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Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography-tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid-ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v / v ). The linear range was 0.11-2.25, 0.012-0.227, and 0.02-0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism.

Lactate dehydrogenase (LDH) is a key enzyme in the last step of glycolysis, playing a role in the pyruvate-to-lactate reaction. It is associated with the prognosis and metastasis of many cancers, including breast cancer. In this study, we investigated the changes in LDH gene expression and lactate concentrations in the culture media during tamoxifen resistance development in the MCF-7 cell line, and examined LDHB promoter methylation levels. An upregulation of 2.9 times of LDHB gene expression was observed around the IC50 concentration of tamoxifen in treated cells, while fluctuation in LDHA gene expression levels was found. Furthermore, morphological changes in the cell shape accompanied the changes in gene expression. Bisulfate treatment followed by sequencing of the LDHB promoter was performed to track any change in methylation levels; hypomethylation of CpG areas was found, suggesting that gene expression upregulation could be due to methylation level changes. Changes in LDHA and LDHB gene expression were correlated with the increase in lactate concentration in the culture media of treated MCF-7 cells.

Cell culture has replaced many in vivo studies because of ethical and regulatory measures as well as the possibility of increased throughput. Analytical assays to determine (bio)chemical changes are often based on end-point measurements rather than on a series of sequential determinations. The purpose of this work is to develop an analytical system for monitoring cell culture based on sequential injection-capillary electrophoresis (SI-CE) with capacitively coupled contactless conductivity detection (C(4)D). The system was applied for monitoring lactate production, an important metabolic indicator, during mammalian cell culture. Using a background electrolyte consisting of 25mM tris(hydroxymethyl)aminomethane, 35mM cyclohexyl-2-aminoethanesulfonic acid with 0.02% poly(ethyleneimine) (PEI) at pH 8.65 and a multilayer polymer coated capillary, lactate could be resolved from other compounds present in media with relative standard deviations 0.07% for intraday electrophoretic mobility and an analysis time of less than 10min. Using the human embryonic kidney cell line HEK293, lactate concentrations in the cell culture medium were measured every 20min over 3 days, requiring only 8.73μL of sample per run. Combining simplicity, portability, automation, high sample throughput, low limits of detection, low sample consumption and the ability to up- and outscale, this new methodology represents a promising technique for near real-time monitoring of chemical changes in diverse cell culture applications., (Copyright © 2013 Elsevier B.V. All rights reserved.)