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.

Conclusion: 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.

Figure 1

ATP-induced changes in [Ca²⁺]i in C6 glioma. (A) Representative response for ATP (100 μM) showing rapid and slow components of decay. (B) Typical ATP response in Ca²⁺-free PSS. (C) ATP response following gadolinium (Gd³⁺) pretreatment (1 μM, for 3 min). (D) ATP response in the presence of SKF96365 (25 μM, pretreatment for 3 min). (E) Quantification of durations of ATP responses for the different treatments (measured at one-half of peak response; N = 4/treatment). Values are mean ± SEM. *p < 0.001 compared with control.

Figure 2

Expression of P2YR linked to calcium stores in rodent C6 glioma. The levels of receptor subtype P2Y₁, P2Y₂, P2Y₄ and P2Y₆ were examined in control and ATP-stimulated C6 cells (100 μM; 4 h exposure). The number of PCR cycles was 35 for all receptor subtypes. GAPDH served as a reaction standard. Data are representatives from 4 independent experiments.

Figure 3

Expression of MCP-1 and IL-8 in C6 cells. (A) Representative levels for MCP-1 (upper row) and IL-8 (middle row) for the different treatments (4 h) applied to C6 glioma. Concentrations of ATP, SKF96365 and gadolinium (Gd³⁺) were the same as used in Ca²⁺ studies (Fig. 1). GAPDH served as a reaction standard (lower row). (B) Overall results (N = 4 experiments) showing relative chemokine expression normalized with GAPDH. Values are mean ± SEM. *p < 0.05 compared with control, **p < 0.05 compared with ATP-treated group.

Figure 4

Production of MCP-1 and IL-8 in C6 glioma. C6 cells were treated with ATP (100 μM) in the presence, or absence, of gadolinium (Gd³⁺, 1 μM) for 48 h. (A) Levels of MCP-1 production (ng/ml) and (B) levels of IL-8 production (pg/ml) for the different treatments. Data are presented as mean ± SEM from 4 independent experiments. * indicates significant difference from control (p < 0.001) and ** indicates significant difference from ATP-treated group (p < 0.05).