Summary of Study ST003547
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 PR002183. The data can be accessed directly via it's Project DOI: 10.21228/M8FK0S 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 | ST003547 |
| Study Title | Analysis of the fate of docosahexaenoic acid in HCT116 colorectal cancer cells cultured at pH 7.4 or 6.5. |
| Study Summary | Cancer cells in acidic tumor regions exhibit a shift from glucose metabolism to the preferential uptake and use of fatty accids. This study aimed to determine the fate of docosahexaenoic acid (DHA) in colon HCT116 cancer cells maintained at pH 7.4 or pH 6.5. The incorporation of triglycerides and/or phospholipids was analysed together with associated changes in the proportion of saturated and monounsaturated fatty acids. We found that DHA heightened the elevated levels of triacylglycerol (TAG) induced by acidic conditions. However, while the abundance of the most prominent Mono-unsaturated Fatty Acid (MUFA), i.e., C18:1 oleate, was directly proportional to the TAG increase in acid-exposed cancer cells, DHA exposure did not further increase the oleate contribution in TAG. The increase in total TAG levels in acidic cancer cells mostly resulted from the incorporation of C22:6 DHA. A more detailed analysis of the composition of TAG revealed that DHA emerges as the predominant constituent of TAG, with 45% of them incorporating at least one DHA, while the TAG content in C18:1 oleate decreased from 46% to 22% in acid-exposed cancer cells. Importantly, while acidic pH and DHA exposure independently increased the amounts of TAG to a similar extent, the PUFA/MUFA ratio in the neutral lipid (NL) fraction was increased by 5-fold in the DHA condition. The PUFA/MUFA ratio eventually peaked in the NL but also phospholipid (PL) fractions of acid-exposed cancer cells when subjected to DHA treatment. Altogether these data indicate that the preferred accumulation of Poly-unsaturated Fatty Acid (PUFA) in acidic cancer cells sensitizes them to lipid peroxidation and associated ferroptosis. In a parallel set of experiments, we showed that this is further supported by the PUFA-induced downregulation of SCD1, an enzyme supporting MUFA synthesis. |
| Institute | UCLouvain |
| Department | IREC |
| Laboratory | Pole of Pharmacology and Therapuetics |
| Last Name | FERON |
| First Name | OLIVIER |
| Address | 57B Avenue Hippocrate B1.57.04 |
| olivier.feron@uclouvain.be | |
| Phone | +3227645264 |
| Submit Date | 2024-10-23 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-11-04 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002183 |
| Project DOI: | doi: 10.21228/M8FK0S |
| Project Title: | Influence of tumor acidosis on the fate of fatty acids in cancer cells. |
| Project Summary: | Cancer cells in acidic tumor regions exhibit a shift from glucose metabolism to the preferential uptake and use of fatty accids. This project aims to analyse the fate of docosahexaenoic acid (DHA, an omega-3 polysunsaturated fatty acid) in cancer cells maintained at physiological pH (7.4) or adpated to an acidic pH (6.5). The accumulation of triglycerides and phospholipids was analysed together with changes in the proportion of saturated and monounsaturated fatty acids. |
| Institute: | UCLouvain |
| Department: | IREC |
| Laboratory: | Pole of Pharmacology and Therapuetics |
| Last Name: | FERON |
| First Name: | Olivier |
| Address: | 57B Avenue Hippocrate B1.57.04 |
| Email: | olivier.feron@uclouvain.be |
| Phone: | +3227645264 |
| Publications: | Nature Communications, pending |
| Contributors: | Sebastien Ibanez |
Subject:
| Subject ID: | SU003676 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | HCT116 |
| Subject Comments: | HCT 116 cell line was isolated from the colon of an adult male with colon cancer. |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | pH | treatment |
|---|---|---|---|---|
| SA387780 | C_L_6.5_1 | HCT116 colon cancer cells | 6.5 | control |
| SA387781 | C_L_6.5_2 | HCT116 colon cancer cells | 6.5 | control |
| SA387778 | DHA_L_6.5_1 | HCT116 colon cancer cells | 6.5 | DHA 50µM |
| SA387779 | DHA_L_6.5_2 | HCT116 colon cancer cells | 6.5 | DHA 50µM |
| SA387784 | C_L_7.4_1 | HCT116 colon cancer cells | 7.4 | control |
| SA387785 | C_L_7.4_2 | HCT116 colon cancer cells | 7.4 | control |
| SA387782 | DHA_L_7.4_1 | HCT116 colon cancer cells | 7.4 | DHA 50µM |
| SA387783 | DHA_L_7.4_2 | HCT116 colon cancer cells | 7.4 | DHA 50µM |
| Showing results 1 to 8 of 8 |
Collection:
| Collection ID: | CO003669 |
| Collection Summary: | Colon HCT116 cancer cells were purchased from ATCC, stored according to the supplier’s instructions and used within 6 months after resuscitation of frozen aliquots. All cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 10 mM D-glucose, 2 mM L-glutamine (or glutamax) and if needed 25 mM of both PIPES and HEPES before adjusting pH to 7.4 or 6.5. Cells were tested for mycoplasma contamination with the MycoAlert™ Mycoplasma Detection kit (Lonza) before being used. Cell number was assessed by cell counting on a hematocytometer with Trypan Blue mortality exclusion dye. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003685 |
| Treatment Summary: | The effects of 25 µM docosahexaenoic acid (DHA) supplementation were compared with those of the vehicle. Treatment was performed for 24 hours in full media with 10% lipid-depleted FBS (#S181L-500, VWR) and BSA-conjugated DHA (#10-2206, Larodan); non conjugated BSA (vehicle) was used as control. DHA was conjugated with BSA in PBS to obtain a fatty acid/BSA ratio of 4:1 (mol:mol). Aliquoted DHA-BSA and free BSA were stored at -80°C under light protection and directly used upon unfreezing by addition of the requested concentrations into the wells. |
Sample Preparation:
| Sampleprep ID: | SP003683 |
| Sampleprep Summary: | An amount of cells containing 10 μg of DNA was homogenized in 700 μL of water with a handheld sonicator and was mixed with 800 μl HCl(1M):CH3OH 1:8 (v/v), 900 μl CHCl3, 200 μg/ml of the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT; Sigma Aldrich) and 3 μl of UltimateSPLASH™ ONE internal standard mix (#330820, Avanti Polar Lipids) and 3 μl of SphingoSPLASH™ I internal standard mix (#330734, Avanti Polar Lipids). After vortexing and centrifugation, the lower organic fraction was collected and evaporated using a Savant Speedvac spd111v (Thermo Fisher Scientific) at room temperature and the remaining lipid pellet was stored at - 20°C under argon. Just before mass spectrometry analysis, lipid pellets were reconstituted in 100% ethanol. Lipid species were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) on a Nexera X2 UHPLC system (Shimadzu) coupled with hybrid triple quadrupole/linear ion trap mass spectrometer (6500+ QTRAP system; AB SCIEX). |
Combined analysis:
| Analysis ID | AN005829 | AN005830 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Shimadzu Nexera X2 | Shimadzu Nexera X2 |
| Column | Waters Xbridge BEH Amide (150 mm × 4.6 mm, 3.5 μm) | Waters Xbridge BEH Amide (150 mm × 4.6 mm, 3.5 μm) |
| MS Type | ESI | ESI |
| MS instrument type | QTRAP | QTRAP |
| MS instrument name | ABI Sciex 6500+ Qtrap | ABI Sciex 6500+ Qtrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | nmol / mg DNA | nmol / mg DNA |
Chromatography:
| Chromatography ID: | CH004430 |
| Chromatography Summary: | Chromatographic separation was performed on a XBridge amide column (150 mm × 4.6 mm, 3.5 μm; Waters) maintained at 35°C using mobile phase A [1 mM ammonium acetate in water-acetonitrile 5:95 (v/v)] and mobile phase B [1 mM ammonium acetate in water-acetonitrile 50:50 (v/v)] in the following gradient: (0-6 min: 0% B - 6% B; 6-10 min: 6% B - 25% B; 10-11 min: 25% B - 98% B; 11-13 min: 98% B - 100% B; 13-19 min: 100% B; 19-24 min: 0% B) at a flow rate of 0.7 mL/min which was increased to 1.5 mL/min from 13 minutes onwards. |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters Xbridge BEH Amide (150 mm × 4.6 mm, 3.5 μm) |
| Column Temperature: | 35°C |
| Flow Gradient: | 0-6 min: 0% B - 6% B; 6-10 min: 6% B - 25% B; 10-11 min: 25% B - 98% B; 11-13 min: 98% B - 100% B; 13-19 min: 100% B; 19-24 min: 0% B |
| Flow Rate: | 0 - 13 min: 0.7 mL/min, 13 - 24 min: 1.5 mL/min |
| Solvent A: | 5% water/95% acetonitrile; 1 mM ammonium acetate |
| Solvent B: | 50% water/50% acetonitrile; 1 mM ammonium acetate |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS005549 |
| Analysis ID: | AN005829 |
| Instrument Name: | ABI Sciex 6500+ Qtrap |
| Instrument Type: | QTRAP |
| MS Type: | ESI |
| MS Comments: | SM, CE, CER, DCER, HCER, LCER were measured in positive ion mode with a product ion of 184.1, 369.4, 264.4, 266.4, 264.4 and 264.4 respectively. TAG, DAG and MAG were measured in positive ion mode with a neutral loss for one of the fatty acyl moieties. PC, LPC, PE, LPE, PG, PI and PS were measured in negative ion mode by fatty acyl fragment ions. Lipid quantification was performed by scheduled multiple reactions monitoring (MRM), the transitions being based on the neutral losses or the typical product ions as described above. The instrument parameters were as follows: Curtain Gas = 35 psi; Collision Gas = 8 a.u. (medium); IonSpray Voltage = 5500 V; Temperature = 550°C; Ion Source Gas 1 = 50 psi; Ion Source Gas 2 = 60 psi; Declustering Potential = 60 V; Entrance Potential = 10 V; Collision Cell Exit Potential = 15 V. The following fatty acyl moieties were taken into account for the lipidomic analysis: 14:0, 14:1, 16:0, 16:1, 16:2, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:4, 22:5 and 22:6 except for TGs which considered: 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 20:5, 22:2, 22:3, 22:4, 22:5, 22:6. |
| Ion Mode: | POSITIVE |
| MS ID: | MS005550 |
| Analysis ID: | AN005830 |
| Instrument Name: | ABI Sciex 6500+ Qtrap |
| Instrument Type: | QTRAP |
| MS Type: | ESI |
| MS Comments: | SM, CE, CER, DCER, HCER, LCER were measured in positive ion mode with a product ion of 184.1, 369.4, 264.4, 266.4, 264.4 and 264.4 respectively. TAG, DAG and MAG were measured in positive ion mode with a neutral loss for one of the fatty acyl moieties. PC, LPC, PE, LPE, PG, PI and PS were measured in negative ion mode by fatty acyl fragment ions. Lipid quantification was performed by scheduled multiple reactions monitoring (MRM), the transitions being based on the neutral losses or the typical product ions as described above. The instrument parameters were as follows: Curtain Gas = 35 psi; Collision Gas = 8 a.u. (medium); IonSpray Voltage = −4,500 V; Temperature = 550°C; Ion Source Gas 1 = 50 psi; Ion Source Gas 2 = 60 psi; Declustering Potential = −80 V; Entrance Potential = −10 V; Collision Cell Exit Potential = −15 V. The following fatty acyl moieties were taken into account for the lipidomic analysis: 14:0, 14:1, 16:0, 16:1, 16:2, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4, 20:5, 22:0, 22:1, 22:2, 22:4, 22:5 and 22:6 except for TGs which considered: 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 20:5, 22:2, 22:3, 22:4, 22:5, 22:6. |
| Ion Mode: | NEGATIVE |
