Changes in Lactate Production, Lactate Dehydrogenase Genes Expression and DNA Methylation in Response to Tamoxifen Resistance Development in MCF-7 Cell Line

Abstract

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.

High Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Correlating the Metabolic Changes of Lactate, Pyruvate and L-Glutamine with Induced Tamoxifen Resistant MCF-7 Cell Line Potential Molecular Changes

“…A noticeable change in MCF-7 cells morphology was observed during the TMX treatment operation as shown in Figure 2 . The observed morphological changes were aligned with changes presented in our previous works [ 14 , 16 ]. The significant changes in cells morphology have been seen at 35 µM.…”

“…Data graphing was accomplished by GraphPad Prism 5.0 software as shown in Figure 3 . The rate of growth of treated cells with TMX decreased around the concentration of 35 μM and 40 μM, which is the IC 50 reported in our study in MCF-7 cells sensitive to TMX [ 14 ], and only cells that started to have molecular and metabolic changes survived the increased concentrations of TMX in comparison to control and low TMX dose groups.…”

“…Lactate dehydrogenase ( LDH ) is involved in the conversion of pyruvate to lactate and vice versa, concomitantly with the inter-conversion of NAD + and NADH [ 13 ]. Experiments in our laboratory by Hamadneh et al [ 14 ], showed a significant increase in LDH-B gene expression during the development of TMX resistance in MCF-7 cells up to 3-fold with gradual increase of TMX doses which is consistent with other published research [ 15 ]. Increased LDH-B gene expression led to a significant increase in lactate level and expected to show a further increase in pyruvate level.…”

“…While significant increase was observed after treatment with 35 µM and 40 µM of TMX Figure 5 B. This can also be attributed by the overexpression of LDH-B that was reported in our laboratory by Hamadenh et al after developing TMX resistant MCF-7 cells, and consequently a significant increase in concentration of produced pyruvate in addition to lactate [ 14 ]. Furthermore, EMT process that noticed in TMX resistant MCF-7 cells was accompanied by an increase in the expression of LDH-B isoenzyme that is responsible for the conversion of lactate to pyruvate and vice versa, even though the absence of expression in some cancer types of BC and prostate cancer due to hypermethylation of the promoter in genes level [ 38 ].…”

“…A significant increase in lactate production was observed after treatment with 35 µM of TMX Figure 5 A. This can be due to either Warburg effect in resistant MCF-7 cells [ 36 ], and/or the increased LDH-B enzyme expression that has a key role in TMX resistant MCF-7 cells [ 14 , 37 ]. For pyruvate, a little fluctuation in its production was noticed before treatment with 35 µM TMX.…”