Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions

Abstract

Folate (vitamin B9) is involved in one-carbon transfer reactions and plays a significant role in nucleic acid synthesis and control of cellular proliferation, among other key cellular processes. It is now recognized that the role of folates in different stages of carcinogenesis is complex, and more research is needed to understand how folate reactions become dysregulated in cancers and the metabolic consequences that occur as a result. ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism expressed in many tissues, is ubiquitously downregulated in cancers and is not expressed in cancer cell lines. The RT4 cell line (derived from papillary bladder cancer) which expresses high levels of ALDH1L1 represents an exception, providing an opportunity to explore the metabolic consequences of the loss of this enzyme. We have downregulated this protein in RT4 cells (shRNA driven knockdown or CRISPR driven knockout) and compared metabolomes of ALDH1L1-expressing and -deficient cells to determine if metabolic changes linked to the loss of this enzyme might provide proliferative and/or survival advantages for cancer cells. In this study, cell extracts were analyzed using Ultra High Performance Liquid Chromatography High Resolution Mass Spectrometry (UHPLC-HR-MS). A total of 13,339 signals were identified or annotated using an in-house library and public databases. Supervised and unsupervised multivariate analysis revealed metabolic differences between RT4 cells and ALDH1L1-deficient clones. Glycine (8-fold decrease) and metabolites derived from S-adenosylmethionine utilizing pathways were significantly decreased in the ALDH1L1-deficient clones, compared with RT4 cells. Other changes linked to ALDH1L1 downregulation include decreased levels of amino acids, Krebs cycle intermediates, and ribose-5-phosphate, and increased nicotinic acid. While the ALDH1L1-catalyzed reaction is directly linked to glycine biosynthesis and methyl group flux, its overall effect on cellular metabolism extends beyond immediate metabolic pathways controlled by this enzyme.

Metabolomics Analysis Reveals Altered Metabolic Pathways and Response to Doxorubicin in Drug-Resistant Triple-Negative Breast Cancer Cells

“…Cells were plated at approximately 80% confluency in 6-well culture plates and were treated with 500, 200, 100, 50, or 0 nM doxorubicin with a final DMSO amount of 0.1% (n = 4 per group). After 24 h of treatment, metabolites were extracted from cell samples as described previously [ 26 , 32 , 33 , 34 ]. Briefly, treatment media were aspirated, and cells were washed with 1 mL of ice-cold PBS.…”

“…Metabolomics data were acquired via previously published UHPLC-HRMS methods [26,[32][33][34][35][36][37]. The analysis utilized a Vanquish UHPLC system coupled to a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an HSS T3 C18 column (2.1 mm × 100 mm, 1.7 µm, Waters Corporation) held at 50 • C. A binary pump was used with water + 0.1% formic acid (A) and methanol + 0.1% formic acid (B) as mobile phases.…”

Metabolomics Analysis Reveals Novel Targets of Chemosensitizing Polyphenols and Omega-3 Polyunsaturated Fatty Acids in Triple Negative Breast Cancer Cells

“…After treatment, metabolites were extracted from cell samples as described previously [91][92][93]. Briefly, treatment media were aspirated, and cells were washed with 5 mL of ice-cold PBS.…”

“…Metabolomics data were acquired via previously published UHPLC-HRMS methods using a Vanquish UHPLC system coupled to a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an HSS T3 C18 column (2.1 × 100 mm, 1.7 µm, Waters Corporation) held at 50 • C [91][92][93][94][95][96][97][98]. A binary pump was used with water + 0.1% formic acid (A) and methanol + 0.1% formic acid (B) as mobile phases.…”