Yan Jing, Zhang Bingbing, Hosseinzadeh Zohreh, Lang Florian
Cell Physiol Biochem. 2016;38(4):1643-51. doi: 10.1159/000443104. Epub 2016 Apr 28.
BACKGROUND/AIMS: Oscillations of cytosolic Ca2+ activity ([Ca2+]i) participate in the orchestration of tumor cell proliferation. [Ca2+]i could be increased by intracellular Ca2+ release followed by store-operated Ca2+-entry (SOCE). [Ca2+]i could be decreased by Ca2+ extrusion via Na+/Ca2+ exchange. Mechanisms accomplishing SOCE include the pore-forming ion channel unit Orai1 and its regulator STIM1, Na+/Ca2+ exchanger isoforms include NCX1. In MCF-7 breast carcinoma cells Orai1 and NCX1 have previously been shown to be modified by pharmacological inhibition of Janus activated kinase JAK2. The present study explored whether SOCE and Na+/Ca2+ exchange are similarly sensitive to pharmacological JAK3 inhibition.
MCF-7 breast carcinoma cells were studied in the absence and presence of the JAK3 inhibitor WHI-P154 (22 µM). [Ca2+]i was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and Na+/Ca2+ exchanger activity from increase of [Ca2+]i following extracellular Na+ removal. Transcript levels were quantified with RT-PCR.
Addition of ATP (100 µM) was followed by a rapid increase of [Ca2+]i, which was significantly blunted by WHI-P154. Thapsigargin-induced intracellular Ca2+ release was not appreciably influenced by WHI-P154. Subsequent SOCE was, however, significantly blunted by WHI-P154. WHI-P154 further significantly decreased Orai1 transcript levels. The increase of [Ca2+]i following extracellular Na+-removal and the NCX1 transcript levels were similarly decreased by WHI-P154.
The JAK3 inhibitor WHI-P154 decreases both, Orai1 and NCX1 transcript levels and thus impairs SOCE and Na+/Ca2+ exchange.
背景/目的：胞质Ca2+活性（[Ca2+]i）的振荡参与肿瘤细胞增殖的调控。细胞内Ca2+释放后通过储存-操纵性Ca2+内流（SOCE）可使[Ca2+]i升高。通过Na+/Ca2+交换进行的Ca2+外排可使[Ca2+]i降低。实现SOCE的机制包括形成孔道的离子通道单位Orai1及其调节因子STIM1，Na+/Ca2+交换异构体包括NCX1。先前已证明在MCF-7乳腺癌细胞中，Orai1和NCX1可被Janus激活激酶JAK2的药理抑制作用所修饰。本研究探讨了SOCE和Na+/Ca2+交换对JAK3药理抑制作用的敏感性是否相似。
在不存在和存在JAK3抑制剂WHI-P154（22 μM）的情况下研究MCF-7乳腺癌细胞。通过Fura-2荧光估计[Ca2+]i，用肌质网Ca2+-ATP酶（SERCA）抑制剂毒胡萝卜素（1 μM）耗尽Ca2+储存后再添加Ca2+后[Ca2+]i的增加来评估SOCE，通过细胞外Na+去除后[Ca2+]i的增加来评估Na+/Ca2+交换活性。用RT-PCR定量转录水平。
添加ATP（100 μM）后[Ca2+]i迅速升高，而WHI-P154使其明显减弱。毒胡萝卜素诱导的细胞内Ca2+释放未受到WHI-P154的明显影响。然而，随后的SOCE受到WHI-P154的显著抑制。WHI-P154进一步显著降低Orai1转录水平。细胞外Na+去除后[Ca2+]i的增加以及NCX1转录水平同样受到WHI-P154的降低。
JAK3抑制剂WHI-P154降低Orai1和NCX1转录水平，从而损害SOCE和Na+/Ca2+交换。
