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首页> 外文期刊>Cellular Physiology and Biochemistry >1?±,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells
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1?±,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells

机译：人子宫内膜石川细胞中1？±，25（OH）2D3敏感的胞质pH调节和糖酵解通量

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>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.
机译：> 背景/目的： 肿瘤细胞增殖被1,25-二羟基维生素D3（1,25（OH） 2 D 3 ），一种甾族激素，主要因其在钙和磷代谢中的作用而闻名。肿瘤细胞的关键特性包括增强的糖酵解通量，葡萄糖的过度消耗和乳酸的形成。由于糖酵解对胞质pH高度敏感，因此维持糖酵解需要输出H + 离子和乳酸，这部分由Na + / H + <交换器（例如NHE1）和单羧酸盐转运蛋白（例如MCT4）。然而，从未报道过1,25（OH） 2 D 3 对这些转运过程的影响。由于细胞质pH值对细胞凋亡的影响，本研究进一步探讨了1,25（OH） 2 D 3 对细胞凋亡以及对细胞凋亡调节激酶AKT，转录因子Forkhead的影响盒O-3（FOXO3A）和B细胞淋巴瘤蛋白质BCL-2。 方法： 在人子宫内膜腺癌（Ishikawa）细胞中，利用（2'，7'-bis-（2-羧乙基）-5- （and-6）-羧基荧光素[BCECF]荧光，铵脉冲后Na + 依赖性碱化作用引起的Na + / H + 交换子活性，使用qRT-PCR检测NHE1和MCT4转录水平，NHE1，MCT4，总和磷酸AKT，总和磷酸FOXO3A和BCL-2蛋白的丰度（通过Western印迹），使用比色酶测定法测定上清液中的乳酸浓度以及使用CytoTox进行细胞死亡定量96 ?? ，膜联蛋白V和碘化丙啶染色 结果： 用1,25（OH） 2 < / sub> D 3 （100 nM）显着增加了细胞质pH（pHi），显着降低了Na + / H + 交换子活性，NHE1和MCT4转录水平以及蛋白质丰度和上清液中乳酸浓度显着增加。含1,25（OH） 2 D 3 （100 nM）的石川细胞的nt进一步触发凋亡，这种作用与AKT和FOXO3A磷酸化的降低以及降低有关。丰富的BCL-2。 结论： 在石川细胞中，1,25（OH） 2 D 3 是一种强大的糖酵解刺激剂，大概是由于胞浆碱化。尽管刺激了糖酵解作用，但是1,25（OH） 2 D 3 会轻微刺激细胞，但会导致自杀性细胞死亡，这可能部分是由于AKT的激活减少而抑制作用减少凋亡转录因子FOXO3A的表达和抗凋亡蛋白BCL-2的下调。

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