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

Jantaratnotai N, Choi HB, McLarnon JG - BMC Cancer (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

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

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.

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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.
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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.

Mentions: Application of ATP (100 μM) to C6 glioma cells resulted in an early phase of increased [Ca2+]i which rapidly declined and was followed by a sustained component of response (Fig. 1A). In the presence of Ca2+-free PSS (0 PSS), ATP administration led to a single phase of [Ca2+]i mobilization representing only the rapid portion of the response (Fig. 1B). These results suggested ATP binding to a P2YR activating an inositol 1,4,5-trisphosphate (IP3)-mediated intracellular release of Ca2+ and a subsequent entry of divalent ion through SOC [21,22]. To test this possibility two inhibitors of SOC, gadolinium and SKF96365, were applied prior to ATP stimulation. The concentrations of gadolinium (at 1 μM) [18] and SKF96365 (at 25 μM) [23,24] were chosen according to ones used in previous studies which were found effective for SOC inhibition. Representative ATP responses are presented with pre-treatment (3 min) with gadolinium (Fig. 1C) or SKF96365 (Fig. 1D). Exposure of C6 to either SOC inhibitor yielded a pattern of ATP response close to that observed in Ca2+-free PSS (Fig. 1B) with attenuation of the prolonged component of [Ca2+]i. Peak amplitudes of [Ca2+]i responses did not appreciably differ between control or the different treatments. Overall results are summarized in Fig. 1E (N = 4/treatment) and show durations (measured at half-maximal of peak value) of ATP responses were similar between Ca2+-free PSS and treatments with the two SOC inhibitors. Respective inhibitions of response durations relative to control ATP response were 44% (0 PSS), 49% (gadolinium) and 55% (SKF96365).


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

Jantaratnotai N, Choi HB, McLarnon JG - BMC Cancer (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807438&req=5

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.
Mentions: Application of ATP (100 μM) to C6 glioma cells resulted in an early phase of increased [Ca2+]i which rapidly declined and was followed by a sustained component of response (Fig. 1A). In the presence of Ca2+-free PSS (0 PSS), ATP administration led to a single phase of [Ca2+]i mobilization representing only the rapid portion of the response (Fig. 1B). These results suggested ATP binding to a P2YR activating an inositol 1,4,5-trisphosphate (IP3)-mediated intracellular release of Ca2+ and a subsequent entry of divalent ion through SOC [21,22]. To test this possibility two inhibitors of SOC, gadolinium and SKF96365, were applied prior to ATP stimulation. The concentrations of gadolinium (at 1 μM) [18] and SKF96365 (at 25 μM) [23,24] were chosen according to ones used in previous studies which were found effective for SOC inhibition. Representative ATP responses are presented with pre-treatment (3 min) with gadolinium (Fig. 1C) or SKF96365 (Fig. 1D). Exposure of C6 to either SOC inhibitor yielded a pattern of ATP response close to that observed in Ca2+-free PSS (Fig. 1B) with attenuation of the prolonged component of [Ca2+]i. Peak amplitudes of [Ca2+]i responses did not appreciably differ between control or the different treatments. Overall results are summarized in Fig. 1E (N = 4/treatment) and show durations (measured at half-maximal of peak value) of ATP responses were similar between Ca2+-free PSS and treatments with the two SOC inhibitors. Respective inhibitions of response durations relative to control ATP response were 44% (0 PSS), 49% (gadolinium) and 55% (SKF96365).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

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

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.

Show MeSH
Related in: MedlinePlus