ATP stimulates chemokine production via a store-operated calcium entry pathway in C6 glioma cells

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 (Ca(2+)) 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 Ca(2+ )[Ca(2+)]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 [Ca(2+)]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 μM) to C6 glioma induced an increase in [Ca(2+)]i with the response exhibiting two components of decay. In the presence of the SOC inhibitors, SKF96365 or gadolinium, or with Ca(2+)-free solution, ATP responses lacked a slow phase suggesting the secondary component was due to SOC-mediated influx of Ca(2+). 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. CONCLUSION: These results suggest ATP-induced Ca(2+ )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.