Summary of Study ST003029

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 PR001882. The data can be accessed directly via it's Project DOI: 10.21228/M8FQ52 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	ST003029
Study Title	Estrogen-mediated inhibition of purine metabolism and cell cycle arrest as a novel therapeutic approach in colorectal cancer cells
Study Summary	Purine metabolism is upregulated in various cancers including colorectal cancer (CRC). While previous research has elucidated the role of Estrogen (E2) in metabolism remodeling and ATP production, its effects on purine metabolism remained unexplored. This study investigates the impact of E2 signalling on purine metabolism in CRC cells. We demonstrate, for the first time, a protective effect of E2 on CRC cells by targeting the purine synthesis pathway through its receptor estrogen receptor α (ERα). A full metabolomic profiling, next generation sequencing (NGS) and integrated OMICS were conducted for HCT-116 cells treated with E2 with and without silencing ERα. Our results revealed an enrichment of the purine metabolic pathway, with 27 genes in the de novo purine synthesis pathway downregulated in E2-treated CRC cells. Besides, E2-induced DNA damage, cell cycle arrest, and apoptosis are ERα-dependent. Our findings suggest potential therapeutic avenues for CRC treatment through antimetabolites targeting purine synthesis, as E2 treatment reduces the expression of relevant metabolites.
Institute	Sharjah Institute for Medical Research
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	2023-11-29
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2024-05-31
Release Version	1

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Project:

Project ID:	PR001882
Project DOI:	doi: 10.21228/M8FQ52
Project Title:	Estrogen-mediated inhibition of purine metabolism and cell cycle arrest as a novel therapeutic approach in colorectal cancer cells
Project Summary:	Purine metabolism is upregulated in various cancers including colorectal cancer (CRC). While previous research has elucidated the role of Estrogen (E2) in metabolism remodeling and ATP production, its effects on purine metabolism remained unexplored. This study investigates the impact of E2 signalling on purine metabolism in CRC cells. We demonstrate, for the first time, a protective effect of E2 on CRC cells by targeting the purine synthesis pathway through its receptor estrogen receptor α (ERα). A full metabolomic profiling, next generation sequencing (NGS) and integrated OMICS were conducted for HCT-116 cells treated with E2 with and without silencing ERα. Our results revealed an enrichment of the purine metabolic pathway, with 27 genes in the de novo purine synthesis pathway downregulated in E2-treated CRC cells. Besides, E2-induced DNA damage, cell cycle arrest, and apoptosis are ERα-dependent. Our findings suggest potential therapeutic avenues for CRC treatment through antimetabolites targeting purine synthesis, as E2 treatment reduces the expression of relevant metabolites.
Institute:	Sharjah Institute for Medical Research
Last Name:	Facility
First Name:	Core
Address:	M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah
Email:	tims-tof@sharjah.ac.ae
Phone:	065057656

Subject:

Subject ID:	SU003143
Subject Type:	Cultured cells
Subject Species:	Colorectal cancer cells

Factors:

Subject type: Cultured cells; Subject species: Colorectal cancer cells (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA328081	E2_2-01-8862	17-β-estradiol
SA328082	E2_2-02-8863	17-β-estradiol
SA328083	E2_3-02-8865	17-β-estradiol
SA328084	E2_1-02-8861	17-β-estradiol
SA328085	E2_3-01-8864	17-β-estradiol
SA328086	E2_1-01-8860	17-β-estradiol
SA328087	control_2-01-8854	Control
SA328088	control_1-02-8853	Control
SA328089	control_2-02-8855	Control
SA328090	control_3-01-8856	Control
SA328091	control_3-02-8857	Control
SA328092	control_1-01-8852	Control

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO003136
Collection Summary:	HCT-116 cells were cultured using a complete RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 2 mM l-glutamine, 1% non-essential amino acids, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum. Depending on each experimental run, HCT-116 cells were seeded and incubated at 37°C in a 5% CO2 environment for 24 hours
Sample Type:	Colerectal cancer cells

Treatment:

Treatment ID:	TR003152
Treatment Summary:	20 nM 17-β-estradiol (E2), diluted in 70% ethanol, (Sigma-Aldrich, St. Louis, MO, USA), was introduced to the cells for 48 hours. Cells designated as control received the same volume of 70% ethanol, serving as the vehicle.

Sample Preparation:

Sampleprep ID:	SP003149
Sampleprep Summary:	To assess the intracellular metabolites, whole metabolites levels were measured after proper treatment. In brief, 1.0 × 106 of HCT-116 cells were collected and cell extracts were washed twice with 5% of mannitol. The pellet was lysed using 400 uL of protease inhibitor dissolved in lysis buffer, allowing it to sit for 10 minutes at room temperature. Subsequently, all samples underwent 30 seconds of vortexing and were sonicated using either the COPLEY sonicator or QSONICA SONICATOR (Qsonica, Newtown, CT, USA) at 30% AMP for 30 seconds until the pellets dissolved. The resulting solution was transferred to new Eppendorf tubes and centrifuged at 14000 rpm for 5 minutes. After centrifugation, the supernatant layer was carefully moved to another Eppendorf tube, to which 400 uL of methanol and 300 uL of chloroform were added. Each mixture was vortexed for 30 seconds and then centrifuged at 14000 rpm for 5 minutes. The upper layer was transferred and stored in a glass vial. For washing, 300 uL of methanol was added to the white disk and lower layer, vortexed until the white disk broke and settled at the bottom of the Eppendorf. Subsequently, samples were centrifuged at 14000 rpm for 3 minutes, and the supernatant was transferred into the same glass vial. To dry the solvent, the samples were processed in the EZ-2 Plus (GeneVac, Ipswich, UK) at 37 ± 1 °C. The dried samples were resuspended with 200 µL (0.1% formic acid in deionized water) and vortexed for 2 minutes for thorough mixing. Finally, the samples were filtered using a hydrophilic nylon syringe filter with a 0.45 µm pore size and returned to the glass insert within LC glass vials for analysis by Q-TOF MS. A quality control (QC) sample was prepared by pooling the same volume (10 uL) from each sample, and all samples were placed in the autosampler at a temperature set at 4℃.

Sampleprep ID:

SP003149

Sampleprep Summary:

To assess the intracellular metabolites, whole metabolites levels were measured after proper treatment. In brief, 1.0 × 106 of HCT-116 cells were collected and cell extracts were washed twice with 5% of mannitol. The pellet was lysed using 400 uL of protease inhibitor dissolved in lysis buffer, allowing it to sit for 10 minutes at room temperature. Subsequently, all samples underwent 30 seconds of vortexing and were sonicated using either the COPLEY sonicator or QSONICA SONICATOR (Qsonica, Newtown, CT, USA) at 30% AMP for 30 seconds until the pellets dissolved. The resulting solution was transferred to new Eppendorf tubes and centrifuged at 14000 rpm for 5 minutes. After centrifugation, the supernatant layer was carefully moved to another Eppendorf tube, to which 400 uL of methanol and 300 uL of chloroform were added. Each mixture was vortexed for 30 seconds and then centrifuged at 14000 rpm for 5 minutes. The upper layer was transferred and stored in a glass vial. For washing, 300 uL of methanol was added to the white disk and lower layer, vortexed until the white disk broke and settled at the bottom of the Eppendorf. Subsequently, samples were centrifuged at 14000 rpm for 3 minutes, and the supernatant was transferred into the same glass vial. To dry the solvent, the samples were processed in the EZ-2 Plus (GeneVac, Ipswich, UK) at 37 ± 1 °C. The dried samples were resuspended with 200 µL (0.1% formic acid in deionized water) and vortexed for 2 minutes for thorough mixing. Finally, the samples were filtered using a hydrophilic nylon syringe filter with a 0.45 µm pore size and returned to the glass insert within LC glass vials for analysis by Q-TOF MS. A quality control (QC) sample was prepared by pooling the same volume (10 uL) from each sample, and all samples were placed in the autosampler at a temperature set at 4℃.

Combined analysis:

Analysis ID	AN004966
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Bruker Elute
Column	Hamilton® Intensity Solo 2 C18 column (2.1 × 100 mm, 1.8 µm)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH003748
Chromatography Summary:	An ultra-high-performance liquid chromatography system; Elute UHPLC (Bruker Daltonik GmbH, Bremen, Germany) . 10 µL aliquot of the sample was injected and the separation was performed on a Hamilton® Intensity Solo C18 column (2.1 × 100 mm, 1.8 µm) (Bruker Daltonik) in a column oven temperature set at 35 ◦C , using solvent A (0.1% formic acid in deionized Water) and solvent B (0.1% formic acid in acetonitrile) with the following gradient elution mode: 0 to 2 min, 1% B; 2 to 17 min, 1–99% B; 17 to 20 min, 99% B; 20 to 20.1 min, 99–1% B; 20.1 to 30 min, 1% B. The flow rate was 0.25 mL/min from 0 to 20 min, 0.35 mL/min from 20 min to 28.3 min, and 0.25 mL/min from 28.3 to 30 min.
Instrument Name:	Bruker Elute
Column Name:	Hamilton® Intensity Solo 2 C18 column (2.1 × 100 mm, 1.8 µm)
Column Temperature:	35◦C
Flow Gradient:	gradient elution mode: 0 to 2 min, 1% B; 2 to 17 min, 1–99% B; 17 to 20 min, 99% B; 20 to 20.1 min, 99–1% B; 20.1 to 30 min, 1% B.
Flow Rate:	The flow rate was 0.25 mL/min from 0 to 20 min, 0.35 mL/min from 20 min to 28.3 min, and 0.25 mL/min from 28.3 to 30 min.
Solvent A:	Water (0.1% Formic Acid)
Solvent B:	ACN (0.1% Formic Acid)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004706
Analysis ID:	AN004966
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The ESI source condition for every injection was as follows: The drying gas flow rate was 10.0 L/min at a temp of 220 ◦C; the capillary voltage was set at 4500 V; the End Plate offset was set at 500 V; the nebulizer pressure of 2.2 bar. The acquisition involved two segments; auto MS scan, which ranged from 0 to 0.3 min for the calibrant sodium formate, and auto MS/MS scan with CID acquisition, which included fragmentation and ranged from 0.3 to 30 min. The acquisition in both segments was performed using the positive mode at 12 Hz. The automatic in-run mass scan range was from 50 to 1300 m/z, the width of the precursor ion was ±0.5, the cycle time was 0.5 sec, and the threshold was 400 cts. Active exclusion was excluded after 3 spectra and released after 0.2 min. For MS2 acquisition the data dependent acquisition (DDA) was used, and the collision energy stepping fluctuated between 100 and 250% set at 20 eV. M/Z measurements were externally calibrated using 10 mM of sodium formate before sample analysis. In addition, sodium formate solution was injected at the beginning of each sample run and used for internal calibration during data processing TRX-2101/RT-28-calibrants for Bruker T-ReX LC-QTOF (Nova Medical Testing Inc.) was injected before sample analysis to check and test the performance of the column, reversed-phase liquid chromatography (RPLC) separation, multipoint retention time calibration, and the mass spectrometer. Also, TRX-3112-R/MS Certified Human serum for Bruker T-ReX LC-QTOF solution (Nova Medical Testing Inc.) was prepared from pooled human blood and injected before sample analysis to check the performance of the LC-MS instruments.
Ion Mode:	POSITIVE