Metabolic Response to XD14 Treatment in Human Breast Cancer Cell Line MCF-7

Abstract

XD14 is a 4-acyl pyrrole derivative, which was discovered by a high-throughput virtual screening experiment. XD14 inhibits bromodomain and extra-terminal domain (BET) proteins (BRD2, BRD3, BRD4 and BRDT) and consequently suppresses cell proliferation. In this study, metabolic profiling reveals the molecular effects in the human breast cancer cell line MCF-7 (Michigan Cancer Foundation-7) treated by XD14. A three-day time series experiment with two concentrations of XD14 was performed. Gas chromatography-mass spectrometry (GC-MS) was applied for untargeted profiling of treated and non-treated MCF-7 cells. The gained data sets were evaluated by several statistical methods: analysis of variance (ANOVA), clustering analysis, principle component analysis (PCA), and partial least squares discriminant analysis (PLS-DA). Cell proliferation was strongly inhibited by treatment with 50 µM XD14. Samples could be discriminated by time and XD14 concentration using PLS-DA. From the 117 identified metabolites, 67 were significantly altered after XD14 treatment. These metabolites include amino acids, fatty acids, Krebs cycle and glycolysis intermediates, as well as compounds of purine and pyrimidine metabolism. This massive intervention in energy metabolism and the lack of available nucleotides could explain the decreased proliferation rate of the cancer cells.

Cells grown in three-dimensional spheroids mirror in vivo metabolic response of epithelial cells

“…Analysis of cell culture media: Metabolites from cell culture medium were extracted as previously reported by acetonitril:methanol 44 . Pellets were analyzed by GC/MS profiling in the same manner as endometabolites 48 . Instead of peak sum normalization, only the internal standard as well as a correction (17:12) for dilution introduced by the NaCl rinsing in 3D cultures was applied for normalization.…”

Metabolic profiling of isolated mitochondria and cytoplasm reveals compartment-specific metabolic responses

“…Based on the protocol of Fiehn ( 2006 ), sample derivatization, gas chromatography–mass spectrometry (GC–MS) analysis and data evaluation were performed as described by Pan et al ( 2016 ). Briefly, the prewarmed pellets were derivatized by shaking with 20 µL 20 mg/mL methoxyamine hydrochloride (Sigma-Aldrich, Germany) in pyridine (Sigma-Aldrich, Germany) at 1200 rpm and 28 °C for 90 min and, after short centrifugation, with 50 µL N -methyl- N -trimethylsilyltrifluoroacetamide (Sigma-Aldrich, Germany) at 1200 rpm and 37 °C for 30 min.…”

“…Alterations of metabolites have been reported in blood plasma, urine of cancer patients and cancer cell lines, providing promising possibilities of biomarker discoveries (Bullinger et al 2007 ; Frickenschmidt et al 2008 ; Asiago et al 2010 ; Shen et al 2013 ; Armitage and Barbas 2014 ; Willmann et al 2015 ). Additionally, metabolomics helps in understanding cellular mechanisms by showing the metabolic status of drug-treated or non-treated cancer cells (Jain et al 2012 ; Budczies et al 2015 ; Pan et al 2016 ). Since metabolomics has been described as the link between genotype and phenotype (Fiehn 2002 ), Stefely et al ( 2016 ) have profiled 174 yeast strains with different single gene knockouts to elucidate the mitochondrial protein functions.…”

Monitoring for Response to Antineoplastic Drugs: The Potential of a Metabolomic Approach

“…Functional endpoints relevant to a response biomarker would include cell death or a reduction in cell proliferation. Unfortunately, most in-vitro studies on drug-induced changes in the metabolome primarily involve a description of changes in the metabolome that result from the drug itself, or it is unclear whether the changes are pharmacologic or due to impairment of tumor cell expansion [ 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ].…”

“…In-vitro experiments occur in defined spaces, where it is possible to see changes in metabolite concentrations in the extracellular space due to fluxes with the intracellular compartment, alterations in cellular metabolism, or due to a massive release from cell lysis. In vivo, the metabolome could be affected by a variety of tumor and host factors, which is why we and others have observed that not all cancer-associated features in the circulating metabolome can be attributed to the metabolic perturbations characteristic of a cancer cell [ 52 , 57 , 58 ]. Indeed, it is well known that in-vivo observations do not recapitulate what is observed in cell line experiments [ 59 ].…”