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Title: Store-operated Ca{sup 2+} entry in rhabdomyosarcoma cells

Abstract

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{sup 2+} activity ([Ca{sup 2+}]{sub i}). Store-operated Ca{sup 2+}-entry (SOCE) that increases intracellular [Ca{sup 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{sup 2+}]{sub i} was detected via Fura-2 fluorescence and SOCE – resulting from [Ca{sup 2+}]{sub i} increase following store depletion with extracellular Ca{sup 2+} removal and inhibition of the sarcoendoplasmatic reticular Ca{sup 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 embryonalmore » (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. - Highlights: • Orai1, STIM1, and NFκB are expressed in RD and RH30 rhabdomyosarcoma cell lines. • Orai1, STIM1, and NFκB are significantly upregulated in the RH30 cell line and leads to a significantly increased SOCE. • Orai1 signaling is involved in SOCE thus contributing to migration, invasion and proliferation.« less

Authors:
 [1];  [1];  [2];  [3];  [2];  [1];  [1];  [4]
  1. Department of Pediatric Surgery & Pediatric Urology, Eberhard-Karls-University, Hoppe-Seyler Straße 3, 72076, Tuebingen (Germany)
  2. Department of Cardiology & Vascular Medicine and Physiology, Eberhard-Karls-University, Gmelinstr.5/Otfried-Mueller-Str.10, 72076, Tuebingen (Germany)
  3. Department of Biochemistry, University of Crete Medical School, GR-71003, Heraklion, Crete (Greece)
  4. (Germany)
Publication Date:
OSTI Identifier:
22606150
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 477; Journal Issue: 1; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; APOPTOSIS; CALCIUM IONS; CELL PROLIFERATION; CONNECTIVE TISSUE; FLUORESCENCE; INHIBITION; METASTASES; PEDIATRICS; POLYMERASE CHAIN REACTION; RADIOTHERAPY; RHABDOMYOSARCOMAS; TRANSCRIPTION; TRANSCRIPTION FACTORS

Citation Formats

Schmid, Evi, E-mail: Evi.Schmid@med.uni-tuebingen.de, Stagno, Matias Julian, Yan, Jing, Stournaras, Christos, Lang, Florian, Fuchs, Jörg, Seitz, Guido, and Department of Pediatric Surgery, University Hospital Marburg, Marburg. Store-operated Ca{sup 2+} entry in rhabdomyosarcoma cells. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.06.032.
Schmid, Evi, E-mail: Evi.Schmid@med.uni-tuebingen.de, Stagno, Matias Julian, Yan, Jing, Stournaras, Christos, Lang, Florian, Fuchs, Jörg, Seitz, Guido, & Department of Pediatric Surgery, University Hospital Marburg, Marburg. Store-operated Ca{sup 2+} entry in rhabdomyosarcoma cells. United States. doi:10.1016/J.BBRC.2016.06.032.
Schmid, Evi, E-mail: Evi.Schmid@med.uni-tuebingen.de, Stagno, Matias Julian, Yan, Jing, Stournaras, Christos, Lang, Florian, Fuchs, Jörg, Seitz, Guido, and Department of Pediatric Surgery, University Hospital Marburg, Marburg. Fri . "Store-operated Ca{sup 2+} entry in rhabdomyosarcoma cells". United States. doi:10.1016/J.BBRC.2016.06.032.
@article{osti_22606150,
title = {Store-operated Ca{sup 2+} entry in rhabdomyosarcoma cells},
author = {Schmid, Evi, E-mail: Evi.Schmid@med.uni-tuebingen.de and Stagno, Matias Julian and Yan, Jing and Stournaras, Christos and Lang, Florian and Fuchs, Jörg and Seitz, Guido and Department of Pediatric Surgery, University Hospital Marburg, Marburg},
abstractNote = {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{sup 2+} activity ([Ca{sup 2+}]{sub i}). Store-operated Ca{sup 2+}-entry (SOCE) that increases intracellular [Ca{sup 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{sup 2+}]{sub i} was detected via Fura-2 fluorescence and SOCE – resulting from [Ca{sup 2+}]{sub i} increase following store depletion with extracellular Ca{sup 2+} removal and inhibition of the sarcoendoplasmatic reticular Ca{sup 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. - Highlights: • Orai1, STIM1, and NFκB are expressed in RD and RH30 rhabdomyosarcoma cell lines. • Orai1, STIM1, and NFκB are significantly upregulated in the RH30 cell line and leads to a significantly increased SOCE. • Orai1 signaling is involved in SOCE thus contributing to migration, invasion and proliferation.},
doi = {10.1016/J.BBRC.2016.06.032},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 477,
place = {United States},
year = {Fri Aug 12 00:00:00 EDT 2016},
month = {Fri Aug 12 00:00:00 EDT 2016}
}