Study Type	MS- comparative metabolomic analysis
Study Summary	Hepatocellular carcinoma (HCC) is the second prominent cause of cancer-associated death worldwide. Usually, HCC is diagnosed in advanced stages, where sorafenib, a multiple target ty-rosine kinase inhibitor, is used as the first line of treatment. Unfortunately, resistance to sorafenib is usually encountered within six months of the treatment. Therefore, there is a critical need to identify the underlying reasons for drug resistance. In the present study, we investigated the proteomic and metabolomics alterations accompanying to sorafenib resistance in hepatocellular carcinoma Hep3B cells by employing ultra-high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). The Bruker Human Metabolome Database (HMDB) library was used to identify the differentially abundant metabolites through MetaboScape 4.0 software (Bruker). For protein annotation and identification, the Uniprot proteome for Homo sapiens (Human) da-tabase was utilised through MaxQuant. The results revealed that 27 metabolites and 18 proteins were significantly dysregulated due to sorafenib resistance in Hep3B cells compared to the parental phenotype. D-alanine, L-proline, o-tyrosine, succinic acid and phosphatidylcholine (PC, 16:0/16:0) were among the significantly altered metabolites. Ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial superoxide dismutase, UDP-glucose-6-dehydrogenase, sorbitol dehydrogenase and calpain small subunit 1 were among the significantly altered proteins. The findings revealed that resistant Hep3B cells demonstrated significant alterations in amino acid and nucleotide met-abolic pathways, energy production pathways and other pathways related to cancer aggressive-ness, migration, proliferation, and drug-resistance. Joint pathway enrichment analysis unveiled unique pathways, including the antifolate resistance pathway and other important pathways that maintain cancer cells' survival, growth, and proliferation. Collectively, the results identified po-tential biomarkers for sorafenib-resistant HCC and gave insights into their role in chemotherapeutic drug resistance, cancer initiation, progression, and aggressiveness, which may contribute to better prognosis and chemotherapeutic outcomes.
Institute	Sharjah Institute for Medical Research
Last Name	Soares
First Name	Nelson
Address	University of Sharjah, Sharjah, UAE
Email	nsoares@sharjah.ac.ae
Phone	+971 65 05 7763
Submit Date	2022-08-31
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2022-09-16
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001454
Project DOI:	doi: 10.21228/M8S71T
Project Title:	Multi-Omics analysis revealed a significant alteration of critical metabolic pathways due to sorafenib-resistance in Hep3B cell lines
Project Type:	MS-comparative metabolomic analysis
Project Summary:	Hepatocellular carcinoma (HCC) is the second prominent cause of cancer-associated death worldwide. Usually, HCC is diagnosed in advanced stages, where sorafenib, a multiple target ty-rosine kinase inhibitor, is used as the first line of treatment. Unfortunately, resistance to sorafenib is usually encountered within six months of the treatment. Therefore, there is a critical need to identify the underlying reasons for drug resistance. In the present study, we investigated the proteomic and metabolomics alterations accompanying to sorafenib resistance in hepatocellular carcinoma Hep3B cells by employing ultra-high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). The Bruker Human Metabolome Database (HMDB) library was used to identify the differentially abundant metabolites through MetaboScape 4.0 software (Bruker). For protein annotation and identification, the Uniprot proteome for Homo sapiens (Human) da-tabase was utilised through MaxQuant. The results revealed that 27 metabolites and 18 proteins were significantly dysregulated due to sorafenib resistance in Hep3B cells compared to the parental phenotype. D-alanine, L-proline, o-tyrosine, succinic acid and phosphatidylcholine (PC, 16:0/16:0) were among the significantly altered metabolites. Ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial superoxide dismutase, UDP-glucose-6-dehydrogenase, sorbitol dehydrogenase and calpain small subunit 1 were among the significantly altered proteins. The findings revealed that resistant Hep3B cells demonstrated significant alterations in amino acid and nucleotide met-abolic pathways, energy production pathways and other pathways related to cancer aggressive-ness, migration, proliferation, and drug-resistance. Joint pathway enrichment analysis unveiled unique pathways, including the antifolate resistance pathway and other important pathways that maintain cancer cells' survival, growth, and proliferation. Collectively, the results identified po-tential biomarkers for sorafenib-resistant HCC and gave insights into their role in chemotherapeutic drug resistance, cancer initiation, progression, and aggressiveness, which may contribute to better prognosis and chemotherapeutic outcomes.
Institute:	Sharjah Institute for Medical Research
Department:	Medicinal Chemistry
Last Name:	Soares
First Name:	Nelson
Address:	University of Sharjah, Sharjah, UAE
Email:	nsoares@sharjah.ac.ae
Phone:	+971 65 05 7763

Subject:

Subject ID:	SU002359
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Not applicable
Cell Biosource Or Supplier:	Sigma-Aldrich (St. Louis, MO)
Cell Strain Details:	Hep3B

Factors:

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

mb_sample_id	local_sample_id	Phenotype
SA217812	A01-01_57_1_1697	Parental
SA217813	A03-02_59_1_1702	Parental
SA217814	A03-01_59_1_1701	Parental
SA217815	A01-02_57_1_1698	Parental
SA217816	A02-01_58_1_1699	Parental
SA217817	A02-02_58_1_1700	Parental
SA217818	B03-02_62_1_1708	Resistant
SA217819	B03-01_62_1_1707	Resistant
SA217820	B01-01_60_1_1703	Resistant
SA217821	B01-02_60_1_1704	Resistant
SA217822	B02-01_61_1_1705	Resistant
SA217823	B02-02_61_1_1706	Resistant

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO002352
Collection Summary:	Cell lines and culture conditions: The Hep3B cell line used in this study was purchased from Sigma-Aldrich (St. Louis, MO). Sorafenib-resistant Hep3B cells were developed from Hep3B cell lines One million Hep3B cells were seeded in T75 flasks and cultured in Dulbecco's Modified Eagle Medium (DMEM) as monolayers. The medium was sup-plemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, St. Louis, #F2442-500ML) and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, #P4333). Cultures were then incubated in 5% CO2 humidified atmosphere at 37°C for 3-4 days until 70-80% confluency was reached. Aseptic techniques were applied to avoid poten-tial contamination, and the confluency and contamination were checked routinely.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR002371
Treatment Summary:	Generation of resistant Hep3B cell line: Resistant Hep3B cells were developed in clinically relevant models. In T-75 flasks, Hep3B cells were seeded overnight and incubated at 37°C. The cells were then treated with sorafenib at the 10 % inhibitory concentration (IC10) (0.4µM) of sorafenib (Biovision, Milpitas, CA, USA #BAY 43-9006) to mimic the clinically consumed low dose of chemotherapeutic drugs by a cancer patient who is then exposed to escalating doses over time. To develop resistance, the survived cells were transplanted to a new flask and then treated with sorafenib at escalating concentrations over six months. Then, sorafenib at concentration IC10 (0.4µM) was persistently retained in the culture media to ensure and maintain resistance. MTT cell viability assay was performed each month to validate the resistance behavior of the cells.

Treatment ID:

TR002371

Treatment Summary:

Generation of resistant Hep3B cell line: Resistant Hep3B cells were developed in clinically relevant models. In T-75 flasks, Hep3B cells were seeded overnight and incubated at 37°C. The cells were then treated with sorafenib at the 10 % inhibitory concentration (IC10) (0.4µM) of sorafenib (Biovision, Milpitas, CA, USA #BAY 43-9006) to mimic the clinically consumed low dose of chemotherapeutic drugs by a cancer patient who is then exposed to escalating doses over time. To develop resistance, the survived cells were transplanted to a new flask and then treated with sorafenib at escalating concentrations over six months. Then, sorafenib at concentration IC10 (0.4µM) was persistently retained in the culture media to ensure and maintain resistance. MTT cell viability assay was performed each month to validate the resistance behavior of the cells.

Sample Preparation:

Sampleprep ID:	SP002365
Sampleprep Summary:	Metabolite extraction: For metabolomics, three biological replicates were used. An equal number of cells were utilized for each sample. The cells were centrifuged separately at 15000 rpm for 10 min to separate the cells and cell-free supernatants. The supernatants were dis-carded, and to each cell pellet (~3x 106), 1 mL of 0.1% formic acid in methanol was added, followed by vertexing for 2 min x 4, interrupted by 15 min incubation on ice. This is followed by sonication using a COPLEY probe-sonicator (QSONICA SONI-CATOR, USA) for 30 seconds with 30% amplifier in an ice bath. Subsequently, the cel-lular debris of each sample was collected by centrifugation at 15000 rpm for 10 min at 4°C. The supernatants were separated and dried at 37 ± 1 °C using EZ-2 Plus (Gene-Vac-Ipswich, UK). Finally, 200 µL of 0.1% formic acid in water was used to resuspend the dried samples, which were then vortexed for 2 min, filtered by a 0.45µm hydro-philic Nylon Syringe Filter and transferred to 200 μL (micro-inserts) in LC vials. The quality control (QC) sample was prepared by pooling the same volume (10 μL) of each sample, and all samples were placed in the autosampler at a temperature set at 4℃ and analyzed with UHPLC-QTOF-MS.

Sampleprep ID:

SP002365

Sampleprep Summary:

Metabolite extraction: For metabolomics, three biological replicates were used. An equal number of cells were utilized for each sample. The cells were centrifuged separately at 15000 rpm for 10 min to separate the cells and cell-free supernatants. The supernatants were dis-carded, and to each cell pellet (~3x 106), 1 mL of 0.1% formic acid in methanol was added, followed by vertexing for 2 min x 4, interrupted by 15 min incubation on ice. This is followed by sonication using a COPLEY probe-sonicator (QSONICA SONI-CATOR, USA) for 30 seconds with 30% amplifier in an ice bath. Subsequently, the cel-lular debris of each sample was collected by centrifugation at 15000 rpm for 10 min at 4°C. The supernatants were separated and dried at 37 ± 1 °C using EZ-2 Plus (Gene-Vac-Ipswich, UK). Finally, 200 µL of 0.1% formic acid in water was used to resuspend the dried samples, which were then vortexed for 2 min, filtered by a 0.45µm hydro-philic Nylon Syringe Filter and transferred to 200 μL (micro-inserts) in LC vials. The quality control (QC) sample was prepared by pooling the same volume (10 μL) of each sample, and all samples were placed in the autosampler at a temperature set at 4℃ and analyzed with UHPLC-QTOF-MS.

Combined analysis:

Analysis ID	AN003715
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	UHPLC-QTOF-MS
Column	Hamilton Intensity Solo 2 C18 (100 mm x 2.1 mm,1.8um)
MS Type	ESI
MS instrument type	Trapped Ion Mobility Q-TOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH002752
Chromatography Summary:	The Elute UHPLC and Q-TOF Mass Spectrometer (Bruker, Bremen, Germany) were utilized for metabolite and peptide detection. The Elute HPG 1300 pumps, Elute Au-tosampler (Bruker, Bremen, Germany), and Hamilton® Intensity Solo 2 C18 column (100 mm x 2.1 mm, 1.8 m beads) were employed using reversed-phase chromatog-raphy. Solvents used for separation were 0.1 % FA in LC grade water (solvent A) and 0.1 % FA in ACN (solvent B). Each metabolite and protein extract were analyzed in duplicate. For metabolomics analysis, the column was kept at 35°C, and each sample was injected twice with an in-jection volume of 10 µL. Sample elution was performed in 30 min gradient starting with 1% ACN for 2 min and then ramped to 99% ACN within 15 min. After that, 99% ACN was kept for 3 min, and then the re-equilibration to 1% ACN was done for 10 min. The flow rate was 0.25 mL/min for 20 min and then 0.35 mL/min for 8.3 min and then the flow rate set at 0.25 mL/min for 1.7 min.
Instrument Name:	UHPLC-QTOF-MS
Column Name:	Hamilton Intensity Solo 2 C18 (100 mm x 2.1 mm,1.8um)
Column Temperature:	35°C
Flow Gradient:	Sample elution was performed in 30 min gradient starting with 1% ACN for 2 min and then ramped to 99% ACN within 15 min. After that, 99% ACN was kept for 3 min, and then the re-equilibration to 1% ACN was done for 10 min
Flow Rate:	was 0.25 mL/min for 20 min and then 0.35 mL/min for 8.3 min and then the flow rate set at 0.25 mL/min for 1.7 min.
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003464
Analysis ID:	AN003715
Instrument Name:	Bruker timsTOF
Instrument Type:	Trapped Ion Mobility Q-TOF
MS Type:	ESI
MS Comments:	For MS2 acquisition in metabolomics analysis, the collision energy was fluctuated between 100-250% of 20 eV and end plate offset of 500V. The acquisition was in two sections: auto MS scan for the calibrant sodium formate in 0-0.3 min, and auto MS/MS for fragmentation, in 0.3 to 30 min. Positive mode at 12 Hz was performed in both acquisition sections . The scan range was 20 to 1300 m/z, the precursor ion’s width of ±0.5, the precursors number of 3 , the cycle time of 0.5 s econds, and the threshold of 400 counts. After three spectra, active exclusion was performed and released after 0.2 min. A timsTOF (Bruker, Bremen, Germany) with an Apollo II electrospray ionization (ESI) source was utilized for the MS analysis with the following parameters: the nebulizer pressure was 2.2 bar, the drying gas flow rate was 10 L/min, the drying tempera-ture was 220°C, and the capillary voltage was 4500 V. In the first 0.3 min of each LC-MS/MS run, the external cali-brant, sodium formate, was injected. Mass calibration was done prior to analysis ac-cording to the manufacturer’s recommendations using external mass calibration (10 mM sodium formate calibrant solution). The performance of the column and the mass spectrometer was tested using a test mixture of (TRX-2101/RT-28-calibrants for Bruker T-ReX LC-QTOF solution from Nova Medical Testing Inc.) to check the performance of reversed-phase liquid chromatography (RPLC) separation and perform multipoint re-tention time calibration, and (TRX-3112-R/MS Certified Human serum for Bruker T-ReX LC-QTOF solution from Nova Medical Testing Inc.) to check the performance of sample preparation protocols as well as LC-MS instruments. This product is prepared from pooled human blood.
Ion Mode:	POSITIVE