Investigation of discriminant metabolites in tamoxifen-resistant and choline kinase-alpha-downregulated breast cancer cells using .sup.1H-nuclear magnetic resonance spectroscopy

Metabolites linked to changes in choline kinase-[alpha] (CK-[alpha]) expression and drug resistance, which contribute to survival and autophagy mechanisms, are attractive targets for breast cancer therapies. We previously reported that autophagy played a causative role in driving tamoxifen (TAM) resistance of breast cancer cells (BCCs) and was also promoted by CK-[alpha] knockdown, resulting in the survival of TAM-resistant BCCs. There is no comparative study yet about the metabolites resulting from BCCs with TAM-resistance and CK-[alpha] knockdown. Therefore, the aim of this study was to explore the discriminant metabolic biomarkers responsible for TAM resistance as well as CK-[alpha] expression, which might be linked with autophagy through a protective role. A total of 33 intracellular metabolites, including a range of amino acids, energy metabolism-related molecules and others from cell extracts of the parental cells (MCF-7), TAM-resistant cells (MCF-7/TAM) and CK-[alpha] knockdown cells (MCF-7/shCK-[alpha], MCF-7/TAM/shCK-[alpha]) were analyzed by proton nuclear magnetic resonance spectroscopy (.sup.1 H-NMRS). Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) revealed the existence of differences in the intracellular metabolites to separate the 4 groups: MCF-7 cells, MCF-7/TAM cells, MCF-7-shCK-[alpha] cells, and MCF-7/TAM/shCK-[alpha] cells. The metabolites with VIP>1 contributed most to the differentiation of the cell groups, and they included fumarate, UA (unknown A), lactate, myo-inositol, glycine, phosphocholine, UE (unknown E), glutamine, formate, and AXP (AMP/ADP/ATP). Our results suggest that these altered metabolites would be promising metabolic biomarkers for a targeted therapeutic strategy in BCCs that exhibit TAM-resistance and aberrant CK-[alpha] expression, which triggers a survival and drug resistance mechanism.