Down-Regulation of Store-Operated Ca2+ Entry and Na+ Ca2+ Exchange in MCF-7 Breast Cancer Cells by Pharmacological JAK3 Inhibition.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
The JAK3 inhibitor WHI-P154 decreases both, Orai1 and NCX1 transcript levels and thus impairs SOCE and Na+/Ca2+ exchange.
Jing
J
Yan
Bingbing
B
Zhang
Zohreh
Z
Hosseinzadeh
Florian
F
Lang
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001943:Breast Neoplasms; D002118:Calcium / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D005260:Female; D016257:Fura-2 / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D053616:Janus Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D061986:MCF-7 Cells; D000071740:ORAI1 Protein / Q000235:genetics / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
D000071740:ORAI1 Protein; D011799:Quinazolines; D019831:Sodium-Calcium Exchanger; D035181:TATA-Box Binding Protein; C509817:TBP protein, human; C113888:WHI P154; C110374:sodium-calcium exchanger 1; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D053616:Janus Kinase 3; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2