title: 1α,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells.
abstract: OBJECTIVE
  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.
authors.forename: Ni
authors.initials: N
authors.lastname: Zeng
authors.forename: Yuetao
authors.initials: Y
authors.lastname: Zhou
authors.forename: Shaqiu
authors.initials: S
authors.lastname: Zhang
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Singh
authors.forename: Bing
authors.initials: B
authors.lastname: Shi
authors.forename: Madhuri S
authors.initials: MS
authors.lastname: Salker
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism*; D000071316:Forkhead Box Protein O3 / Q000378:metabolism; D006019:Glycolysis / Q000187:drug effects*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000032:analysis; D027501:Monocarboxylic Acid Transporters / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D027682:Cation Transport Proteins; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D027501:Monocarboxylic Acid Transporters; D009124:Muscle Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C406962:SLC16A4 protein, human; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D019344:Lactic Acid; D051057:Proto-Oncogene Proteins c-akt
keywords: Cytosolic pH regulation; Glycolysis; MCT4; Monocarboxylate transporter; NHE1; Na+/H+ exchanger; Tumor cells; Vitamin D