Sequestration of 9-Hydroxystearic Acid in FAHFA (Fatty Acid Esters of Hydroxy Fatty Acids) as a Protective Mechanism for Colon Carcinoma Cells to Avoid Apoptotic Cell Death

Abstract

Hydroxy fatty acids are known to cause cell cycle arrest and apoptosis. The best studied of them, 9-hydroxystearic acid (9-HSA), induces apoptosis in cell lines by acting through mechanisms involving different targets. Using mass spectrometry-based lipidomic approaches, we show in this study that 9-HSA levels in human colorectal tumors are diminished when compared with normal adjacent tissue. Since this decrease could be compatible with an escape mechanism of tumors from 9-HSA-induced apoptosis, we investigated different features of the utilization of this hydroxyfatty acid in colon. We show that in colorectal tumors and related cell lines such as HT-29 and HCT-116, 9-HSA is the only hydroxyfatty acid constituent of branched fatty acid esters of hydroxyfatty acids (FAHFA), a novel family of lipids with anti-inflammatory properties. Importantly, FAHFA levels in tumors are elevated compared with normal tissue and, unlike 9-HSA, they do not induce apoptosis of colorectal cell lines over a wide range of concentrations. Further, the addition of 9-HSA to colon cancer cell lines augments the synthesis of different FAHFA before the cells commit to apoptosis, suggesting that FAHFA formation may function as a buffer system that sequesters the hydroxyacid into an inactive form, thereby restricting apoptosis.

The Contribution of Cytosolic Group IVA and Calcium-Independent Group VIA Phospholipase A2s to Adrenic Acid Mobilization in Murine Macrophages

“…Phospholipid classes were separated twice with chloroform/methanol/28% (w/w) ammonium hydroxide (60:37.5:4, v/v/v) as the mobile phase, using plates impregnated with boric acid [41]. The bands corresponding to the different lipid classes were scraped off from the plate, and fatty acid methyl esters were obtained from the various lipid fractions by transmethylation with 0.5 M KOH in methanol for 60 min at 37 • C [42][43][44][45]. Analyses were carried out using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass-selective detector operated in an electron impact mode (EI, 70 eV).…”

“…The apparatus was also equipped with an Agilent 7693 autosampler and an Agilent DB23 column (60 m length × 0.25 mm internal diameter × 0.15 µM film thickness) (Agilent Technologies, Santa Clara, CA, USA). Data analysis was carried out with the Agilent G1701EA MSD Productivity Chemstation software, revision E.02.00 [42][43][44][45].…”

Phospholipid Arachidonic Acid Remodeling During Phagocytosis in Mouse Peritoneal Macrophages

“…After addition of internal standards, lipids were separated by thin-layer chromatography, as described in the preceding paragraph. The bands corresponding to the different lipid classes were scraped off from the plate, and fatty acid methyl esters were obtained from the various lipid fractions by transmethylation with 0.5 M KOH in methanol for 60 min at 37 • C [41][42][43]. Analysis was carried out using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass-selective detector operated in electron impact mode (EI, 70 eV), equipped with an Agilent 7693 autosampler and an Agilent DB23 column (60 m length × 0.25 mm internal diameter × 0.15 µm film thickness) (Agilent Technologies, Santa Clara, CA, USA).…”

“…Analysis was carried out using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass-selective detector operated in electron impact mode (EI, 70 eV), equipped with an Agilent 7693 autosampler and an Agilent DB23 column (60 m length × 0.25 mm internal diameter × 0.15 µm film thickness) (Agilent Technologies, Santa Clara, CA, USA). Data analysis was carried out with the Agilent G1701EA MSD Productivity Chemstation software, revision E.02.00 [41][42][43].…”

Phospholipid arachidonic acid remodeling in macrophages: role of plasmalogen species

“…FAMEs were obtained from the various lipid fractions by transmethylation with 0.5 M KOH in methanol for 60 min at 37 ºC (215,(217)(218)(219)(220). Analysis was carried out using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass-selective detector operated in electron impact mode (EI, 70 eV), equipped with an Agilent 7693 autosampler and an Agilent DB23 column (60 m length x 0.25 mm internal diameter x 0.15 µm film thickness).…”