title: ATP stimulates chemokine production via a store-operated calcium entry pathway in C6 glioma cells.
abstract: BACKGROUND
  Glioma present as one of the most challenging cancers to treat, however, understanding of tumor cell biology is not well understood. Extracellular adenosine triphosphate (ATP) could serve as a critical signaling molecule regulating tumor development. This study has examined pharmacological modulation of calcium (Ca2+) entry through store-operated channels (SOC) on cellular expression and production of immune-cell mobilizing chemokines in ATP-stimulated C6 glioma cells.
  
  
  METHODS
  Calcium spectrofluorometry was carried out to measure mobilization of intracellular Ca2+ [Ca2+]i following ATP stimulation of rat C6 glioma cells. Pretreatment with two inhibitors of SOC, SKF96365 or gadolinium, was used to examine for effects on [Ca2+]i. RT-PCR was performed to determine effects of purinergic stimulation on C6 cell expression of metabotropic P2Y receptors (P2YR) and the chemokines, monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8). ELISA was carried out to measure production of MCP-1 and IL-8 with ATP stimulation of glioma cells.
  
  
  RESULTS
  Application of ATP (at 100 microM) to C6 glioma induced an increase in [Ca2+]i with the response exhibiting two components of decay. In the presence of the SOC inhibitors, SKF96365 or gadolinium, or with Ca2+-free solution, ATP responses lacked a slow phase suggesting the secondary component was due to SOC-mediated influx of Ca2+. RT-PCR confirmed expression of purinergic P2Y-subtype receptors in C6 cells which would serve as a precursor to activation of SOC. In addition, ATP-stimulated C6 cells showed enhanced expression of the chemokines, MCP-1 and IL-8, with SKF96365 or gadolinium effective in reducing chemokine expression. Gadolinium treatment of ATP-stimulated C6 cells was also found to inhibit the production of MCP-1 and IL-8.
  
  
  CONCLUSIONS
  These results suggest ATP-induced Ca2+ entry, mediated by activation of SOC in C6 glioma, as a mechanism leading to increased cellular expression and release of chemokines. Elevated levels of MCP-1 and IL-8 are predicted to enhance the mobility of tumor cells and promote recruitment of microglia into developing tumors thereby supporting tumor growth.
authors.affiliation: Department of Anesthesiology, Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, 2176 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. scnjt@mahidol.ac.th
authors.forename: Nattinee
authors.initials: N
authors.lastname: Jantaratnotai
authors.forename: Hyun B
authors.initials: HB
authors.lastname: Choi
authors.forename: James G
authors.initials: JG
authors.lastname: McLarnon
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D018925:Chemokines / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005910:Glioma / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D017628:Microglia / Q000473:pathology; D009361:Neoplasm Invasiveness; D011379:Prognosis; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics
chemical_list: C491766:Ccl2 protein, rat; D018932:Chemokine CCL2; D018925:Chemokines; D016209:Interleukin-8; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D002118:Calcium