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BackgroundKetogenic diets (KDs) or short-term fasting are popular trends amongst supportive approaches for cancer patients. Beta-hydroxybutyrate (3-OHB) is the main physiological ketone body, whose concentration can reach plasma levels of 2–6 mM during KDs or fasting. The impact of 3-OHB on the biology of tumor cells described so far is contradictory. Therefore, we investigated the effect of a physiological concentration of 3 mM 3-OHB on metabolism, proliferation, and viability of breast cancer (BC) cells in vitro.MethodsSeven different human BC cell lines (BT20, BT474, HBL100, MCF-7, MDA-MB 231, MDA-MB 468, and T47D) were cultured in medium with 5 mM glucose in the presence of 3 mM 3-OHB at mild hypoxia (5% oxygen) or normoxia (21% oxygen). Metabolic profiling was performed by quantification of the turnover of glucose, lactate, and 3-OHB and by Seahorse metabolic flux analysis. Expression of key enzymes of ketolysis as well as the main monocarboxylic acid transporter MCT2 and the glucose-transporter GLUT1 was analyzed by RT-qPCR and Western blotting. The effect of 3-OHB on short- and long-term cell proliferation as well as chemo- and radiosensitivity were also analyzed.Results3-OHB significantly changed the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in BT20 cells resulting in a more oxidative energetic phenotype. MCF-7 and MDA-MB 468 cells had increased ECAR only in response to 3-OHB, while the other three cell types remained uninfluenced. All cells expressed MCT2 and GLUT1, thus being able to uptake the metabolites. The consumption of 3-OHB was not strongly linked to mRNA overexpression of key enzymes of ketolysis and did not correlate with lactate production and glucose consumption. Neither 3-OHB nor acetoacetate did interfere with proliferation. Further, 3-OHB incubation did not modify the response of the tested BC cell lines to chemotherapy or radiation.ConclusionsWe found that a physiological level of 3-OHB can change the energetic profile of some BC cell lines. However, 3-OHB failed to influence different biologic processes in these cells, e.g., cell proliferation and the response to common breast cancer chemotherapy and radiotherapy. Thus, we have no evidence that 3-OHB generally influences the biology of breast cancer cells in vitro.Electronic supplementary materialThe online version of this article (10.1186/s40170-018-0180-9) contains supplementary material, which is available to authorized users.

Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by a progressive loss of human corneal endothelial cells (HCEC) and excess deposition of extracellular matrix (ECM). As FECD pathophysiology involves Rho‐associated kinase (ROCK) signalling activation, we investigated the effect of various ROCK inhibitors on the expression of ECM proteins and proteolytic enzymes using ex vivo and in vitro models. Methods: Endothelial cell‐Descemet membrane lamellae (DM) from donor corneas (n = 20) and FECD patients (n = 100) undergoing Descemet membrane endothelial keratoplasty were used as ex vivo model. A human corneal endothelial cell line (HCEC‐12) served as in vitro model. Rho‐ROCK signalling pathway components and the effects of the ROCK inhibitors ripasudil (10/30 μM), Y‐27632 (10/30 μM), and netarsudil (1 μM) on the expression of ECM proteins and proteolytic enzymes were analysed, with or without addition of transforming growth factor β1 (TGFβ1, 5 ng/ml), using quantitative real‐time PCR and Western Blot analysis. Results: Constitutive expression of components of the Rho‐ROCK signalling pathway was higher in DM specimens from FECD patients compared to DM from normal donors. Stimulation with all three ROCK inhibitors significantly downregulated the expression of ECM genes, such as collagen 3 alpha 1 (COL3A1) and fibronectin 1 (FN1), in both FECD and normal DM as well as in HCEC‐12, compared to untreated controls, with ripasudil showing the most significant effect. Downregulation of COL1A1 and FN1 by ripasudil was verified on protein level as well. Ripasudil also suppressed the TGFβ‐induced expression and phosphorylation of downstream molecules of the TGFβ pathway (SMAD2) as well as of the Rho‐ROCK pathway (MLC2). Ripasudil further upregulated gene expression of proteolytic enzymes, e.g. matrix metalloproteinase 1 (MMP1) and MMP3. Conclusions: These findings suggest that inhibition of ROCK signalling in FECD patients may have a beneficial effect by suppressing expression and stimulating turnover of abnormal ECM components.