Store-operated Ca(2+) entry in rhabdomyosarcoma cells.
Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, has an intrinsic or early-acquisition of resistance to chemo- and radiation therapy. Molecular determinants pivotal for RMS migration, metastatic invasion, cell proliferation, and survival are incompletely identified. Migration and cell proliferation were shown to correlate with cytosolic Ca(2+) activity ([Ca(2+)]i). Store-operated Ca(2+)-entry (SOCE) that increases intracellular [Ca(2+)] is accomplished by Orai1, a pore-forming ion channel unit, the expression of which is stimulated by the transcription factor NFκB. The present study explored the expression of Orai1 and its regulators STIM1 and NFκB in human rhabdomyosarcoma cell lines and analyzed their impact on cell proliferation and migration. For the study human rhabdomyosarcoma cell lines RD (embryonal) and RH30 (alveolar) were analyzed for Orai1, STIM1, and NFκB transcription by RT-PCR and their corresponding proteins in Western blot. [Ca(2+)]i was detected via Fura-2 fluorescence and SOCE - resulting from [Ca(2+)]i increase following store depletion with extracellular Ca(2+) removal and inhibition of the sarcoendoplasmatic reticular Ca(2+) ATPase - detected with thapsigargin. Cell migration was analyzed in transwell and mitotic cell death with the clonogenic assay. In summary, Orai1, STIM1, and NFκB are expressed in embryonal (RD) and alveolar (RH30) rhabdomyosarcoma. SOCE inhibitor BTP2, Orai1 inhibitor 2-APB, or NFκB inhibitor wogonin virtually abrogated (BTP2, 2-APB) or significantly reduced (wogonin) SOCE. Moreover, SOCE inhibitors 2-APB and BTP2 and wogonin significantly inhibited migration and proliferation of both, RD and RH30 cells. These results suggest that Orai1 signaling is involved in SOCE into rhabdomyosarcoma cells thus contributing to migration, invasion and proliferation.
Department of Pediatric Surgery & Pediatric Urology, Eberhard-Karls-University, Hoppe-Seyler Straße 3, 72076, Tuebingen, Germany. Electronic address: Evi.Schmid@med.uni-tuebingen.de.
Evi
E
Schmid
Department of Pediatric Surgery & Pediatric Urology, Eberhard-Karls-University, Hoppe-Seyler Straße 3, 72076, Tuebingen, Germany.
Matias Julian
MJ
Stagno
Department of Cardiology & Vascular Medicine and Physiology, Eberhard-Karls-University, Gmelinstr.5/Otfried-Mueller-Str.10, 72076, Tuebingen, Germany.
Jing
J
Yan
Department of Biochemistry, University of Crete Medical School, GR-71003, Heraklion, Crete, Greece.
Christos
C
Stournaras
Department of Cardiology & Vascular Medicine and Physiology, Eberhard-Karls-University, Gmelinstr.5/Otfried-Mueller-Str.10, 72076, Tuebingen, Germany.
Florian
F
Lang
Department of Pediatric Surgery & Pediatric Urology, Eberhard-Karls-University, Hoppe-Seyler Straße 3, 72076, Tuebingen, Germany.
Jörg
J
Fuchs
Department of Pediatric Surgery & Pediatric Urology, Eberhard-Karls-University, Hoppe-Seyler Straße 3, 72076, Tuebingen, Germany; Department of Pediatric Surgery, University Hospital Marburg, Marburg, Germany.
Guido
G
Seitz
D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D005453:Fluorescence; D016257:Fura-2; D006801:Humans; D017136:Ion Transport; D016328:NF-kappa B / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics; D000071740:ORAI1 Protein / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012208:Rhabdomyosarcoma / Q000378:metabolism* / Q000473:pathology; D000071737:Stromal Interaction Molecule 1 / Q000235:genetics
D016328:NF-kappa B; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D012333:RNA, Messenger; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium; D016257:Fura-2
Cell proliferation; Migration; Orai1; Rhabdomyosarcoma; SOCE