1α,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells

Abstract

Background/Aims: Tumor cell proliferation is modified by 1,25-Dihydroxy-Vitamin D3 (1,25(OH)₂D₃), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H⁺ ions and lactate, which is in part accomplished by Na⁺/H⁺ exchangers, such as NHE1 and monocarboxylate transporters, such as MCT4. An effect of 1,25(OH)₂D₃ on those transport processes has, however, never been reported. As cytosolic pH impacts on apoptosis, the study further explored the effect of 1,25(OH)₂D₃ on apoptosis and on the apoptosis regulating kinase AKT, transcription factor Forkhead box O-3 (FOXO3A) and B-cell lymphoma protein BCL-2. Methods: In human endometrial adenocarcinoma (Ishikawa) cells, cytosolic pH (pHi) was determined utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na⁺/H⁺ exchanger activity from Na⁺ dependent realkalinization after an ammonium pulse, NHE1 and MCT4 transcript levels using qRT-PCR, NHE1, MCT4, total & phospho AKT, total & phospho-FOXO3A and BCL-2 protein abundance by Western blotting, lactate concentration in the supernatant utilizing a colorimetric enzyme assay and cell death quantification using CytoTox 96^®, Annexin V and Propidium Iodide staining. Results: A 24 hours treatment with 1,25(OH)₂D₃ (100 nM) significantly increased cytosolic pH (pHi), significantly decreased Na⁺/H⁺ exchanger activity, NHE1 and MCT4 transcript levels as well as protein abundance and significantly increased lactate concentration in the supernatant. Treatment of Ishikawa cells with 1,25(OH)₂D₃ (100 nM) further triggered apoptosis, an effect paralleled by decreased phosphorylation of AKT and FOXO3A as well as decreased abundance of BCL-2. Conclusions: In Ishikawa cells 1,25(OH)₂D₃ is a powerful stimulator of glycolysis, an effect presumably due to cytosolic alkalinization. Despite stimulation of glycolysis, 1,25(OH)₂D₃ stimulates slightly but significantly suicidal cell death, an effect presumably in part due to decreased activation of AKT with decreased inhibition of pro-apoptotic transcription factor FOXO3A and downregulation of the anti-apoptotic protein BCL-2.

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“…Equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel in Glycine-Tris buffer, electro-transferred onto polyvinylidene difluoride membranes and blocked with 5% nonfat milk in TBST at room temperature. The membranes were incubated with primary anti-SGK1 (phospho S422) antibody (1:1,000; Abcam, Cambridge, UK), anti-SGK1 antibody (1:1,000; Cell Signaling Technology), anti-NHE1 antibody (1:1,000; Sigma-Aldrich) [39], and anti-GAPDH antibody (1:1,000; Cell Signaling Technology) at 4°C overnight. After washing (TBST), the blots were incubated with secondary anti-rabbit antibody conjugated with horseradish peroxidase (1:2,000; Cell Signaling Technology) for 2 h at room temperature.…”

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“…Propidium iodide (PI, Sigma-Aldrich) uptake was taken as a measure of cell membrane permeability and annexin V-FITC (Immunotools, Friesoythe, Germany) binding as a measure of cell membrane scrambling with phosphatidylserine translocation at the cell surface, as described previously 63 , 64 . To this end, cells were incubated in 100 µl Ringer solution containing 5 mM Ca 2+ and 1 µl annexin V-FITC After incubation, the cells were centrifuged at 1000 r.p.m.…”

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“…Cell Apoptosis Study. Propidium iodide (PI) (Sigma-Aldrich) or 7-aminoactinomycin D (7-AAD) uptake was taken as a measure of cell membrane permeability, and annexin V−FITC binding was used as a measure of cell membrane scrambling with phosphatidylserine translocation at the cell surface, as described previously 14 . 15 To this end, the drug-treated cells were incubated with annexin V−FITC and PI (MCF-7) or annexin V−FITC and 7-AAD (MCF-7/Adr) to assess cell apoptosis and necrosis.…”