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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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℃.