Colon cancer cell differentiation by sodium butyrate modulates metabolic plasticity of Caco-2 cells via alteration of phosphotransfer network

Abstract

The ability of butyrate to promote differentiation of cancer cells has important implication for colorectal cancer (CRC) prevention and therapy. In this study, we examined the effect of sodium butyrate (NaBT) on the energy metabolism of colon adenocarcinoma Caco-2 cells coupled with their differentiation. NaBT increased the activity of alkaline phosphatase indicating differentiation of Caco-2 cells. Changes in the expression of pluripotency-associated markers OCT4, NANOG and SOX2 were characterized during the induced differentiation at mRNA level along with the measures that allowed distinguishing the expression of different transcript variants. The functional activity of mitochondria was studied by high-resolution respirometry. Glycolytic pathway and phosphotransfer network were analyzed using enzymatical assays. The treatment of Caco-2 cells with NaBT increased production of ATP by oxidative phosphorylation, enhanced mitochondrial spare respiratory capacity and caused rearrangement of the cellular phosphotransfer networks. The flexibility of phosphotransfer networks depended on the availability of glutamine, but not glucose in the cell growth medium. These changes were accompanied by suppressed cell proliferation and altered gene expression of the main pluripotency-associated transcription factors. This study supports the view that modulating cell metabolism through NaBT can be an effective strategy for treating CRC. Our data indicate a close relationship between the phosphotransfer performance and metabolic plasticity of CRC, which is associated with the cell differentiation state.

Fig 1. Caco-2 cell culture sensitivity and differentiation response to sodium butyrate (NaBT).

Cells were incubated in the presence of various concentrations of NaBT for 24, 48 or 72 hours. (A): The viability of cells was analyzed by MTT assay. (B): Cytotoxicity was estimated by measurement of lactate dehydrogenase release after treatment with NaBT. (C) Alkaline phosphatase (ALP) activity assay was used to estimate differentiation status of the cells. Data are presented as mean ± SEM (n = 3–5). *P < 0.01; **p < 0.001 (ANOVA followed by Tukey post-hoc test).

Fig 2

Confocal microscopy of untreated and sodium butyrate (NaBT)-treated Caco-2 cells. Morphological changes of cells occur after treatment of cells for 48h with 1mM NaBT. Cells were stained with MitoTracker (red), anti-whole tubulin (green) and DAPI (blue). For all the above, representative images are shown. Scale bars: 50 μm.

Fig 3. Semi-quantitative densitometric analysis of pluripotency-associated transcription factors.

(A): Densitometric band intensity measurements from S3A Fig showing the relative changes between NaBT-treated (1mM, 48h) Caco-2 cells (NaBT) and without treatment (Ctrl) (B): Densitometric band intensity measurements from S4A Fig showing relative changes between primary colorectal tumor samples (T) and adjacent normal tissue samples (Ctrl). Data are presented as mean ± SEM (*p < 0.05, Student’s t test).

Fig 4. Quantitative RT-PCR analysis of pluripotency-associated transcription factors OCT4A and SOX2.

(A): Relative gene expression in Caco-2 cells treated for 48h with 1 mM sodium butyrate (NaBT) or without treatment (Ctrl). (B): Relative gene expression in primary colorectal tumor samples (T) and adjacent normal tissue samples (Ctrl). Data are presented as mean ± SEM (*p < 0.05, Student’s t test).

Fig 5. 48-hour pre-treatment with sodium butyrate induced an increase in oxidative metabolism of Caco-2 cells.

(A): Oxygen consumption rates in the absence of sodium butyrate in the respiratory medium were measured using high-resolution respirometry. (B): Oxygen consumption rates in the presence of sodium butyrate. (C): Parameters of mitochondrial respiration obtained using mitochondrial stress test protocol. (D): ATP concentration measured by UPLC. (E): Effect of glycolysis and OXPHOS inhibition on the ATP/ADP ratio. All data are presented as mean ± SEM (n = 3–5; *p < 0.05, Student’s t test). AntA–antimycin A, CS–citrate synthase, DOG– 2-deoxyglucose, NaBT–sodium butyrate, Olig–oligomycin, Rot–rotenone.

Fig 6. Effect of sodium butyrate on the activity of main glycolytic enzymes.

(A, B): Lactate dehydrogenase activity (C,D): Pyruvate kinase activity. (E,F):Hexokinase activity. B,D,F–cells were grown in glutamine-free medium. All data are presented as mean ± SEM (n = 3–5; *p < 0.05, ANOVA followed by Turkey post hoc test). Gluc–glucose, NaBT–sodium butyrate.

Fig 7. Rearrangement of phosphotransfer system after treatment of Caco-2 cells with sodium butyrate.

(A): Schematic representation of main pathways analyzed. (B): Creatine kinase activity. (C): Adenylate kinase activity. All data are presented as mean ± SEM (n = 3–5; *p < 0.05, ANOVA followed by Tukey post hoc test). AK–adenylate kinase, CK–creatine kinase, G-6-P–glucose-6-phosphate, Gluc–glucose, Glut–glutamine, NaBT–sodium butyrate, TCA–tricarboxylic acid.