Wanandi S I, Yustisia I, Neolaka G M G, Jusman S W A
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.
Department of Biochemistry, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia.
Braz J Med Biol Res. 2017 Jul 20;50(8):e6538. doi: 10.1590/1414-431X20176538.
Cancer stem cells reside in a distinct region within the tumor microenvironment that it is believed to play a fundamental role in regulating stemness, proliferation, survival, and metabolism of cancer cells. This study aimed to analyze the effect of extracellular alkalinization on metabolism and survival of human CD24-/CD44+ breast cancer stem cells (BCSCs). BCSCs were cultured in alkalinized DMEM-F12 and incubated at 37°C, 5% CO2, and 20% O2 for 30 min, 6, 24, and 48 h. After each incubation period, we analyzed the modulation of various mRNA expressions related to pH and cellular metabolic regulation using the qRT-PCR. Metabolic state was measured using colorimetric and fluorometric assays. To examine cell proliferation and apoptosis, we used trypan blue and annexin V/propidium iodide assay, respectively. This study demonstrated that alkalinization could stimulate extracellular carbonic anhydrase (CAe) activity, as well as CA9 and HIF1α expression. Under alkaline pH and HIF1α regulation, glucose consumption, extracellular lactate production, and LDH activity of BCSCs were upregulated while O2 consumption was downregulated. These metabolic shifts seemed to promote apoptosis and suppress the proliferation of BCSCs. To conclude, modulation of the extracellular environment through alkalinization could change the metabolic states of BCSCs, which in turn affect the cell survival.
癌症干细胞存在于肿瘤微环境中的一个独特区域，据信该区域在调节癌细胞的干性、增殖、存活和代谢方面发挥着重要作用。本研究旨在分析细胞外碱化对人CD24-/CD44+乳腺癌干细胞（BCSCs）代谢和存活的影响。将BCSCs培养于碱化的DMEM-F12中，并在37°C、5% CO2和20% O2条件下孵育30分钟、6小时、24小时和48小时。在每个孵育期后，我们使用qRT-PCR分析与pH和细胞代谢调节相关的各种mRNA表达的调节情况。使用比色法和荧光法测定代谢状态。为了检测细胞增殖和凋亡，我们分别使用了台盼蓝和膜联蛋白V/碘化丙啶检测法。本研究表明，碱化可刺激细胞外碳酸酐酶（CAe）活性以及CA9和HIF1α表达。在碱性pH和HIF1α调节下，BCSCs的葡萄糖消耗、细胞外乳酸生成和LDH活性上调，而氧气消耗下调。这些代谢变化似乎促进了凋亡并抑制了BCSCs的增殖。总之，通过碱化调节细胞外环境可改变BCSCs的代谢状态，进而影响细胞存活。
